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MICROBIAL PRODUCTION OF GLUCOSE OXIDASE AND ITS COMMERCIAL APPLICATIONS

SHAZIA KHURSHID

Session: 2003-2008 Reg. No. 06-Ph.D-GCU-CHEM-03

DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE, PAKISTAN

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"MICROBIAL PRODUCTION OF GLUCOSE OXIDASE AND ITS COMMERCIAL APPLICATIONS"

Submitted to G C University Lahore in partial fulfillment of the requirement for the award of the degree of

DOCTOR OF PHILOSOPHY IN CHEMISTRY BY SHAZIA KHURSHID Session: 2003-2008

Reg. No. 06-Ph.D-GCU-CHEM-03

DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE, PAKISTAN

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ABSTRACT

Glucose oxidase (EC 1.1.3.4) is an important enzyme that oxidizes glucose to gluconic acid. It is present in all aerobic organisms and has become a very useful enzyme for its wide applications especially in food industry and in clinical analysis. The most important application for GOX is the determination of glucose using biosensor technology. GOX belongs to a large group of enzymes oxido reductase and is also called as glucose aerodehydrogenase Glucose oxidase was produced from different microorganisms. Both fungi and bacteria produce glucose oxidase during fermentation. The present project was planned for the optimum production of glucose oxidase by Aspergillus niger and its utilization for estimation of glucose and for the production of calcium, gluconate, gluconic acid and its derivatives. The project was divided into two parts, in the first part production of glucose oxidase from Aspergillus niger was investigated and the second part consists of commercial applications of glucose oxidase. Here the aim was to improve GOX production using mutagenesis of A. niger, to optimize the conditions of fermentation, screen fungal strains producing highest GOX activity, and to medium composition. Mutagenesis was carried out on several strains at different time intervals. GOX enzyme purified by (NH4)2SO4 precipitation technique was dialysed and subjected to gel filtration chromatography. The enzyme was found to be intracellular. Five strains of A. niger isolated from grapes, bread, potato, pickle and sugar beet sources were screened for maximum GOX production. It is clear from our results that the A. niger strain isolated from potato was best for GOX production. This strain showed the maximum enzyme activity in medium containing 10% (w/v) glucose and at pH 5.5. Different conditions like the fermentation period, varying concentrations of urea, MgSO4.7H2O, CaCO3 and KH2PO4 were optimized by conducting different experiments. The maximum activity of glucose oxidase was recorded after 48 hours of continuous shaking fermentation of optimum growth medium containing 3.5% (w/v) CaCO3, 0.2% (w/v) Urea, 0.4% (w/v) KH2PO4 and 0.01% (w/v) MgSO4.7H2O. It was observed that addition of Urea, CaCO3, and KH2PO4 in the medium enhanced the GOX production whereas addition of MgSO4.7H2O decreased the GOX production. The GOX was found

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to be quite active upto 60oC with optimum temperature at 30oC. The batch fermentation volume of 50 ml at 100 rpm speed shaker was found to be the optimum for GOX production. Among mutant, it was found that mutant (9) had maximum activity and growth. The UV induced mutation gave a stable and viable culture for hyper production of GOX as the production was enhanced. Then the enzyme was purified by (NH4)2SO4 precipitation technique, Dialysis and Gel filtration chromatography. It was observed that enzyme activity was increased by increasing (NH4)2SO4 concentration. Enzyme activity also increased by Dialysis and Gel filtration chromatography from 11.90 to 37.24 µ/ml. Purification was 11.55 folds than simple precipitation at this final step. In the second part of project two commercial applications of GOX were investigated i.e. estimation of glucose by standardization of conditions using GOX and the production of calcium gluconate, gluconic acid and its derivatives using GOX. In the first application the three enzymes GOX, mutarotase (EC # 5.1.3.3) and peroxidase (EC # 1.11.1.) were produced, extracted and purified for the preparation and optimization of glucose estimation kit. The enzyme concentrations of 5 µL mutarotase, 15 µL glucose oxidase and 10 µL of peroxidase with chromagen Guaiacol added before peroxidase, proved to be best for estimations of glucose in blood samples. The sensitivity of the best kit was as low as 50 mg/dL glucose. The wavelength of 470 nm was best for the test. The results were comparable with standard kit of Medisense Abbott (UK). In the second application, calcium gluconate and gluconic acid and its derivatives were produced by glucose oxidase from Aspergillus niger. The time course during fermentation showed that the calcium gluconate production was maximum at 48 hours after conidial inoculation. The cultural conditions optimized for maximum calcium gluconate production were, glucose concentration 10% (w/v), pH 5.5, 7% (w/v) CaCO3, 0.2% (w/v) urea 0.15% (w/v) KH2PO4 concentration at 35oC. Different nitrogen, phosphate and metal carbonate sources were also optimized. The present study also described the production of gluconic acid and its derivatives on the laboratory scale. Gluconic acid and its metal salts such as sodium, magnesium, copper and nickel gluconates were synthesized from calcium gluconate which was produced by fermentation. The gluconic acid was released by the action of oxalic acid and sulphuric acid on calcium gluconate. Sulphuric acid gave better yields i.e. (90%) as compared to oxalic acid (80%). So the organic acid was obtained by

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H2SO4 in the present work because it was cheap and readily available in local market. Metal gluconates were also produced by both the double decomposition and gluconic acid methods respectively. It is clear from the study that the gluconic acid method gave greater yields compared to the double decomposition method. This project will help in the commercial production of products using GOX in Pakistan.

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CONTENTS

Chapter 1. 1.1 1.2 1.3 1.4 1.5 1.6 Page No.

Introduction

Classification of enzymes Fermentation Glucose oxidase (GOX) General characteristics of GOX Applications of glucose oxidase Commercial applications of glucose oxidase

1-29

1 2 4 5 7 10 10 18 22 23 24 25 26 29 30-67 30 42 48 60 68-94 68 68 73 75 75

1.6.1 The estimation of glucose in diabetic mellitus patients 1.6.2 Production of calcium gluconate, gluconic acid and its derivatives by GOX method 1.7 Aspergillus niger: (selected specie)

1.7.1 Success of Aspergillus niger in fermentation 1.7.2 Mutagenesis 1.7.3 Mutagenesis by U.V. Pictures of A. niger Aim of work 2. 2.1 2.2 2.3 2.4 3. 3.1 Review of Literature Production of glucose oxidase (GOX) Properties of glucose oxidase Application of GOX Enzyme source Materials and Methods (A) Production of glucose oxidase from Aspergillus niger

3.1.1 Screening for the fungal strain producing the highest GOX activity along with optimizing carbon source and pH 3.1.2 Optimization of fermentation conditions for max GOX production 3.1.3 Enzyme kinetics 3.1.4 Enhanced GOX Production by UV mutation

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3.1.5 Purification of glucose oxidase 3.2 Commercial applications of glucose oxidase

76 78

3.2.1 Estimation of glucose by standardization of conditions using GOX 78 3.2.2 Production of calcium gluconate, gluconic acid and its derivatives by GOX Method Standard curve of glucose 4. 4.1 Results and Discussions Production of glucose oxidase from A. niger 84 86 95-183 95 95 138 144 146 149 154 155 169 181 184 188 189 190-212

4.1.1 Screening for microbial strain producing the highest GOX activity along with optimizing pH and carbon source. 4.1.2 Optimization of other fermentation conditions for maximum GOX production. 4.1.3 Enzyme kinetics 4.1.4 Enhance GOX production by UV mutation 4.1.5 Purification of GOX 4.2 Commercial applications of glucose oxidase

4.2.1 Estimation of glucose by standardization of conditions 4.2.2 The Production of calcium gluconate, gluconic acid and its derivatives by GOX method. 4.3 Production of gluconic acid and its derivatives. Summary Recommendations Future extension of research project References

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List of Tables

Table No. 3.1 3.2 3.3 3.4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 Composition of fermentation medium. Composition of each 250 ml flask. Composition of growth medium Composition of each 250 ml flask Effect of glucose concentration on glucose oxidase production by A. niger (source bread) Effect of source potato concentration on glucose oxidase production by A. niger (potato source) Effect of glucose concentration on glucose oxidase production by A. niger (source grapes) Effect of glucose concentration on glucose oxidase production by A. niger (source pickle) Effect of glucose concentration on glucose oxidase production by A. niger (source sugar beet) Effect of pH on glucose oxidase production by A. niger (source bread) Effect of pH on glucose oxidase production by A. niger (source potato) Effect of pH on glucose oxidase production by A. niger (source grapes) Effect of pH on glucose oxidase production by A. niger (source pickle) Effect of pH on glucose oxidase production by A. niger (source sugar beet) Effect of fermentation period on GOX production. Effect of CaCO3 concentration on GOX production. Effect of urea concentration of GOX production. Effect of KH2PO4 concentration on GOX Production. Effect of MgSO4.7H2O concentration on GOX production. Effect of temperature on GOX production. Effect of aeration on GOX production. Different colony restrictor used in A. niger Intracellular glucose oxidase activity in shake flask cultures Page No. 69 70 85 85 98 102 106 110 114 119 123 127 131 135 139 140 141 142 143 144 146 147 148

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4.20

GOX activity in crude extract s

149 150 151 151 152 154 155 156 157 158 158 159 160 161 161 162 163 165 166 167 171 172 173 174 175 177 178 179 180

4.21 Precipitation of protein by ammonium sulphate 4.22 4.23 4.24 4.25 4.26 4.27 4.28 4.29 4.30 4.31 4.32 4.33 4.34 4.35 4.36 4.37 4.38 4.39 4.40 4.41 4.42 4.43 4.44 4.45 4.46 4.47 4.48 GOX purification by ammonium sulphate precipitation technique GOX purification by dialysis Analysis of gel filtration chromatography for GOX Summary of GOX Purification Summary of GOX purification Mutarotase activity in crude extract Mutarotase purification by (NH4)2SO4 precipitation technique Mutarotase purification by dialysis Analysis of gel filtration chromatogtraphy mutarotase Summary of mutarotase purification Analysis of peroxidase in crude extracts Summary of peroxidase purification by (NH4)2SO4 precipitation technique Dialysis Analysis of gel filtration chromatography for peroxidase Summary of peroxidase purification Analysis of results for glucose estimation to standardize the conditions (addition of enzymes at same time) Comparison of with standard kit. Determination of sensitivity of our local kit Effect of fermentation period on calcium gluconate production Effect of pH on calcium gluconate production. Effect of glucose concentration on calcium-gluconate production Effect of different carbonates on calcium gluconate production Effect of different concentration of calcium carbonate on calcium gluconate production Effect of different nitrogen sources on calcium gluconate production Effect of urea concentration on calcium gluconate production. Effect of different phosphate sources on calcium gluconate production Effect of different concentration of KH2PO4 on the production

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of calcium gluconate 4.49 4.50 Yield of gluconic acid from calcium gluconate Yields of metal gluconates 181 182

List of Figures

Figure # Page No. 1.1 1.2 3.1 3.2 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 Structural representation of the GOX reaction Pictures of A. niger Standard curve for bovine serum albumin for protein estimation Standard curve of glucose Effect of glucose concentration on mycelium mass Effect of glucose concentration on mycelium mass (dry) Effect of glucose concentration on enzyme activity Effect of glucose concentration on total protein Effect of glucose concentration on specific enzyme activity Effect of glucose concentration on mycelium mass Effect of glucose concentration on mycelium mass (dry) Effect of glucose concentration on enzyme activity Effect of glucose concentration on total protein Effect of glucose concentration on specific enzyme activity Effect of glucose concentration on mycelium mass Effect of glucose concentration on mycelium mass (dry) Effect of glucose concentration on enzyme activity Effect of glucose concentration on total protein Effect of glucose concentration on specific enzyme activity Effect of glucose concentration on mycelium mass Effect of glucose concentration on mycelium mass (dry) Effect of glucose concentration on enzyme activity Effect of glucose concentration on total protein Effect of glucose concentration on specific enzyme activity 05 26 73 87 99 99 100 100 101 103 103 104 104 105 107 107 108 108 109 111 111 112 112 113

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4.21 4.22 4.23 4.24 4.25 4.26 4.27 4.28 4.29 4.30 4.31 4.32 4.33 4.34 4.35 4.36 4.37 4.38 4.39 4.40 4.41 4.42 4.43 4.44 4.45 4.46 4.47 4.48 4.49 4.50 4.51

Effect of glucose concentration on mycelium mass Effect of glucose concentration on mycelium mass (dry) Effect of glucose concentration on enzyme activity Effect of glucose concentration on total protein Effect of glucose concentration on specific enzyme activity Effect of pH on mycelium mass Effect of pH on mycelium mass (dry) Effect of pH on enzyme activity Effect of pH on total protein Effect of pH on specific enzyme activity Effect of pH on mycelium mass Effect of pH on mycelium mass (dry) Effect of pH on enzyme activity Effect of pH on total protein Effect of pH on specific enzyme activity Effect of pH on mycelium mass Effect of pH on mycelium mass (dry) Effect of pH on enzyme activity Effect of pH on total protein Effect of pH on specific enzyme activity Effect of pH on mycelium mass Effect of pH on mycelium mass (dry) Effect of pH on enzyme activity Effect of pH on total protein Effect of pH on specific enzyme activity Effect of pH on mycelium mass Effect of pH on mycelium mass (dry) Effect of pH on enzyme activity Effect of pH on total protein Effect of pH on specific enzyme activity Effect of fermentation period on GOX production

115 115 116 116 117 120 120 121 121 122 124 124 125 125 126 128 128 129 129 130 132 132 133 133 134 136 136 137 137 138 139

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4.52 4.53 4.54 4.55 4.56 4.57 4.58 4.59 4.60 4.61 4.62 4.63 4.64 4.65 4.66 4.67 4.68 4.69 4.70 4.71 4.72 4.73 4.74 4.75 4.76 4.77

Effect of CaCO3 concentration on GOX production Effect of urea concentration of GOX production Effect of KH2PO4 concentration on GOX production Effect of MgSO4.7H2O concentration on GOX production Effect of temperature on GOX production Intracellular glucose oxidase activity in shake flask culture Precipitation of protein by ammonium sulphate Analysis of gel filtration chromatography for GOX Analysis of gel filtration chromatography for GOX Analysis of gel filtration chromatography for GOX Standard curve of -D-glucose for mutarotase analysis Mutarotase purification by (NH4)2SO4 precipitation technique Analysis of gel filtration chromatography mutarotase Analysis of gel filtration chromatography for peroxidase Comparison with standard (human) kit. Determination of sensitivity of sell prepared kit. Effect of fermentation period on calcium gluconate production Effect of pH on calcium gluconate production. Effect of glucose concentration on calcium-gluconate production Effect of different carbonates on calcium gluconate production Effect of different concentration of calcium carbonate on calcium gluconate production Effect of different nitrogen source on calcium gluconate production. Effect of urea concentration on calcium gluconate production. Effect of different phosphate sources on calcium gluconate production Effect of different concentration of KH2PO4 on the production of calcium gluconate Effect of temperature on glucose oxidation rate

140 141 142 143 145 149 150 153 153 154 156 157 159 163 167 168 171 172 174 175 176 177 178 179 180 181

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Chapter No. 1

INTRODUCTION

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INTRODUCTION

Enzymes are the catalysts of biological systems. They have extraordinary catalytic power often far greater than that of synthetic catalyst (Nelson and Cox, 2000). Chemically most enzymes are proteins with molecular masses ranging from thousands into millions of Daltons. Two remarkable properties of enzymes are their extraordinary specificity, their amazing efficiency and they may speed up the reactions by factors of up to 1020. Each enzyme possesses a region known as the active site and the substrate binds it self with this active site.

1.1

Classification of enzymes

The commission on enzyme appointed by the International Union of Biochemistry (IUB) (1961) classified enzymes into six main classes.

1. Oxidoreductases

These enzyme catalyze oxidation-reduction reaction e.g. glucose oxidase, dehydrogenase and peroxidase.

2. Transferases

These enzymes bring about an exchange of functional group such as phosphate or acyl between two compounds e.g. transferases etc.

3. Hydrolases

These enzymes catalyze hydrolysis. Common examples are proteases, amylases, Lipases etc.

4. Lyases

These enzymes catalyze the addition of ammonia, water or carbondioxide to double bonds or removal of these to form double bonds. An example is the conversion of fumaric acid to maleic acid in the presence of fumarase enzyme.

5. Isomerases

These enzyme catalyze the transfer of groups within molecules to yield isomeric forms of the substrate e.g. phosphor-glyceromutase.

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6. Ligases

These enzymes link two molecules together through the breaking of high energy bonds e.g. acetyl-S-COH, acarboxylase and succinic thiokinase.

1.2

Fermentation

Fermentation is a process that is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of ATP (Adenosine triphosphate) by glycolysis. During this process pyruvate is metabolised to various different compounds. Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and heterolactic fermentation is the production of lactic acid as well as other acids and alcohols. It is the process of releasing energy from a carbohydrate without oxygen by producing alcohol or lactic acid. In common usage fermentation is a type of anaerobic respiration, however a more strict definition exists which defines fermentation as respiration under anaerobic conditions with no external electron acceptor. This process does not necessarily have to be carried out in an anaerobic environment, however. For example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption. Sugars are the common substrates of fermentation, and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be produced by it, such as butyric acid and acetone. Yeast famously carries out fermentation in the production of ethanol in beers, wines and other alcoholic drinks, along with the production of large quantities of carbon dioxide. Anaerobic respiration in mammalian muscle under periods of intense exercise (which has no external electron acceptor) is, under the strict definition, a type of fermentation.

Ø Uses of fermentation

Ancient fermented food processes, used for making bread, wine, cheese, curds, idli, dosa, etc. can be dated to more than 6,000 year ago. They were developed long before man had any knowledge of the existence and involvement of the microorganisms. Also, improved fermentation is a powerful economic goal for semi-industrialized countries, to produce local fuels like bio-ethanol and medicines.

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·

The primary benefit of fermentation is the conversion of sugars and other carbohydrates, e.g., converting juice into wine, grains into beer, carbohydrates into carbon dioxide to leaven bread, and sugars in vegetables into preservative organic acids.

·

Food fermentation has been said to serve five main purposes (Steinkraus 1995). (1) Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates. (2) Preservation of substantial amounts of food through lactic acid, alcohol, acetic acid and alkaline fermentations. (3) Biological enrichment of food substrates with protein, essential amino acids, essential fatty acids, and vitamins. (4) (5) Detoxification during food-fermentation processing. A decrease in cooking times and fuel requirements.

·

Fermentation has some uses exclusive to foods. It can produce important nutrients or eliminate antinutrients. Food can be preserved by fermentation, since this process uses up food energy and can make conditions unsuitable for undesirable microorganisms. For example, in pickling the acid produced by the dominant bacteria inhibits the growth of all other microorganisms. Depending on the type of fermentation, some products (e.g., fusel alcohol) can be harmful to people's health. In alchemy, fermentation is often the same as purification, meaning to allow the substance to naturally rot or decompose.

Ø Role of fermentation in pharmaceuticals and biotechnology industry

There are 5 major groups of commercially important fermentation: (1) (2) Microbial cells or biomass as the product, e.g. bakers yeast, lactobacillus, etc. Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase, and glucose oxidase.

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(3)

Microbial metabolites :

(i) Primary metabolites: ethanol, citric acid, glutamic acid, lysine, vitamins, polysaccharides. (ii) Secondary metabolites: all antibiotic fermentation

(4) (5)

Recombinant products: insulin, HBV, vacine, interferon, GCSF, streptokinase Biotransformations: phenyl acetyl carbinol, steroid biotransformation.

1.3

Glucose oxidase (GOX)

Glucose Oxidase (-D-glucose: Oxygen, 1-Oxidoreductase, EC 1.1.3.4) is an enzyme that oxidizes glucose to gluconic Acid. It is present in all aerobic organisms and normally functions in conjunction with catalase. It has become a very useful enzyme for its wide applications, especially in food industry and in clinical analysis (Bucke, 1983). Glucose oxidase belongs to the large group of enzymes oxido reductases and is also called glucose aerodehydrogenase (Witteveen et al; 1992) Glucose oxidase is a flavo enzyme that catalyzes the oxidation of -D-glucose to -D gluconolactone with H2O2 is also formed in this reaction (White et al, 1964). The reaction catalyzed by glucose oxidase is -D-glucose + Enzyme-FAD Enzyme-FADH2 + O2 H2O2

Catalase

Enzyme ­FADH2 + -D-gluconolactone Enzyme-FAD + H2O2

H2O + 1/2 O2 (Worthington, 1988).

The reaction can be divided into, a reductive and an oxidative step. In the reductive half reaction, GOX catalyses the oxidation of -D-glucose to D-glucono--lactone which is non-enzymatically hydrolyzed to gluconic acid. Subsequently the flavine adenine dinucucleotide (FAD) ring of GOX is reduced to FADH2 (Witt et al., 2000). In the oxidative half reaction the reduced GOX is re oxidised by oxygen to yield hydrogen peroxide.

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CH2OH O OH H H OH H HO H OH -D-glucose

H

Gox

CH2OH O H H H O OH HO H OH D-glucono--lactone

H

CH2OH

H2O

H HO

OH

H OH

H

COOH

H OH Guconic acid

GOX-FAD

GOX-FADH2

H2O 2

Oxidative half reaction

O2

Figure 1.1

Structural representation of the GOX reaction

The hydrogen peroxide is cleaved by catalase (EC 1.11.1.6; CAT) to produce water and oxygen, (Beltrame et al., 2004). Witteveen et al; (1992) stated that in A. niger the enzyme lactonase (EC 3.1.1.17) was responsible for catalysing the hydrolysis of glucono-lactone to gluconic acid, although the presence of lactonase was not necessary since the hydrolysis step does occur spontaneously but at a lower rate.

1.4

General characteristics of GOX

The molecular weight of GOX ranges from approximately 130 kDa (Kalisz et al., 1997) to 175 kDa (Eriksson et al., 1987). The GOX enzyme is highly specific for the -anomer of D-glucose, while the -anomer does not appear to be a suitable substrate. Low GOX activities are exhibited when utilizing 2-deoxy-D-glucose, D-mannose and D-galactose as substrates. Inhibitors of GOX include p-chloromecuribenzoate, Ag+, Hg2+,Cu2+, hydroxylamine, hydrazine, phenylhydrazine, dimedone and sodium bisulphate (Kusai et al; 1960). Nakamura and Fujiki (1968) performed comparative studies on the GOX enzymes of A. niger and P. amagasakiense. The molecular weights of the GOX enzymes from the 2 organisms were determined to be 150 kDa for the P. amagasakiense and 152 kDa for A. niger. The carbohydrate and amino acid compositions of the enzymes were also

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investigated and compared, which indicated that similar carbohydrates were contained in both enzymes which consisted mainly of glucose, mannose and hexosamine. The A. niger GOX contained more mannose and hexosamine than that of P. amagasakiense, but less glucose. The overall carbohydrate content was found to be 16% for A. niger and 11% for P. amagasakiense. The amino acid contents of both enzymes showed that the A. niger GOX contained more histidine, arginine and tyrosine and less lysine and phenylalanine than the P. amagasakiense GOX (Nakamura and Fujiki, 1968). The optimum pH ranges for the A. niger GOX and P. amagasakiense GOX production, were shown to be 3.5-6.5 and 4.05.5, respectively. A. niger GOX was found to have a broader pH range than the P. amagasakiense GOX. Glucose oxidase has a molecular weight of 160,000 a.m.u. (Tsuge et al, 1975) and consists of two identical polypeptide chain subunits having nearly equal molecular weights linked by disulphide bonds (O'Malley and Weaver, 1972) and it is highly specific for -D-glucose (Bentley, 1966). Each subunit of the glucose oxidase contains one mole of Fe and one mole of FAD (Flavin adenine dinucleatide) and it contains 74% protein, 16% natural sugar and 2% amino sugars (Tsuge et al, 1975). The Glucose oxidase enzyme in its purest form is pale-yellow powder. Dried enzyme samples are stable at 0°C for upto 2 years. White 0.1 to 0.2% aqueous solutions are stable for one week at 5°C and its enzyme activity was lost on heating at 39°C (Sidney and Northon, 1955). Glucose oxidase is being produced mostly by microorganisms such as Penecillium notatum Pencillium Chryosporium, Aspergillus niger and Botrytis cinerea (Liu et al; 1998). The glucose oxidase enzyme is produced from Aspergillus niger in the submerged culture with continuous shaking (Fiedurck and Gromada, 1996). Waste mycelium of Aspergillus niger are used to produce the glucose oxidase. There is also effect of metal ions on the glucose oxidase activity. At higher salt concentrations, the metal chlorides and metal gluconates show various degrees of inhibition (Yang et al, 1996). The glucose oxidase from Aspergillus niger is an intracellular enzyme present in the mycelium of the organism and it is released from the mycelium by means of cell disruption techniques (Zetelaki and Vas, 1968). The tertiary structure of glucose oxidase from Aspergillus niger was determined by X-ray crystallography and results were drawn from experiments on

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electrical communication between the enzyme and the electrode. (Hecht et al, 1993). Glucose oxidase is commercially prepared from two fungal sources. Aspergillus niger and Penicillium amagasakiense (Rogalski et al 1988). Fiedurck et al (1986) found after screening different fungi that Aspergillus niger is the best source for the production of this enzyme.

1.5

Applications of glucose oxidase

In Food industries, Glucose Oxidase is used to remove the oxygen from beverages, powdered eggs and as a source of hydrogen peroxide in Food preservation. Reduction of non enzymatic browning in potato chips and French fries with glucose oxidase has been observed (Jiang and Oraikul, 1989). It has also been observed that glucose oxidase-glucose system shows antibacterial effect on food poisoning organisms (Tiina et al, 1989).

The importance of GOX comes from its wide range of applications in many fields.

GOX is of considerable commercial importance due to its applications in food science, clinical chemistry and biotechnology Raba and Mottola (1995) reported that GOX is the most widely used enzyme as an analytical reagent due to its application in the determination of glucose concentration in biological fluid.

Wilson and Turner (1992) also attributed the success of GOX as a diagnostic reagent to the enzyme's relative specificity. Raba and Mottola (1995) reviewed glucose oxidase as an analytical reagent and stated that the glucose/GOX system was a convenient model for method development especially in the area of biosensors.

GOX is of interest in relation to antibacterial properties in honey. It catalyses glucose to form gluconic acid and Hydrogen Peroxide (H2O2), the main agents responsible for antibacterial activity in most honeys.

GOX, usually in combination with CAT is used to stabilize colour and flavour in beer, fish, tinned foods, and soft drinks by the removal of oxygen (Crueger and Crueger, 1990).

GOX is used to remove glucose during the manufacture of egg powder, preventing browning during dehydration caused by the Maillard reaction (Crueger and Crueger, 1990).

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GOX has found its applications in the baking industry, providing slight improvements to the crumb properties in bread and croissants (Rasiah et al., 2005). GOX was also widely used to produce gluconic acid that was used as a mild acidulant in the metal, leather and food industries and also produced its metal salts (Crueger and Crueger, 1990; Nakao et al., 1997 and Klein et al., 2002). GOX has also main role in gluconic acid production (Godjevargova, et al 2004) and (Petruccioli, et al; 1997).

The most important application for GOX is the determination of glucose using biosensor technology (Wilson and Turner, 1992). Commercial diagnostic kits for the determination of glucose in blood, serum and plasma are supplied in colorimetric kits (Wilson and Turner, 1992). GOX together with CAT or HRP has a range of applications in the food industry for glucose determination and as an antioxidant. The GOX-CAT enzyme system was used by (Isaksen and Adler-Nissen, 1997) to scavenge oxygen in mayonnaises with different oxidative susceptibility. The investigation proved that the GOX-CAT enzyme system could be used to retard the lipid oxidation in mayonnaise stored at 5°C and 25°C, in mayonnaises containing pure soybean oil and where up to half the vegetable oil had been supplemented with fish oil. The enzyme system was responsible for scavenging the oxygen during glucose oxidation thereby decreasing the availability of the oxygen for lipid metabolism.

(Parpinello et al. 2002) conducted preliminary studies into the use of the GOXCAT enzyme system to control the browning of apple and pear purees by removing 99% of the oxygen content. Oxygen is known to be a key factor in the browning of fruit purees, and the enzyme system was shown to have the capability to control the non-enzymatic browning during fruit processing and purée storage.

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-D-Glucose

-D-Glucono- -lactone

FAD

GOX

FADH2

1/ 2 O 2

H2O 2

CAT

1/ 2 O 2

H2O

-D-Gluconic acid GOX has found application in the textile industry producing hydrogen peroxide for bleaching process (Tzanov et al., 2002). (Tzanov et al. 2002) covalently immobilised GOX on alumina and glass supports, resulting in higher enzyme recoveries. Maximum hydrogen peroxide

concentrations of 0.35 gL-1 and 0.24 gL-1 were reached after 450 minutes for GOX immobilised on the glass and alumina supports respectively (20g glucose in 50ml 0.1 M acetate buffer, pH 5, 35°C, and aerated at 5 L/min). The alumina support proved to be more stable at the operational conditions and could be used for three consecutive runs. The hydrogen peroxide produced was tested for bleaching scoured woven cotton fabric and was found to be comparable to the standard bleaching processes. No stabilizers were needed since the gluconic acid produced acted as a stabilizing agent. In pharmaceutical and analytical biochemistry it is used for quantitative determination of glucose in biological fluid (Kunst, 1984). In manufacturing of glucose biosensors as a new tool for analysis (Fortier et al 1988 Ciucu, 1989). The production of fructose from sucrose can be achieved by treatment with glucose oxidase after preincubation with invertase. The resulting gluconate can be separated more easily from fructose as it would be case with glucose.

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The use of glucose oxidase in clinical analysis is increasing nowadays. This enzyme has been recommended for glycemia determination because it is more specific and less toxic than O-toluidine method (Regider and Perez, 1985).

This enzyme is also used to determine capillary glucose in screening of gestational diabetes (Mesiggi et al, 1988). The enzyme is utilized to extend the shelf life of fish (Field et al, 1986). Its coupling reaction to peroxidase and a chromogen, glucose "dip-sticks and kits" became available for the screening of blood/urine glucose and showed its utility in glucose estimation (Worthington 1988).

Glucose oxidase has potential use in the wine industry, where it can lower the alcohol content of wine through the removal of some of the glucose (by converting it to D-glucono-1,5-lactone), which would otherwise be converted to alcohol. Tests showed that the glucose oxidase treatment of wine must could reduce the potential alcohol content of wine by about 2%. In addition, glucose oxidase is able to inhibit wine spoilage through its bactericidal effect on acetic acid bacteria and lactic acid bacteria during the fermentation process. The

bactericidal effect of the enzyme means fewer preservatives need to be added to the wine. Some strains of Saccharomyces cerevisiae have been genetically

engineered to carry the glucose oxidase gene itself. In view of the biotechnological and diagnostic importance of this enzyme it was deemed obligatory to investigate the properties of the enzyme.

1.6

i.e. 1. 2.

Commercial applications of glucose oxidase

In the present research projects two commercial applications of GOX were investigated

The estimation of glucose in diabetes mellitus patients. The production of calcium gluconate, gluconic acid and its derivatives using glucose oxidase.

1.6.1 The estimation of glucose in diabetes mellitus patients.

The disease involved in the elevation of blood glucose concentration is known as diabetes mellitus. It is a very common disease now days. It is a metabolic problem and is prevalent in many parts of the world. One basic aspect of diabetes is an abnormality of glucose

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metabolism as due to insufficient action of insulin, either to its absence or to resist in action, blood glucose level in diabetes becomes so elevated that the glucose "spills over" into urine, providing a convenient diagnostic test for the disease (Voet, et al; 1999). Froesch and Reynolds (1956) gave clinical biochemists the first specific method for determining the glucose levels in biological fluid by an enzymatic method. Later in the year Keston (1956 Teller, 1956), suggested the use of a coupled enzyme system in which the H2O2 formed in the enzymic oxidation of glucose was destroyed by peroxidase in the presence of suitable chromogenic oxygen acceptor. The chromogen being in direct proportion to the amount of glucose organally present, could then be determined colorimetrically. Saifer and Gersten Feld (1958) in America introduced a method of glucose estimation based on the above principle using O-dianisidine as oxygen acceptor. The method involved incubation at 37°C for 30 minutes. Middleton and Griffiths (1957) introduced a method in which o-tolidine replaced o-dianisidine. It has the great advantage of rapidity, developing a blue colour at room temperature, and incubation is unnecessary. In the process of determination of glucose concentration in biological sample involved three enzymes i.e. mutarotase, glucose oxidase and peroxidase. The overall reaction mechanism is as follows: -D-glucose -D-glucose 2H2O2

Mutarotase Glucose Oxidase

-D-glucose -D-gluconolactone + H2O2

Peroxidase

2H2O + O2.

Mutarotase/aldose-1-epimerase (EC 5.1, 3.3) involved in the mutarotation of certain sugars. All the known mutarotase substrates contain a reducing pyranose ring in C ­ 1 conformation with equatorial hydroxyls at C ­ 2 and C ­ 3 The C ­ 4 hydroxyls can be axial and various equatorial substituents are possible at C -5 (Bentley, 1962). Keston (1954) investigated the occurrence of GOX enzyme in animal tissues such as rat liver and small intestine, kidneys of porcine, rabbit, rat, bovine and lamb. The production and purifications of mutarotase have been reported from Penicillium notatum, Escherichia coli (Bailey et al 1969) and A niger (Kinoshita et al 1981). Glucose oixdase

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(EC. 1.1.3.4) is a flavoenzyme. Its use for antibiotic products has increased interest in glucose oxidase as well as its utility in glucose estimation.

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Worthington (1988) reported the coupling reaction to peroxidase and a chromogen, glucose "dip sticks ad kits" become available for the screening of blood/urine glucose. The molecule of GOX comprises about 74% protein, 16% neutral sugar and 2% amino sugars (Tsuge et al, 1975). Peroxidase (EC. 1.11.1.7) belong to class oxidoreductases It consist of an iron, porphyrin ring containing enzyme that catalyzes the redox reaction between hydrogen peroxide as an electron acceptor and substrate by means of O2 liberation, (Brill, 1966). Peroxidase enzyme occurs in nearly all plants, animals and microorganisms. Peroxidase is present in radish tomato, soybean, potato, carrot, bananas, dates, and strawberry in plants but horse radish is the rich source among all the others. Peroxidase is found in spleen, lungs, mammary and thyroid glands, bone marrow and intestine, usually it is rare in animal kingdom (Burnette, 1977; Low and Dupis, 1966; Reed 1975; Harris and Loew 1996). The horse radish peroxidase is highly specific sensitive and very stable. And with a chromogenic donor has proven very useful for the assay system, releasing H2O2 in the determination of glucose by GOX reported by Hames and Hooper (2001). These enzymes are widely used in biochemical laboratories as well as in clinical diagnostic for the preparation of glucose estimation kits. The enzymes and their kits for glucose estimation are being imported at high cost foreign exchange is required. The Economy of Pakistan can not afford high cost materials so there is a need to optimize the conditions for such methods. Present project was planned to develop the technology to optimize the conditions for glucose estimation using these three enzymes and to prepare low priced local kits as compared to imported kits. The three enzymes glucose oxidase, mutarotase and peroxidase were produced/extracted and purified for the preparation and optimization of glucose estimation kit.

1.6.1.1

Diabetes mellitus

Diabetes mellitus (DM) is a syndrome; it is not a single disease, but rather a group of metabolic disorders sharing the common underlying feature of hyperglycemia. Hyperglycemia in diabetes results from defects in insulin secretion, insulin action, or most commonly both. The chronic hyperglycemia and attendant metabolic deregulation may be associated with secondary damage in multiple organ systems, especially the

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kidneys, eyes, nerves, and blood vessels. Diabetes affects an estimated 16 million people in the United States, as many as half of whom are undiagnosed. Each year, an additional 8,00,000 individuals develop diabetes in this country, and 54,000 die from diabetes related causes. Diabetes is a leading cause of end-stage renal disease, adult-onset blindness, and non-traumatic lower extremity amputations in the United States. For individuals born in the United States in 2000, the estimated lifetime risk of being diagnosed with diabetes mellitus is one in three for males and two in five for females. The risk is two to five times higher in the African, American, Hispanic, and Native American communities, compared to non-Hispanic whites. Worldwide, more than 140 million people suffer from diabetes, making this one of the most common noncommunicated diseases. The number of affected individuals with diabetes is expected to double by 2025. (Ramzi, et al 2002). The countries with the largest number of diabetics are India, Pakistan, China and the United States.

1.6.1.2

Diagnosis

Ø Blood glucose values are normally maintained in a very narrow range, usually 70 to 120mg/dL. The diagnosis of diabetes is established by noting elevation of blood glucose concentration by any one of three criteria: 1. A random glucose > 200mg/dL, with classical signs and symptoms. 2. A fasting glucose >126mg/dL, on more than one occasions. 3. An abnormal oral glucose tolerate test (OGTT), in which the glucose is >200 mg/dL 2 hours after a standard carbohydrate load. Ø Levels of blood glucose proceed along a continuum. Individuals with fasting glucoses less than 110 mg/dL, or less 140 mg/dL following an OGTT, are considered to be euglycemic. However, those with fasting glucose greater than 110 but less than 126, or OGTT values greater than 140 but less than 200, are considered to have impaired glucose tolerance (IGT). Individuals with IGT have a significance risk of progressing to overt diabetes over time, with upto 5%-10% advancing to diabetes mellitus (DM) per year. In addition, those with IGT are at risk for cardiovascular disease, due to the abnormal carbohydrate metabolism as

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well as the co-existence of other risk factors such as low HDL, hyper triglyceridemia, and increased plasminogen activator inhibitor-1 (PAI-1).

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1.6.1.3

Classification of diabetes mellitus

Ø The elevated blood glucose associated with diabetes results from absence or inadequate pancreatic insulin secretion. The disease states underlying the diagnosis of diabetes mellitus are now classified into four categories (Katzung, 2004). · · · · Type 1, "Insulin-dependent diabetes" Type-2, "Non insulin-dependent Diabetes" Type-3, "Other" and Type-4, "gestational diabetes mellitus"

Diabetes mellitus is a metabolic problem and is prevalent in many parts of world. In developed countries, the incidence rate is 5% and an equal number is liable to develop the disease (Mckee, T., and Mckee, J., 1966). One fundamental aspect of diabetes is an abnormality of glucose metabolism due to insufficient action of insulin. Owing either to its absence or resist once action (Murray et al, 2000). Blood glucose concentration in diabetes becomes so elevated that glucose "spills over" into urine, providing a convenient diagnostic test for the disease (Voet et al, 1999). The three enzymes are involved in the process of determination of glucose level; · · · Mutarotase Glucose oxidase Peroxidase

1.6.1.4

Mutarotase

Mutarotase/aldose-1-epimerase catalyzes the mutarotation of -D-glucose to -Dglucose. (Kenstone, 1954) reported the occurrence of this enzyme in animal tissues such as rat liver and small intestine, kidneys of porcine, rabbit, chicken, rat, bovine and lamb. It was shown that the substrate specificity (D-glucose, D-xylose,

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D-galactose and L-arabionose) was similar to that for the bacterial enzyme. Although a close examination of the relative rates with the different sugars, suggested that the specificities of the mammalian and bacterial enzymes were not identical (Bentley, 1962). Production and purification of mutarotase have since been reported from Penicillium notatum, Escherichia coli and Aspergillus niger (Kinoshita et al; 1981). All the known mutarotase substrates contain a reducing pyranose ring in C-1 conformation with equatorial hydroxyls at C-2 and probably C-3. The C-4 hydroxyls can be axial and various equatorial substituents are possible at C-5. Toyoda et al., (1982) isolated the enzyme mutarotase from 40g kidney cortex and purified by using DEAE-celllose chromatography, polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight of 41,000, Km for -glucose at pH 7.4 and 25°C was 19 mM, optimum pH 6.5-7.5, optimum temperature 30-37°C. Bailey et al (1969) investigated the isolation and purification and mutarotase from bovine kidney cortex by ammonium sulfate precipitation, hydroxylaptite chromatography, DEAE-cellulose chromatography, Bio gel P-100 and sephadex G-150. The specific activity of mutarotase was 198 U/mg, 9,570 U/mg after sephadex G-150 while 25,000 U/mg after DEAE-cellulose chromatography. Toyoda et al., (1983) examined mutarotase from kidney, liver and small intestine of rats, which were separated by DEAE-cellulose chromatography. Liver mutarotase was further separated by hydroxylapatite chromatography. Types I from kidney and type-II from the liver were purified by isoelectric focusing on thin layer polyacrylamide gel. Miwa et al., (1984) studied that hog kidney mutarotase has an affinity for sephadex G-100 equilibrated and eluted with 5m mol/L EDTA buffer (pH 7.4). The affinity was reduced depending upon the presence of 0.15 mL/L NaCl and 0.2 mol/L glucose. Kinoshita et al. (1981) used Aspergillus niger ATCC 6274 for mutarotase production from 45 stock cultures. Mutarotase was purified 115-fold with a yield of 2.6%.

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1.6.1.5

Peroxidase

Peroxidase is an enzyme catalyzing the oxidation by hydrogen peroxide of a number of substrates such as ascrobate, cytochrome-C and the leuco forms of many dyes. A representative reaction is shone blew; H2O2 + DH2 (DH2 =Leuco dye; D=dye) Horseradish Peroxidase (HRP) exists in the form of several isozymes, all containing heme as the prosthetic group. The enzyme has a molecular weight of approximately 40,000 KDa. The ability of HRP to catalyze the oxidation of a number of organic compounds by hydrogen peroxide, resulting in the formation of colored end-products, is utilized in several methods of determination of glucose and galactose in biological fluids. Also, recently a method has been described for the determination of plasma uric acid utilizing uricase and HRP. Peroxidase has wide applications in health sciences as a diagnostic tool. The thyroid peroxidase are widely used to diagnose human autoimmune thyroid desease (Nord, 1953). A variety of enzymes are being utilized in ELISA (Enzyme Linked Immunosorbent Assay) kits among which peroxidase are widely used to prepare "Antibody-Enzyme or Antispecies Antibodies-Enzyme conjugates". Due to its high turn over rate, rapid availability, ease of conjugation and better sensitivity, (Kemeny and Challocombe, 1989). The use of highly specific, sensitive and very stable horseradish peroxidase with a chromogenic donor has proven very useful for the assay system, providing H2O2 in the determination of glucose oxidase. The time required for a single test is about 10 minutes (Okuda, et al, 1977). Some characteristics features of Peroxidase enzyme given below; (Wikipeda).

Peroxidase

2H2O + D

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·

Peroxidases are a large family of enzymes. Now a majority of peroxidase protein sequences can be found in the Peroxi Base database. Peroxidases typically catalyze a reaction of the form: ROOR + electron donor (2 e-) + 2H+ ROH + ROH

·

For many of these enzymes, the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or redox-active cysteine or selenocysteine residues.

·

The nature of the electron donor is very dependent on the structure of the enzyme. For example, horseradish peroxidase can use a variety of organic compounds as electron donors and acceptors. Horseradish peroxidase has an accessible active site and many compounds can reach the site of the reaction.

·

For an enzyme such as cytochrome-C peroxidase, the compounds that donate electrons are very specific because there is a much closed active site (Wikipedia).

While the exact mechanisms have yet to be elucidated, peroxidases are known to play a part in increasing plant's defense against pathogens. Peroxidases are sometimes used as histological marker. Cytochrome-C peroxidase is used as a soluble, easily purified model for cytochrome-C oxidase. Glutathione peroxidase is a peroxidase found in humans, which contains selenocysteine. It uses glutathione as an electron donor and is active with both hydrogen peroxide and organic hydroperoxide substrates. Peroxidases are hemeproteins, that metabolize hydrogen peroxide in different ways. The dismutation of hydrogen peroxide to water and oxygen as catalyzed by catalase doesn't need an additional hydrogen donating substrate as peroxidase does. It seems to be very likely that the different metabolic routes of hydrogen peroxide degradation correspond to differences in plant metabolism. Peroxidase is the most studied plant enzymes due to their abundance in plant tissues. The many physiological functions of peroxidase i.e. as lignifying enzyme, as predominant stress enzyme, as indolylacetic oxidase and many others are well described and understood. However, the high diversity of in vitro substrates and the existence of many different isoenzymes make very difficult to

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postulate a definitive physiological role for plant isoperoxidases. Peroxidases are widely used for genetic, physiological and pathological studies. One of the main physiological aspects of plant peroxidase research deals with their role in plant growth and development. Growth and development of plant is influenced by many factors: plant genome, nutrition, environmental factors and presence of growth regulators. Civello et al., (1995) extracted a peroxidase from strawberry fruit; it was partially purified. The enzyme was partially purified by means of (NH4)2SO4 precipitation, molecular exclusion chromatography and cationic exchange chromatography. The purification fold achieved was near 35. The activity of enzyme was 0.314 U/mL in crude extract and 1.576 U/mL after (NH4)2SO4 precipitation. Jen et al., (1980) isolated and purified homogenous tomato peroxidase isozyme by including hydrophobic chromatography. Enzyme activity and total protein were 364 U/mL and 110.5 mg respectively in crude extract while these were 290 U/mL and 40.7 mg after ammonium sulfate precipitation. Prestamo (1989) obtained peroxidase from kiwifruit, precipitated with ammonium sulfate and purified by DEAEcellulose chromatography. The protein contents and specific activity in crude extract was obtained 518 mg/mL and 4.94 U/mg respectively. 1.6.2 Production of calcium gluconate, gluconic acid and its derivatives by GOX method Reaction for production of gluconic acid is catalyzed by glucose oxidase, oxidation of the aldehydic group on the C-1 of -D-glucose to a carboxyl group resulted in the production of glucono--lactone (C6H10O6) and hydrogen peroxide. Glucose--Lactone is further hydrolyzed to gluconic acid either spontaneously or by lactone hydrolyzing enzyme while hydrogen peroxide is decomposed to water and oxygen by peroxidase. The conversion process could be purely chemical too, but the most commonly involved method is the fermentation process in which Aspergillus niger is widely used. GOX is used to convert glucose to gluconic acid by simple oxidation reaction (Sumitra. et al 2006 Fig. A, B, C):

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O

OH

H H H H

OH OH OH OH CH2OH

Fig (A)

Gluconic Acid (C6H12O7)

HO O OH OH OH O

Fig (B):

Glucono--Lactone

-D-glucose

Gox

FAD

H2O 2 Catalase

Glucose- -Lactone Spontaneous Lactonase Gluconic Acid

FADH2 1/ 2 O 2

1/ 2 O 2

H2O

Glucose + ½ O2

Gox

Gluconic Acid

Fig (C): Oxidation of glucose by GOX using A. niger

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Nearly 100% of the glucose is converted into gluconic acid under the appropriate conditions. Due to the presence of CaCO3 in the fermentation broth the gluconic acid produced immediately converted into its salt i.e. calcium gluconate which is the stable product. Later on gluconic acid can be obtained from calcium gluconate. Calcium gluconate i.e. D-gluconic acid calcium salt (C12H22CaO14) is one of the most important salt, which occurs as a white crystalline or granular powder without taste or odour. It is used for the production of gluconic acid.

CH2OH (CHOH)4 COO Ca COO (CHOH)4 CH2OH

Calcium gluconate Gluconic acid is a colourless, odourless, non corrosive, mildly acidic, less irritating, non toxic, easily biodegradable, non velotile organic acid with 196.16 molecular mass, 3.7 pKa and 1.24 g/ml density (Eric et al 1956). The chemical structure of gluconic acid consists of a six-carbon chain with five OH groups terminating in carboxyl group. This latter group can lose a hydrogen ion and thus turns the molecule into an acid. The IUPAC name of gluconic acid is 2, 3, 4, 5, 6-pentahydroxy hexanoic acid.

OH HO OH OH OH OH O

D-gluconic acid It cannot form cyclic structure. It is abundantly available in plants, fruits, rice, meat, diary products, wine (up to 0.25%), honey (up to 1%) and in vinegar (Sumitra et al 2006). The oxidation reaction: Glucose

1/ 2 O 2 Gox

gluconic acid

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O

C

H 1/ 2 O 2 Gox

COOH (CHOH)4 CH2OH

(HCOH)4 H2COH

(gluconic acid)

Calcium gluconate is also used in food, textile, pharmaceutical, chemical and leather industries (Pedrosa et al, 2000). It can be used orally, intramuscularly and intravenously for calcium deficiency (Ray and Banik, 1999). Gluconic acid salts are produced by three methods:

(i)

(1984).

Chemical method

In this method oxidation of glucose is done by a hypo-chlorite solution (Kundu and Das

(ii)

Electrical method

In this method electrolytic oxidation of glucose solution is carries out with bromide (Ambekar et al; 1965).

(iii)

Fermentation method

In this method oxidation of glucose is done in fermentation medium containing specific microorganism, glucose and other ingredients (Lee et al 1998). Gluconic acid was considered to be the product of incomplete oxidation of glucose to gluconic acids, later studies showed that enzyme activities of oxidases and dehydrogenase is responsible for oxidizing glucose to gluconic acid (Pons et al 2000). The microbial production of gluconic acid and its salt i.e. calcium gluconate can be seen as a more economical way in comparison to the other methods. Trager and Qazi (1991) investigated the contribution of GOX to gluconic acid production at increased dissolved oxygen concentration. It was found that Aspergillus Species, were more suitable for the production of calcium gluconate. (Yomod et al 1992). High yield of calcium gluconate was obtained by using A. niger (Rosenberg et al 1992; Bajaree and kannika, 1993). Lee et al (1998) prepared gluconic acid and its salt by A. niger. Butkhewitsch (1923) produced gluconic acid by A. niger strain in the presence of calcium carbonate. The rate at which glucose is converted to gluconic acid is more rapid in the presence of undissolved CaCO3 than in the presence of free acid. The use of an excess of CaCO3 retards the fermentation since the calcium gluconate formed tends to crystallize out and prevents free contact of the medium with the mold. If the free gluconic acid is not

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neutralized, the fermentation capacity of the mold may be inhibited after prolonged exposure to the acid. So the precipitation of calcium gluconate while the excess of CaCO3 prevented the formation of free gluconic acid otherwise the acidity inactivate the GOX (Prescott and Dunn, Industrial microbiology 1997) and (Sumitra et al 2006). In the present research project Gluconic acid, and its derivatives (metal salts) such as sodium, magnesium, copper, nickel and cobalt gluconates were synthesized from calcium gluconate which were earlier produced by fermentation process by the GOX method using A. niger as fermentative organism.

1.7

Aspergillus niger selected specie

Ø Aspergillus niger is a fungus and one of the most common species of genus Aspergillus. It causes black mould on certain types of fruit, vegetables and bread and is a common contaminant of food. Classification of A. niger: Domain Kingdom Phylum Class Order Genus Specie Eukaryota Fungi Ascomycota Eurotiomycetes Eurotiales Aspergillus Aspergillus niger

Ø Micheliex link in 1809 described the structure of Aspergillus niger. Its hyphae are seplate and hyaline. The conidiophores originate from the based foot cell located on the supported hyphae and terminate in a vesicle at the Apex. In Aspergillus niger the condiophore is long, smooth, colourless or brown. The Phialides is biseriate, the Vesicle is round with the radiate head. The industrial importance of Aspergillus niger is not limited to its more than 35 native products but also to the development and commercialization of the new products which are derived by modern molecular biology techniques (Davies, 1991). During the past few years numerous studies have been conducted on Aspergillus niger. It is consider the most important fungi for production and secretion of protein. It demonstrates many advantages such as:

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Aspergillus niger has a long history of usage within the fermentation industry and is generally regarded as safe (GRAS) in accordance with the food and drug administration (FDA). This often facilitates the path toward regulatory improval of the production system.

Aspergillus niger is a prodigious exporter species of homologous proteins and is able to produce certain enzymes in quantities of kilogram per cubic meter under the right conditions.

Aspergillus has a useful production system for heterologous protein derived from other filamentous fungi. The fermentation industries are very familiar with the conditions required to maximize production of homologous proteins in Aspergillus. Thus it provides a good starting point for the identification of physicochemical influences that are likely to be of greatest importance to heterologous protein production and secretion using a similar strain.

Aspergillus is also capable of producing extra cellular proteases. Such proteases could not only damage the product directly but also contaminate the final products and require an additional purification step.

The production of fungal toxins should also be considered on using Aspergillus strains (Bosch et al, 1995). Aspergillus niger has some uses as the organism itself, in addition to its products of fermentation e.g. due to its ease of visualization and resistance to several antifungal agents, Aspergillus niger is used to test the efficacy of preservative treatments (Jong and Gantt, 1987). Also the production of chymosin using A. niger was extensily studied by Dun coleman et al; (1991). Bovine chymosin production increased upto 1g/l after gene expression in A. niger.

1.7.1 Success of Aspergillus niger in fermentation

Ø The success of Aspergillus niger for industrial production of biotechnological products is largely due to the metabolic versatility of this strain. A. niger is well known to produce variety of organic acids, enzymes, plant growth regulators, mycotoxins and antibiotics. Many useful enzymes are being produced using industrial fermentation of A. niger. For example, A. niger glucoamylase is used in

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the production of high fructose corn syrup, and pectinases which are used in cider and wine clarification. -galactosidase, an enzyme that breaks down certain complex sugars, is a component of Beano and other medications which the manufacturers claim can decrease flatulence. Another use for A. niger within the biotechnology industry is in the production of magnetic isotope-containing variants of biological macromolecules for NMR analysis (Davies, 1991). Ø During the past few years numerous studies have been conducted on A. niger, presumably the most important fungi for production and secretion of protein. A. niger also cultured for the extraction of the enzymes glucose oxidase (GOX) and Alpha-galactosidase (AGS). Glucose oxidase is used in the design of glucose biosensors, due to its high affinity for -D-glucose (Staiano et al, 2005) alphagalactosidase is produced by Aspergillus niger; it is used to hydrolyze alpha 1-6 bonds found in melibiose, raffinose, and stachyose. Ø The employment of A. niger as a host organism for production and secretion of homologous and heterologous proteins demonstrates many advantages such as: Aspergillus is capable of carrying out efficient post translational modifications of products, e.g. glycosylation. This is especially important for some proteins derived from eucaryotes. Aspergillus species are effective secretors of proteins, often in a native, correctly folded form. They tend not to accumulate large quantities of the protein intracellularly, in form of inclusion bodies, as some bacteria and yeast do. Aspergillus has a useful production system for heterologous proteins derived from other filamentous fungi. Transformation stability is relatively high; therefore the threat of revertants is less pronounced (Bosch et al., 1995).

1.7.2 Mutagenesis

Mutagenesis is a term that refers to the deliberate production of genetic variability through the use of various forms of energy (neutrons, gamma rays, X-rays) or various chemical treatments. Mutations can also arise because mistakes can be made during DNA replication that results in the incorporation of an incorrect base. The chemical nature of any given base can be altered either by environmental or chemical means. Once altered,

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these changes may then be propagated by further DNA replication. Finally, large scale changes can sometimes occur in the form of DNA insertions and/or deletions. Mutation is a random event, it requires the production of very large numbers of individuals in the hope that one or more organisms will carry the desired mutation. It is not possible to direct this process and the changes induced in the DNA are not known. Mutagenesis can only modify the genes of an organism. Filamentous fungi are capable of producing large amounts of specific enzymes although the concentrations are relatively too low for commercial exploitation in the naturally occurring wild type strains, so the improvement in enzyme production can be achieved by mutation and selection program in fungi (Bisawaet et al., 1990). Mutation selection program for isolation of hyper producing strains which are less effective to end product inhibition have also been reported (Coughlan, 1992). Mutagensis is still a cost effective procedure. Mutagensis is the source of all genetic variations but no single mutagenic treatment could give all possible type of mutations. Mutations are classically divided into two types: (i) (ii) Physical e.g. Ultraviolet, gamma, X- ray irradiation Chemical e.g. NTG ( nitroso trimethyl guanidine). EMS (ethyl methane sulfate), MNNG (N-methyl N- nitro N- nitrosoguanidine) (Rowlands, 1984). Gene cloning is one of the latest and modern technology which is also being used to introduce mutation in wild type strains, but it was proved to be expensive. So mutagenesis using chemical and physical treatments is the most popular and affordable to obtain the mutant strain at low cost.

1.7.3 Mutagenesis by UV

The treatment with UV radiation was carried out to select a strain with improved glucose oxidase activity. UV radiations in the range of 200- 300nm are absorbed by nucleic acids especially at 254 nm. It can produce change in DNA molecule i.e. breakage of DNA strands, alteration of bases and breakage of dimers nitrogen bases and can cause major deletions in DNA molecules (Conn and Stumph, 1994). So that mutants can be screened with maximum production of enzyme due to over expression of genes involved in the enzyme production.

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All the Eukaryotes have DNA repair mechanism, which can eliminate radiations- induced mutation causing the formation of genetic mutant (Brown, 1992). An industrial strain improvement plays a key role in the commercial development of many fermentation processes. This practice of strain improvement by mutagenesis with UV is highly developed technique.

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Figure 1.2

Pictures of A. niger

(A)

Growth of A. niger in Petridish.

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(B)

Microscopic View of Fungus Showing Hyphae & Vesicles

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(C)

Microscopic View of A. niger Showing Conidiophore Containing Conidia

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Aim of work

The aim of this work was to improve the production of glucose oxidase. The selected Aspergillus niger strains should be able to produce the enzyme with higher amount. This work was focused on the following two main objectives:

(1)

a) b)

Production of glucose oxidase

Screening of different native strains for optimal production of enzyme. Changes in medium composition i.e. optimization of the culture conditions for maximum GOX production. c) Enhance GOX production by UV mutant A. niger strain.

It involves

(2)

(a)

Applications of glucose oxidase:

The project was also designed to produce glucose oxidase from A. niger for its ultimate use in diagnostic kits i.e. for estimation of glucose. (b) An important object of the work was to develop a process for the production of calcium gluconate, gluconic acid and its derivatives from GOX.

It involves

The goal of the present research project was to enhance the GOX production, and its utilization for the maximum production of calcium gluconate, gluconic acid, its derivatives. The goal will use to develop and standardize the glucose estimation kit depending on our own sources and in view of the economy of Pakistan. Therefore, the purpose of this project was to develop and encourage the local technology and skill and also to save substantial amount of foreign exchange being drained on the import of GOX enzyme based kits. Hence this project will help the commercial production of these items in Pakistan.

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Chapter No. 2

LITERATURE REVIEW

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REVIEW OF LITERATURE

Several of valuable enzymes have been developed for a variety of commercial uses. Glucose Oxidase is also an important and frequently used commercial enzyme. Although GOX's have been isolated from many fungal sources that from A. niger is found to be the best for production of this enzyme (Fiedurek et al; 1986). Glucose oxidase is widely used to determine the glucose concentration in biological samples. An antimicrobial effect of this enzyme has also been reported. Glucose oxidase is used to remove oxygen from packed food materials i.e. used as antioxidant and plays a role in gluconic acid production. Glucose oxidase is used in the food industry as well as in clinical analysis (Bucke, 1983).

2.5

Production of glucose oxidase (GOX)

Glucose oxidase was first isolated from mycelia of A. niger and Penicillium glaucum by Muller (1928). By 2009, the industrial production of GOX was carried out using both A. niger and P. amagaskiense. Besides these two fungi, many other microorganisms were reported as GOX producers. Coulthard et al; (1942) demonstrated the presence of GOX in Penicillium notatum and extracted the enzyme in highly purified form. Keilin and Hartee (1947) reinvestigated the reports of Muller (1928) and Coulthard et al; (1942) also studied the structure physical properties and enzyme kinetics. They found in P. notatum that cytochrome system was not involved in intracellular oxidation of glucose to gluconic acid as endogenous respiration of the mycelium was cyanide-sensitive, but that the oxidation of glucose by the mycelia was not inhibited by cyanide. Attempts to modify the specificity of GOX by repeated subcultivation on fructose medium were unsuccessful. Since the first isolation and characterization of glucose oxidase by Muller (1928), much work has been done to optimize the process of GOX production through either genetic manipulation of the host strain or improvement of cultivation conditions. The main studies concerning the production of GOX were carried out using A. niger as follows. James and Myrbork (1951) found that the press juice for enzyme preparation of A. niger contained an enzyme system which had absorbed oxygen and classified it as an oxidase. He made an important observation that addition of glucose to the extract caused

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a great increase in the amount of oxygen consumed. He further found that during the absorption of oxygen, in the presence of glucose, gluconic acid was also produced. The enzyme catalyzed this reaction is therefore called glucose oxidase. They found that enzyme was highly specific for -D glucose while manose and galactose showed little specificity as substrates. They explained that glucose oxidase is not a dehydrogenase since it does not accelerate the reduction of methylene blue. Bentley (1959) obtained enzyme from mold cultures was originally named glucose oxidase. The antibacterial activity of some penicillium notatum culture filtrates was later shown to be due to H2O2 formed by action of enzyme. It catalyzes the removal of two hydrogen atoms from -D-glucopyranose forming as the primary product -Dgluconolactone. Pazur and Ando (1959) developed a rapid and effective method for glucose oxidase purification based on ammonium sulphate fractionation and chromatography on DEAEcellulose. It is effective for separating the glucose oxidase from catalase. Kusai et al; (1960) first time discovered the glucose oxidase in Penicillium amagasakiense and were also able to get the enzyme in crystalline form. Zetelaki and Vas (1968) have investigated the effect of aeration and agitation on the GOX production by A. niger in a 5 liters stirred tank bioreactor. They found that the maximum enzyme production was achieved at 700 rpm. Further increases in agitation speed resulted in neither a higher growth rate nor higher activity. The usage of pure oxygen resulted in an increase of mycelial dry weight of about 15 fold and the GOX production was doubled compared to the aerated culture. Zetelaki (1970) found an acceleration of the growth and GOX production of the A. niger increased with the sugar consumption in the cultivation broth. Doubling the pressure in the bioreactor (i.e. doubling the solubility of oxygen) resulted in a faster synthesis of enzyme and a higher rate of growth in the early stage of cultivation. Nakamatsu et al; (1975) examined the various microorganisms belonging to genus Penicillium for the productivity of glucose oxidase, GOX enzyme was found in these strains. The best glucose oxidase producer P. pupurogenum was isolated from a natural source. This microorganism produced 32,000 units ml-1. broth of glucose oxidase in a

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submerge culture for 3 days. That value was about ten times of P. amagasakience which is known to be an excellent glucose oxidase producer. Mischak et al; (1985) reported that glucose oxidase is produced in response to the addition of D-glucose the growth medium. Kelly and Adinarayna (1986) purified and characterized the glucose oxidase from Ligninolytic cultures of Phanerochaete chryrosporium. Enzyme was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. Kim et al; (1988) identified enzyme metabolite produced by Talaromyces flavus as glucose oxidase and it was found to be useful in the biocontrol of Verticillium dahliae. As described earlier the process of GOX production can be optimized by the improvement of cultivation conditions and medium composition. Different media have been used for A. niger cultivation during GOX production. Some examples are as follow: Zetelaki and Vas (1968) reported the medium composition gl-1 for A. niger 1026/5 strain as sucrose, 50-70; Ca(NO3)2 . 4H2O 2.0, Citric Acid 7.5; KH2PO4 0.25; MgSO4.7H2O 0.25; FeCl3.6H2O, 0.1; CSL 20.0. Zetelaki (1970) reported as sucrose, 50; Ca(NO3)2 2.0, Citric acid 7.5; kH2PO4, 0.25; KCl, 0.25; MgSO4.7H2O 0.25, FeCl3.6H2O, 0.01 and CSL, 20.0 for A. niger. Nakamatsu et al; (1975) cultivation medium composition gl-1 is glucose, 40; NaNO3. 2.0; KCl, 0.5; KH2PO4. 1.0, MgSO4. 7H2O, 0.5; FeSO4.7H2O, 0.01, YE. 2.0 pH. 6.0 for A. niger and Penicillium spp strains. Petruccioli and Federici, (1993) used glucose, 80.0; Peptone, 3.0; NaNO3, 5.0; kH2PO4. 1.0; FeSO4.7H2O, MgSO4.7H2O, 0.5; CaCO3 35.0 for P. variable P16 strain. Petruccioli et al; (1994) (1995) reported composition as glucose. 80.0, Peptone 3.0, NaNO3, 5.0, KCl, 0.5; kH2PO4, 1.0; FeSO4.7H2O, 0.01; CaCO3, 35.0; pH 6 for P. variable P16 strain. In the present project medium composition used was somewhat similar to Petruccioli et al; (1993) with some modifications i.e. urea was used instead of peptone and no KCl was added in the medium.

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Nakamatsu et al; (1975) studied the effect of different complex carbon sources as well as different nitrogen sources on GOX production. Among eight sources of widely differing natural carbon sources, they found that beet molasses was the best carbon source to support growth and GOX production. On the other hand nitrate and urea gave a better GOX yield than ammonium salts. Van Dijken and Veenhins (1980) used cytochemical staining technique to establish the location of GOX in microbodies. Under normal condition of glucose oxidase production with A. niger, glucose oxidase behaves as an intracellular enzyme and it is release from the mycelium by means of cell disruption techniques. Petruccioli et al; (1994) studied the GOX production by Penicillium variable P16 immobilized in different carriers. Among different carriers, polyurethane proved to be the best for GOX production and production continued for 7 repeated batches. Fiedurck et al; (1994) explained a new method for the biosybnthesis of glucose oxidase by A. niger conidia immobilized on seeds of wheat, rye, barley, peas and mustards. They showed that highest production was reached on the wheat carrier (1.3 µml-1). Some culture conditions, temperature and agitation. Speed, the amount of support was optimized to improve the growth and enzyme biosynthesis by the immobilized mycelium. Hatzinikolaou and Macris (1995) reported factors regulating production of glucose oxidase by A. niger. They examined the factors effecting the production of intra and extra cellular GOX and found that, molasses as best carbon source that enhanced the enzyme activity upto 5.7ml-1 CaCO3 was also identified as strong inducer of glucose oxidase activity. Lawrence et al; (1995) obtained transgenic potato plants that expressed a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. They found that H2O2 levels were elevated in both leaf and tuber tissues of these plants. Willis (1996) explained that GOX from A. niger is an intracellular enzyme present in the mycelium of the organism. He grew A. niger by submerged culture fermentation in a mineral medium containing a source of organic nitrogen and carbohydrate. They assayed the GOX activity in the filtrate and purified it by means of (NH4)2SO4 precipitation and DEAE-cellulose chromatography.

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Lu et al; (1996) showed that waste mycelium of A. niger from the production of NaG, CaG and MgG (G = gluconate) can be used to produce glucose oxidase. The effects of metal ions on the glucose oxidase activity were also studied. Gromada and Fiedurek (1996) reported the effect of some medium components and metabolic inhibitors on glucose oxidase production by mutant A. niger strains studied in shake flask experiments. Altering the composition of the basal medium, particularly substitution of (NH4)2 HPO4 for NaNO3, and a lack of Mg+2 ions caused an increase in GOX activity. A significant increase (68.3%) in intracellular GOD activity was found in the presence of sodium orthovanadate (1mM). Yang et al; (1996) explained the method of production of GOX by waste mycelium of A. niger and the effects of metal ions on activity of GOX. The results showed that the waste mycelium from production of NaG, KG, CaG2, MgG2 (G = gluconate) was used to produce GOX. The effects of metal ions on GOX activity were also studied, calcium gluconate increased while MgG2 reduced the productivity. Chu et al; (1997) increased the production of Glucose oxidase by catabolites during fermentation through membrane dialysis fermentation. The enzyme production by this method is two times greater than that of process without dialysis and total enzyme activity was increased by 30-50%. Kapat et al; (1998) analyzed the effects of agitation and aeration on the production of glucose oxidase from a recombinant strain of Saccharomyces-cerevisiae, optimization of the speed of agitation and the rate of aeration in a stirred tank fermentor was carried out to achieve maximum GOX production. The maximum activity of extracellular GOX was achieved when the speed of agitation was 420 rpm and rate of aeration was 0.25 vvm respectively. Liu et al; (1999) observed the synthesis of glucose oxidase by A. niger and noted that CaCO3 induced the synthesis of enzyme and CaCl2 inhibited it, while the biosynthesis was promoted by MnCO3, thioglucolic acid, pyroracemic acid and gluconic acid. Ray and Banik (1999) concluded that glucose (15%) and urea (0.14%) induced glucose oxidase synthesis and optimum yield of calcium gluconate. KH2PO4 (0.2%) and MgSO4 (0.06%) stimulated glucose oxidase activity and calcium gluconate production.

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Rothberg et al; (1999) analyzed the optimal concentration of dissolved oxygen in order to maximize the intracellular GOX formation in A. niger. They showed that a dissolved oxygen concentration at 3% of saturation and at total pressure of 1.2 bar was optimal for maximizing intracellular GOX activity. Enshasy et al; (1999) evaluated the effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake flask cultures and its relation to GOX excretion by recombinant A. niger NRRL 3. It was shown that culture conditions resulting in the formation of smaller fungal pallets with an increased mycelial yield result in higher yield of exocellular glucose oxidase. Fiedurck and Gromada (2000) carried out the work on production of catalase and glucose oxidase by A. niger using unconventional oxygenation of culture. They showed that maximal oxygen concentration occurred in 50ml of medium containing 0.2g wet mycelium and 0.2% (w/v) glucose at pH 5.0. Liu et al; (2001) showed the effects of various metal ions on the simultaneous production of glucose oxidase and catalase by A. niger, CaCO3 induced high synthesis of both enzymes. The production of both enzymes was growth associated. Finally a model of growth and product formation was proposed. Park et al; (2002) reported that the initial moisture content, cultivation time, inoculum size and concentration of basal medium was optimized in solid state fermentation (SSF) for the production of xylamase by an A. niger mutant using statistical experimental design. The cultivation time and concentration of basal medium were the most important factors affecting xylamase activity. Cultivation time of 5 days, 65% initial moisture content and ten times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in solid state fermentation. Kristin (2002) demonstrated the basic principles of enzyme kinetics using the enzyme glucose oxidase coupled with peroxidase. A series of reaction were arranged that vary the enzymes concentration, substrate concentration and temperature of the reaction kinetics of glucose oxidase in each series is observed by monitoring the rate of the colour change in each beaker. Zubair et al; (2002) showed that the activity of glucose oxidase becomes maximum after 36 hours of fermentation for the medium containing 2.0% (w/v) rice polishings, 0.3%

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(w/v) urea, 4% (w/v) KH2PO4 and 0.05% CaCO3 at pH 4 and 30C°. They found that addition of these enhanced the GOX production while MgSO4.7H2O addition decreased it. Liu et al; (2003) studied the response surface methodology to optimize the speed of agitation and the rate of aeration for the maximum production of glucose oxidase by A. niger. A quadratic model for glucose oxidase production was most probable. The quadratic response to agitation was the most significant positive effect. The maximum activity of GOX was monitored when the speed of agitation and rate of aeration were 756 rmin-1 and 0.9 vv-1 m-1, respectively. Mudeppa et al; (2003) studied the thermal inactivation of glucose oxidase from A. niger both in the absence and presence of additives. Additives such as lysozyme NaCl and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7- fold respectively from its initial value at 60°C due to charge neutralization by NaCl and lysozyme while K2SO4 enhanced the thermal stability by hydrophobic interactions. The maximum GOX production can also be achieved by mutation and selection program in fungi i.e. by recombinant strains besides the improvement of cultivation and medium conditions (Saddler, 1982). Mutagenesis and selection i.e. random screening is a cost effective procedure and for reliable short term strain development is frequently the method of choice. Mutagenis is the source of all genetic variations. There are various methods for mutation e.g. physical i.e. U.V., gamma and x-rays irradiation and chemical e.g. NTG (nitrosotrimethyl guanidine), EMS (ethylmethane sulfate), MNNG (N-methyl N-nitro N-nitrosoguanidine) described by Rowlands (1984). Gene cloning is one of the latest and modern technologies which are also being used to introduce the mutation in the wild type strains. Bisawaet et al; (1990) and Singh et al; (1995) examined that the improvement in enzyme expression can be achieved by mutation and selection program in fungi. According to Rowland (1984) the mutagenic procedures can be optimized in term of type of mutagen and dose. Fiedurck and Gromada (1997) found that mutagenesis using UV enhanced the activity of glucose oxidase. These procedures were adopted because sometimes the natural host

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organism did not prove to be ideal for GOX over-expression due to three main reasons. First, GOX expression and excretion depends on the presence of glucose and pH of the culture medium higher than 4.5. GOX production leads to gluconic acid and hydrogen peroxide production which complicate the cell culture techniques. These problems can be solved by substituting the natural GOX promoter with another potent and glucose independent Aspergillus promoter (Kopetzki et al; 1994). Second, GOX is usually cellwall associated, thus making purification more difficult. Third, purified Aspergillus GOX is often contaminated with host cell enzymatic impurities such as catalase, amylase and cellulose which interfere with its applications. To overcome these disadvantages, GOX expression and secretion were studied in Saccharomyces-cerivisiae (De Baetselier et al; 1991). According to Hellmuth et al; (1995), the production of GOX by recombinant strains has been carried out to improve the secretion of GOX into medium. Mainly two strategies for the over expression of homologous genes are possible: 1. 2. Amplification of the gene copy number. Expression of the gene under the control of strong regulatory elements.

Com and Stumph (1994) studied mutagenesis through UV radiation to improve the GOX activity. UV radiation in the range of 200-300 nm is absorbed by nucleic acid and is especially damaging at 254 nm. It can produce lesions in DNA molecule including breakage of DNA strand, alteration of bases, formation of dimers among nitrogen (thiamin) bases and can cause major deletion in DNA molecules. Therefore, such mutants can be screened for enhanced production of enzyme due to over expression of genes involved in the enzyme production or due to mutations regulatory gene elements. Brown (1992) showed that DNA repair mechanisms exist in most eukaryotes, which can eliminate radiation induced mutations causing the formation of genetic mutant. Pluschkell et al; (1996) explained the kinetics of GOX excretion by recombinant A. niger. The majority of GOX was produced during rapid growth in the first phase of the cultivation. During the second phase of the cultivation; excretion of GOX occurred at a slower rate, although the majority of GOX produced during the first phase was excreted during the second phase of the cultivation. At the end, about 90% of the total glucose oxidase produced was recovered from culture medium.

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Suresh et al; (1999) studied a UV induced mutant strain of A. niger (CFRI-1105-49) overproduced a starch hydrolyzing enzyme different from amylase, suggested significant applications in starch processing industries. Singh et al; (2001) studied A. niger ORS-4.410, a mutant of A. niger ORS-4 by repeated irradiation with UV rays. Comparison of gluconic acid production of the parent and mutant strain showed a significant increase in gluconic acid production that was 87% higher than the wild type strain. Paula et al; (2003) reported the mutants of A. niger N402, induced by UV mutagenesis, were selected and tested for resistance or sensitivity to 5-fluorocytosine also increased citric acid production. Khattab and Bazaraa (2005) reported the enhancement of extracellular GOX production by screening, mutagenesis and protoplast fusion of various strains of A. niger. In the present project the production of glucose oxidase is improved by optimizing the cultivation conditions and fermentation medium composition alongwith screening of fungal strain. Mudy, Wang and their co-workers (2006) studied recombinant A. niger glucose oxidase expressed in Trichroderma resei has capability to be a new recombinant host for A. niger GOX production. Clarke et al; (2006) studied the location of GOX during production by A. niger. Enzyme location impacts significantly on enzyme recovery. The production of the enzyme glucose oxidase by A. niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each sample. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cells, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cells, it was predominantly

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associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38% (w/v), respectively. Singh et al; (2006) A. niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 obtained was 149% higher than that for wild-type strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 hour, the mutant strain had consistent levels of GOX production (2.62 +/- 0.51 Uml-1). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62 +/- 0.51 Uml-1) using HCF-TM compared with the crude form of cane molasses (0.762 +/- 0.158 Uml-1). Simpson et al; (2007) isolated purified and partially characterized a novel glucose oxidase (GOX), a flavoenzyme, from Penicillium sp. Maximum activities of 1.08 U mg-1dry weight intracellular and 6.9 U ml-1 extracellular GOX were obtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI's of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148 kda, consisting of two equal subunits with molecular weight of 70 kda. The enzyme displayed a temperature optimum between 25 and 30 °C, and an optimum pH range of 6­8 for the oxidation of -D-glucose. The enzyme was stable at 25°C for a minimum of 10 hours, with a half-life of approximately

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30 min at 37 °C without any prior stabilisation. The lyophilized enzyme was stable at -20 °C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5 U mg-1; Km, 18.4 mM; kcat, 741 s-1 and kcat km-1, 40 s-1 mM-1. Stability at room temperature, good shelf-life without stabilization and the neutral range for the pH optimum of GOX contribute to its usefulness in current GOX-based biosensor applications. Silvia et al; (2006) reported that glucose oxidase (GOX) is a glycoprotein that finds wide application in food industry and clinical analysis. The gene encoding the GOX from Penicillium variabile P16 was expressed in Pichia pastoris X 33 using the methanol inducible AOX1 promoter. Among 11 transformants resistant toward high zeocin concentrations, six Mut+ strains were screened in shaken flasks and the strain X33 c9, producing 0.33 U ml-1 of heterologous GOX after 11 days of fermentation, was selected. Recombinant GOX (ca. 50 U ml-1) was produced in a 3-l fermenter under not optimized conditions, recovered and purified in order to characterize and to compare it with the native one. The GOX from P. pastoris had a molecular weight of 82 kDa. Comparison of carbohydrate moieties showed a slight over-glycosylation of the GOX from Pichia over the native enzyme (17 and 14%, respectively). pH behavior of the recombinant enzyme, in terms of both activity and stability, was similar to that of the native one; on the other hand, a certain difference was observed in optimal temperature for activity and in thermal stability. P. pastoris appears to be a good expression system for GOX production. Haq et al; (2006) studied the effect of volume of culture medium on enhanced citric acid productivity by a mutant culture of A. niger. Black strap molasses, a by-product of sugar industries is easily and abundantly available for its exploitation as a carbon source in fermentation process. The parental culture of A. niger was improved by mutation using ultraviolet radiations (UV) and N-menthyl N-nitro guanidine i.e. mutagen MNNG. Six UV and eight MNNG treated mutant strains were isolated after extensive screening and optimization. Mutant strain of A. niger MNNG-2 showed enhanced citric productivity (87.60 gl-1) over the metal strain Btl-45 (19.53 gl-1) in case of mutant MNNG-7). All kinetic parameters including yield coefficients and volumetric rates revealed the hyper-

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reducibility of citric acid by mutant MNNG-2 using blackstrap molasses as the basal medium in stirred reactor. Dimitris et al; (2007) purified two glucose oxidase GOX isoforms using electrophoretic homogeneity from the mycelium extract GOXI and the extracellular medium GOXII of A. niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kda and pI values of 3.7 and 3.6 for GOXI and GOXII respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent K

m

values on glucose, as well as pH and

temperature activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX. Shrikant et al; (2007) investigated purification of glucose oxidase from A. niger and that of -galactosidase from Kluyveromyces lactis using polyethylene glycol (PEG)-sodium sulfate aqueous two phase system (ATPS) in the presence of PEG-derivatives, i.e. PEGCoomassie brilliant blue G-250 and PEG-benzoate, PEG-palmitate and PEG-TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG-benzoate and PEG-palmitate enriching -galactosidase in the salt phase whereas in the case of glucose oxidase, PEG-Coomassie brilliant blue G-250 derivative worked as a better affinity ligand for other proteins. A 19-fold purification was obtained with the PEG dye

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derivative after has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation showed strong affinity interaction of PEG-glucoside with the enzyme. In the present project the production of glucose oxidase was improved by optimizing the cultivation conductor and fermentation medium composition alongwith UV mutation of A. niger.

2.2

Properties of glucose oxidase

Classification

Some important characteristics of GOX from A.niger are:

Glucose oxidase belongs to large group of enzyme called oxidoreductase also called glucose aerodehydrogenase (Sidney and Northon 1955 and Witteveen et al; 1992)

Molecular weight

160,000 da reported by Tsuge et al; (1975).

Composition

According to O'Malley and Wcaver (1972) the GOX consists of two identical poly peptides chain subunits (80,000 da) covalently linked by disulfide bonds. Each subunit contains one mole of Fe and one mole of FAD (Flavin-adeninedinucleotide). While Tsuge et al; (1975) examined the molecule to be approximately 74% (w/w) protein, 16% (w/w) neutral sugar and 2% (w/w) amino sugars. They indicated that the FAD is replaceable with FHD (Flavin hypoxanthinedinucleotude) without loss of activity.

Specificity

The enzyme is highly specific for -D glucose. The -anomer is not acted upon 2-deoxyD-glucose, D-mannose and D-galactose, exhibit low activities as substrate investigated by Bentley (1966) and (Kunst 1984).

Optimum pH

Optimum pH for enzyme is 5.5 with broad range of 4-7 reported by Bright and Appleby (1969) and Weibel and Bright (1971).

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Inhibitors of enzyme

Ag+, Hg+2, Cu+2 act as inhibitors of enzyme production investigated by Nakamura and Ogura (1968). While Swobada et al; (1969) showed that FAD binding was inhibited by several nucleotides.

Stability

Dry preparations are stable for many years under cold conditions Sidney and Northon (1955).. Samples of the dried enzyme are stable at 0°C upto 2 years. Aqueous solutions (0.1 to 0.2%) are stable for 1 week at 5°C. The activity GOX is lost on heating at temperature greater than 39°C. The purest enzyme preparations are pale yellow powder

Reaction catalyzed

Enzyme ­ FADH2 + -D ­ gluconolactone Enzyme ­ FAD + H2O2 (Worthington, 1988)

-D-glucose + Enzyme ­ FAD enzyme ­ FADH2 + O2

Keilin and Hartee (1947) investigated the glucose oxidase from both A. niger and Penicillium notatum with respect to its structure, physical properties and enzyme kinetics and came to the following conclusions. 1. The enzyme catalyzed the oxidation of glucose to gluconic acid by molecular oxygen, (O2), which is reduced to H2O2. C6H12O6 + H2O + O2 2. Glucose oxidase C6H12O7 + H2O The enzyme, which is a flavoprotein, shows in the oxidized state the characteristic absorption spectrum with bands at 377 and 455 mu. On addition of glucose the enzyme was almost completely decolorized and bands became hardly visible. They disappeared completely in presence of Na2SO4. The prosthetic group was alloxazine adenine dinucleotide and was demonstrated by removing the prosthetic group from D-amino acid oxidase and regenerating the activity of latter by the addition of boiled glucose oxidase. 3. From the absorption constant at 455 mu. The minimum molecular weight was estimated to be about 75,000 kda. The molecular weight calculated from the sedimentation velocity and diffusion constant was 152,000 da, which showed that the molecule of enzyme contains two prosthetic nuclei.

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4.

The enzyme did not fluorescence in ultraviolet light within the pH limits of its catalytic activity (pH 2-8). Outside this range it lost its activity and became fluorescent.

5.

The enzyme showed a pronounced specificity for the glucose. About fifty sugars and their derivatives tested, four of them oxidized at about 1% of the rate of glucose oxidation (mannose, xylose, 6-methyl glucose and 4,6dimethyl-glucose), where the remaining were virtually or completely unattacked.

Bentley et al; (1963) has reviewed the general properties of GOX, they reported that GOX oxidizes glucose to D-glucolactone and H2O2. D-gluconolactone gone hydrolysis to gluconic acid. They examined that enzyme was highly specific for -D-glucose. The anomer was not acted upon 2-deoxy-D-glucose, D-mannose and D-galactose had exhibit low activities as substrate. Gibson et al; (1964) reported that glucose oxidase is a FAD dependent glycoprotein catalyzing the oxidation of -D-glucose to glucono-1,5-lactone. It removes hydrogen from glucose and reduces itself. The reduced form of glucose oxidase is then reoxidized by molecular oxygen. The developed hydrogen peroxide is decomposed by catalase to water and oxygen. Pa Zur and Kleppe (1964) stated that the glucose oxidase from A. niger contains two flavin adenine di-nucleotide (FAD) moieties per mol-1 and had a molecular weight of approximately 150,000 da. The optimum pH for the enzyme was 5.5 and isoelectric point was 4.2. The enzyme was capable of oxidizing D-aldohexoses and mono-deoxy-Dglucose at varying rates. Differences in the rates of oxidation of the compounds have been interpreted to indicate that the structural features of the substrates of particular importance, in the enzyme reaction, are a pyranose ring in the chain conformation, an equatorially oriented hydroxyl group at position 3. These features are probably involved in the enzyme substrate complex formation. Willis (1966) showed that glucose oxidase from A. niger is an intracellular enzyme present in the mycelium of the organism. Weibel and Bright (1971) examined that the enzyme worked with broad range of pH, usually. They showed that optimum pH of glucose oxidase was 5.5.

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Greenfield and Laurence (1975) immobilized glucose oxidase and catalase on number of inorganic supports both individually and simultaneously. The effect of support type, immobilization technique and concentration of enzyme in the immobilizing solution on the initial activity of the immobilized enzyme was measured. The effect of operating pH and temperature on the initial activity and the storage stability was studied. Moderate activity levels can be achieved with inexpensive clay supports using relatively crude mixtures of glucose oxidase/fungal catalase, immobilized by the glutaraldehyde coupling procedure. Sasaki et al; (1982) studied the application of hydrophobic-ionic chromatography to microbial glucose-oxidase. The enzyme was absorbed on an Amberlite CG-50 column. Truge et al; (1984) studied the inactivation of glucose oxidase by the cationic detergent, hexadecyltri-methyl ammonium bromide. Glucose oxidase was inactivated completely at pH, 7.0. (0.1 M sodium phosphate buffer) by the cationic detergent. Attia et al; (1987) studied the physical and chemical properties of glucose oxidase from Penicillium charysogenum. Results showed that GOX is highly specific for -Dglucopyranose removal of CH2O group from pentose reduced the rate of oxidation to zero besides that hexoses like glactosemannose and and fructose were noted. It was that GOX contained 17% (w/w) carbohydrate residues in which mannose was most abundant (i.e. 12%) followed by glucosamine 3.4% and glactose 1.5%. Ye et al; (1988) reported the influence of additives (polyhydric alcohol, polyethyleneglycol and salts) on the thermostability of glucose oxidase at 60°C in aqueous medium. The results obtained revealed a stabilizing effect in the presence of polydric alcohol and for most of the polyethylene-glycol used. Kriechbaum et al; (1989) observed that glucose oxidase grom A. niger is a homodimer with a molecular weight of 150 to 180 KDa. It contains two tightly bound FAD molecules. Dissociation of the subunits only occurs under denaturation conditions and is accompanied by the loss of the cofactor FAD. The aminoaid sequence for the 583 residues protein has been derived from the DNA sequence independently. Ciulu (1989) worked out a simple rapid and precise amperometrical method to study the physico-chemical properties of glucose oxidase and catalase covalently immobilized on surface of the dialysis membrane.

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Takegewa et al; (1989) stated that 30% N-linked sugar chains of the GOX of A. niger contributed to high solubility of GOX in water. Grazillo et al; (1995) explained that GOX produced by Penecillium variab P16, purified by ion exchange and gel filteration chromatography. They reported that the molecular weight of the native enzyme was 126,000 KDa and that of the subunit was 62000 KDa, which indicated that the native enzyme was a dimer. The enzyme was active at pH. 6.0 and 55°C and highly specific for -D-glucose. Two isoenzymes, with pH values of 4.8 ­ 4.9, were detected on analytical isoelectric focusing gels. The GOX was inhibited by Ag+ and Cu+2 ions severely and by NaF to a lower extent. Rando et al; (1997) observed the glucose oxidase production by a newly isolated strain of Penicillium pinophilum. The sucrose was used as carbon source. They reported that the enzyme was specific for D-glucose. GOX showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mm. The enzyme was unstable at temperature above 40°C in the range pH 2-4 and at a pH above 7. The optimum pH was determined in the range pH 4-6. Liu et al; (1998) investigated that H2O2 producing enzyme glucose oxidase of Botrytis cinerea fungus was purified by anion exchange chromatography and chromato focussing. They found that enzyme has its optimum pH at 7.5 and an isoelectric point of 4.2. Analysis showed that GOX was specific for -D-glucose. The expression of the GOX of Botrytis cinerea was induced by low glucose concentration in the culture medium. In this respect, the GOX of B. cinerea was similar to the GOX of P. chrysosporium and differ from the glucose oxidases of Aspergillus and Penicillium, which required high glucose concentration for expression. Stosz et al; (1998) did work on localization of GOX with immunocytochemistry in the biocontrol fungus Talaromyces flavus. They found that the production of enzyme GOX was involved in the biological control of verticillium will be Talamyces flavus. The immuno staining revealed that GOX was both intracellular and extracellular and extremely stable enzyme, retaining 13% of its original activity after 2 weeks at 25°C. Sukhacheva et al; (2004) reported a method for isolation and purification of extra cellular glucose oxidase from Penicillium funiculosum 433. The enzymatic preparation was produced with a yield of 56% and a specific activity of 3730 per mg protein. The

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ezyme displayed a high thermostability to metal ions, and performance across a wide pH range. Bhatti et al; (2006) isolated an intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of A. niger NFCCP. The enzyme was partially purified to a yield of 28.4% and specific activity of 135 U mg-1 through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a Km value of 25 mmol L-1. The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GODcatalyzed D-glucose oxidation was 40 °C. The enzyme displayed a high thermostability having a half-life (t1/2) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol-1, and free energy of denaturation (G*) of 103.63 kJ mol-1. These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays. Karmali et al; (2004) investigated a simple and direct assay method for glucose oxidase (EC 1.1.3.4) from A. niger and Penicillium amagasakiense using fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of D-glucose at carbon 1 into D-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ([2]H[2]O). The intensity of the D-glucono-1,5-lactone band maximum at 1212 cm [1] due to C-O stretching vibration was measured as a function of time to study the kinetics of D-glucose oxidation. The extinction coefficient of D-glucono-1,5-lactone was determined to be 1.28 mM[-][1]cm[-][1]. The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V[m][a][x], K[m], k[c][a][t], and k[c][a][t]/K[m]) determined by Line weaver-Burk plot were 433.78 59.87 U mg protein, 10.07 1.75 mM, 1095.07 151.19s[-][1], and 108.74 s[-][1] mM[-][1], respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36 42.83 U mg[-][1] protein, 6.47 0.85 mM, 1187.77 108.16s[][1], and 183.58 s[-][1] mM[-][1] for V[m][a][x], K[m], k[c][a][t], and k[c][a][t]/K[m], respectively. Therefore, this spectroscopic method is highly suited to assay for glucose

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oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.

2.3

Application of GOX

Keilin and Hartee (1947) reported the use of glucose oxidase for the determination of glucose in biological materials and for the study of glucose producing system by monometric methods. It was based upon the specificity of GOX for glucose. He found the enzyme was highly specific for -D-glucose. Shearer et al; (1947) reported the cytotoxicity with antibody-glucose oxidase conjugates specific for human colonic-cancer. 1 g fractions of normal sera and antisera to CEA and HT-29 were prepared and conjugated to glucose oxidase, an enzyme which permits specific iodination of cell membranes in the presence of glucose iodide and lactoperoxidase. Cytotoxicity was evaluated with several established tumor cell lines in vitro by measuring the uptake of I125-iodoeoxyuridine after 24 hours in tissue culture. Specific iodination and cytotoxicity were observed with both enzyme-antibody conjugates, but the enzyme-antibody conjugate specific for CEA was less effective than that for HT-29. Cho and Bailey (1977) used glucose oxidase and glycoamylase combindly for conversion of maltose to gluconic acid and obtained high yield of gluconic acid. Dockrell and Play fair (1984) investigated that marine malaria parasite Plasmodium voelii was killed by a system in which this parasite was incubated with glucose and glucose oxidase generating hydrogen per oxide. Collings et al; (1985) used the GOX in quantitation of HLA-DR expressions by cells involved in the skin lesions of tuberculoid and lepromatous leprosy. Rama Samy et al; (1985) showed that lignin degradation activity was found in glucose oxidase producing strains. Regidor and Perez (1985) recommended the glucose oxidase method for glycemia tests. It is performed directly on serum. This method is the most specific and less toxic than the ortho-toluidine method.

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Paynter et al; (1986) compared HPLC and GOX assay system for determination of glucose contents in biomass samples and both were found in agreement with each other. Field et al; (1986) recommended glucose oxidase for extending shelf life of fish. The enzyme system (GOX + catalase) was applied as a dip and immobilized in algin blankets. Merigge et al; (1988) suggested the use of glucose oxidase in capillary glucose determination in the screening of gestational diabetes. Ueno et al; (1988) used glucose oxidase to determine the effects of reactive oxygen metabilites on erythro-protein production in renal carcinoma cells. Sandholm et al; (1988) reported the antibacterial effect of the glucose oxidase and lactoperoxidase system against mastitis pathogens. This system using optimum glucose/glucose oxidase ratios resulted in the complete killing of mastitis pathogens with the exception of P. aeroginosa. Kojuharova et al; (1988) used combined immobilized preparations of GOX and catalase in the oxidation of D-glucose and the preparation of calcium gluconate. These enzymes were immobilized in hydrogel and were able to retain 62% of the activity of mobilized enzyme. Fortier et al; (1988) investigated a fast and easy preparation of an amperometric glucose biosensor with the help of glucose oxidase. Electrochemical polymerization of pyrole in aqueous KCl solution containing glucose oxidase produced adherent films at platinum electrode surface. Such coated electrodes were prepared in 20 minutes and were able to determine glucose in the range 0-100 mM. Tiina et al; (1989) investigated the antibacterial effect of the glucose oxidase-glucose system on food poisoning organism i.e. Staphylococusaureus, Yersinia enterocolitica Bacillus cereus, Compylobacter ejuni and Listeria monocytogenes using automated turbidometry. The results indicated a clear inhibition of growth with 0.5-1.0 mg ml-1 glucose and 0.5-1.04ml-1 glucose oxidase. The most resistant pathogens were Campylobacterjejuni and Listeria monocytogenes. Morsan et al; (1989) stated that tooth pastes containing amylglucosidase/glucose oxidase could be used to reduce plaque. Sankaran et al; (1989) prepared a spray- dried, sugar free egg powder using glucose oxidase and catalase co immobilized on cotton cloth. Glucose oxidase and catalase were

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co immobilized on polyethylenimine coated cotton cloth by absorption followed by cross-linkage with glutaraldehyde. It could serve as an efficient and easily retrievable system for the repeated desugaring of eggmelange in a batch reactor. Ciucu (1989) prepared an electrode for glucose using glucose oxidase and catalase covalentely bonded on the surface of a dialysis membrane. The time of measurements was about 15-20 seconds for the kinetic method of the initial stop and one minute for the steady-state method the sensor responded linearly to glucose in the range 10-2 to 10-4 moles. Jiang and Ooraikul (1989) reported the use of glucose oxidase for reduction of non enzymatic browning in potato chips and French fries. A GOX with activity of 750 units per gram was used to reduce non enzymatic browning in potato chips and French fries. Wang et al; (1989) have prepared GOX modified carbon fiber electrodes by electrochemical deposition of the enzyme in the presence of platinum complex. The electrode is incorporated as an electrochemical detector in a flow system exhibiting a very rapid response to dynamic changes in the glucose concentration. Cohen et al; (1989) demonstrated the use of GOX in electrochemical analysis of oxygen content in samples of whole human saliva. James and Myrbork (1951) showed that enzyme preparation of A. niger contained an enzyme system which had absorbed oxygen and classified it as an oxidase. They also found that addition of glucose to the extract caused a great increase in the amount of oxygen consumed. They observed that during the absorption of oxygen, in the presence of glucose, gluconic acid was produced and the enzyme called glucose oxidase catalyzed this reaction. Glucose + O2 GOX Gluconic acid They showed that enzyme was highly specific for -D-glucose while mannose and galactose showed little activity as substrates. Robert and Reddy (1988) showed that GOX was the predominate source of hydrogen peroxide (H2O2) produced in the ligninolytic cultures of Phanerochaete chrysosporium. They found that GOX catalyzed the oxidation of -D-glucose to -D-gluconolactone and H2O2 in the presence of oxygen (O2). In a subsequent step -D-gluconolactone is hydrolyzed to D-gluconic acid non enzymatically.

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Petruccioli et al; (1995) reported that GOX produced by a strain of Penicillium (Penecillium variable P16), in a model system to control the activities of tomato lipoxygenase (LPO), polyphenol oxidase (PPO), and peroxidase (POD). They showed that GOX inhibited all three enzymes at the typical pH of tomato fruit (i.e.4.3). Metosh (1998) reported that various species of A. niger expressed GOX that catalyzes the formation of gluconolactone from glucose with reduction of molecular oxygen to hydrogen-peroxide. The basic function of this enzyme was the production of H2O2 for use in lignin degradation catalyzed by lignin peroxidase. Hames and Hooper (2001) reported that it was possible to assay the enzyme (GOX) that catalyzed the reaction by linking (or coupling) it to a second enzyme reaction that involved a characteristics absorbance change. They showed that GOX was often used to measure the concentration of glucose in the blood of diabetic patients, did not result in a change in absorbance upon conversion of substrates (glucose, oxygen and water) to products (gluconic acid + H2O2). However, the H2O2 produced was acted on by a second enzyme, peroxidase, which converted a colourless compound into a coloured one (chromogen) whose absorbance was easily measured. The activity of GOX was measured accurately by adjusting the peroxidase and its co-substrates in excess so as not to be the rate-limiting step of the linked assay. It was examined that the rate of production of coloured chromogen was proportional to the rate of production of H2O2, whose production in turn was proportional to the activity of GOX. Kinoshita et al; (1981) selected A. niger ATCC 6274 as an mutarotase producer from 45 stock cultures. Mutarotase was purified 115 folds to homogeneity from cell extracts with a yield of 2.6%. The time required for glucose determination with a GOX reagent was significantly reduced by the addition of mutarotase. Okuda et al; (1977) produced a modification, utilizing mutarotase of an enzymatic colorimetric system determining D-glucose with D-GOX and peroxidase. The time required for the assay of D-gluocose in aqueous solutions is about 10 minutes and the lower limit is 0.4 g of D-glucose.

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Wong et al; (1981) investigated that peroxidase, in the presence of H2O2, aminoantipyrine and chromotropic-acid catalyzed the formation of a deep blue compound having absorption with maximum at 590 nm wavelength. By coupling GOX, peroxidase and the chromogen it was possible to measure glucose enzymatically at levels of 10-100 g having short incubation of 5 minutes. Kaplan (1957) studied that due to marked affinity and specificity for -D-glucose; GOX is being used to determine the true glucose value of plasma/serum as well as other biological material. Williams (1976) reported that GOX has also been used to label antibodies used in the detection of tumor markers antigens and viral antigens apart from the measurement of glucose. Fiedurck et al; (1998) reported the production of gluconic acid, extracellular GOX and catalase in submerged culture by a number of biochemical mutants. Optimization of stirred speed, time, cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. 300 rpm appeared to be optimum to ensure good growth and best GOX production but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Miron et al; (2002) studied the production of GOX and gluconic acid by A. niger. The gluconic acid that is produced from glucose by means of enzyme action can therefore be considered as a useful source of carbon for growth, and does not interfere with the biosynthesis of GOX, inspite of being a product of its activity. Mukhopadhyay et al; (2005) studied the production of gluconic acid from whey by free and immobilized A. niger in a polyurethane foam. They found that addition of small amount of glucose (0.5%) in the whey medium enhanced the production of gluconic acid by 140% over the unsupplemented medium. Immobilized mycelia produced 92g of gluconic acid from one litre of whey medium, containing 9.5% (w/v) lactose and 0.5% glucose against 69g by free mycelia. Immobilized mycelia can be reused. Sumitra et al; (2006) investigated the microbial production of gluconic acid along with its properties and applications. They showed that gluconic acid is a mild organic acid derived from glucose by a simple oxidation reaction. The reaction is facilitated by the

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enzyme glucose oxidase (fungi such as A. niger) and glucose dehydrogenase (bacteria such as Gluconobacter). Vincent Marks (1996) reported a simple, accurate, rapid method of determining glucose especially in blood, C.S.F. and urine, using glucose-oxidase and peroxidase. He also studied a comparison between glucose and non-glucose reducing fractions before, during and after insulin administration is made, in which it was shown that non-glucose reducing substances in blood are diminished by insulin over a prolonged period. Wang et al; (2000) studied a micro-machined capillary electrophoresis chip used for simultaneous measurements of glucose, ascorbic acid, acetaminophen and uric acid. Fluid control was used to mix the sample and enzyme glucose oxidase. The enzymatic reaction a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide occurs along the separation channel. The enzymatically liberated neutral peroxide species was separated electrophoretically from the anionic uric and ascorbic acids in the separation channel. The three oxidizable species were detected at different migration times by goldcoated thick-film amperometric detector. Glucose can be detected within less than 100 seconds and detection of all electro active constituents is carried out within 4 minutes. Measurement of glucose in the presence of acetaminophen, a neutral compound are accomplished by comparing the responses in the presence and absence of GOX in the running buffer. The reproducibility of the on-chip glucose measurements was improved by using uric acid as an internal standard. Factors influencing the performance, including the GOX concentration, field strength, and detection potential were optimized such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate) as well as for introducing of high speed clinical micro-analyzers based on multi-channel chips. Pereira et al; (2004) studied a novel amperometric glucose sensor developed on the facilitated proton transfer across micro-interfaces between two immiscible electrolyte solutions. The protons were generated as a result of the dissociation of gluconic acid produced during the enzymatic degradation of glucose by GOX. The characteristics of the glucose sensor were investigated using several experimental conditions, namely, the concentration of ligand and enzyme.

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Liu et al; (2005) studied a micro-flow chemiluminescence (CL) system invivo for glucose determination by the on line micro-dialysis sampling. The sol-gel method was introduced to co-immobilize horse radish peroxidase and glucose oxidase on the inside surface of the micro-flow cell which was fabricated in polymethyl methacrylate (PMMA). The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H2O2, then H2O2 oxidizing luminal to produce CL in presence of horse radish peroxidase. The microdialysis probe was utilized for sampling in the rabbit blood. The glucose level in blood of the rabbit was on line monitored with good results. Pezzotti et al; (2005) investigated D-glucosaminic acid (2-amino-2 deoxy-D-gluconic acid) a component of bacterial lipopolysaccharides and a chiral synthon, easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2 deoxy-D-glucose) catalyzed by GOX. Pezzotti and Therisod (2006) studied several aldonic acids (d-mannonic, d-galactonic, d-xylonic, 2-deoxy-d-arabinohexonic (2-deoxy-d-gluconic) prepared on a scale of several grams by a simple oxidation catalyzed by GOX in pure water. Ikeda et al; (2006) analysed gluconic acid production using an enzymatic hydrolysate of waste office antomation paper in a culture of A. niger. In repeated batch cultures using flasks; saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yield were 60% (SM) and 67% (GM) with 80-100 gl-1 of gluconic acid. When pure oxygen was supplied the production rate increased to 4 times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM. Roukas et al; (2004) explained the production of citric and gluconic acid by A. niger ATCC 10577 in solid state fermentation. Citric acid yield 8% and gluconic acid yield 63% obtained at the moisture level of 75% initial pH 7.0, temperature 30°C, and fermentation time 15 days. Addition of 60% methanol into substrate increased the concentration of citric and gluconic acid.

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Sankpal et al; (2000) worked on the bioconversion of glucose to gluconic acid at low pH using A. niger. A glucose solution (100gl-1) was made to flow through capillaries of vertical fabric support, used for immobilization and it was oxidized to gluconic acid at the interface. Conditions of temperature, humidity, air flow and glucose feed rate had been optimized. The system ran continuously for a period of 61 days utilizing the entire available glucose. The emerging broth contained a product concentration of 120-140 gl-1 of gluconic acid which was higher than the expected (max of 109g gluconic-acid/100g glucose) as a result of evaporative concentration during the downward flow. Mutlu et al; (1997) developed a Clark type, amperometric multienzyme electrode employing glucose oxidase and mutarotase. It was used to determine the enzymatic activity of a model enzyme, invertase, in soluble and immobilized forms. A calibration curve with linear range up to 70 U of invertase activity corresponding to the 6.0 na s-1 of electrode response sketched. It was shown that, multienzyme electrodes can be utilized as a very rapid, reliable and sensitive tool for the determination of enzymatic activity either in a free or immobilized form. Mizutani et al; (1997) demonstrated that glucose and sucrose can simultaneously determined by the use of an enzyme sensor system consisting of a glucose-sensing electrode based on a lipid-modified glucose oxidase and a measuring cell that contains an invertase/mutarotase-coimmobilized layer. From the current response of the enzyme electrode after the addition of a glucose/sucrose mixture, the concentrations of the two kinds of sugars can be separately determined: the concentration of glucose (0. 2 mM-3 mM) is determined from the steady-state current increase obtained from 2 to 6s after the addition of the mixture, and that of sucrose (10 mM-60 mM), from the rate of current increase from 8 to 20 s after the addition. The relative standard deviations are, 1.7% (w/v) for glucose; and 3.1% for sucrose (n = 10). The system can be applied to the rapid determination of glucose and sucrose in food samples. Guemas et al; (2000) reported that glucose and sucrose were measured with an amperometric method by using the flow injection analysis technique. A carbon paste electrode with a renewable surface containing glucose oxidase, horseradish peroxidase,

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and ferrocene were used in combination with the soluble enzymes invertase and mutarotase. The effect of invertase, mutarotase, and ascorbic acid on the electrode response was examined. Glucose and sucrose concentrations were determined with <3% errors. The proposed method for glucose and sucrose measurements was validated in real samples of fruit juices. The results were also compared with those obtained with the ultraviolet method. Zajoncova et al; (2004) developed a new biosensing flow injection method for the determination of alpha-amylase activity the method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, cocrosslinked by gelatin-glutaraldehyde together with bovine serum albumine. AlphaGlucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-Dglucose by mutarotase. Beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol-1 maltose, which means 2 nkat ml-1 in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3: 1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml-1. A linear range of current response was 0.1-3 mmol-1 maltose, with a response time of 35 s. The biosensor was stable four at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 0C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months). Ohdan et al; (2007) worked to establish the enzymatic process to produce amylose from cellobiose Incubation of cellobiose with cellobiose phosphorylase and alpha-glucan phosphorylase in the presence of maltotetraose and a catalytic amount of inorganic phosphate at 45Co for 16 h resulted in the production of linear alpha-1,4-glucan with a 19.3% (w/v, against cellobiose weight). The yield was successfully improved (32.4%) when mutarotase and glucose oxidase were added to remove glucose in the reaction mixture. The average molecular weight of the product was controlled from 42 to 720 kda

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by changing the initial molar ratio of cellobiose to maltotetraose. The combined use of two different phosphorylases could be a useful tool in converting beta-1,4-linkedpolysaccharide into alpha-1,4-linked-polysaccharide. Shlyahovsky et al; (2007) developed a hybrid systems composed of a glucose oxidase (GOX)/peroxidase-mimicking DNAzyme, and microperoxidase-11 (MP-11)/anti-

thrombin aptamer.The hybrid systems were employed as amplifying labels for the colorimetric or chemiluminescence detection of enzyme functions, and thrombin analysis, respectively. In the GOX/DNAzyme system, the GOX-mediated oxidation of glucose led to the formation of H2O2, and this activated the oxidation of ABTS to a coloured product, or to the generation of chemiluminescence in the presence of luminol. The MP-11/anti-thrombin aptamer enabled the amplified analysis of thrombin by the MP11-mediated generation of chemiluminescence in the presence of luminol/H2O2. Chisari et al; (2007) extracted polyphenol oxidase and peroxidase from two different varieties of strawberry fruit (Fragaria x ananassa D, cv. 'Elsanta' and Fragaria vesca L, cv. 'Madame Moutot') and characterized them using reliable spectrophotometric methods in all cases, the enzymes followed Michaelis-Menten kinetics, showing different values of peroxidase kinetics parameters between the two cultivars: K-m = 50.68 +/- 2.42 mM ('Elsanta') and 18.18 +/- 8.79 mM ('Madame Moutot') mM and V-max = 0.14 +/- 0.03 U/g ('Elsanta') and 0.05 +/- 0.01 U/g ('Madame Moutot'). The physiological pH of fruit at the red ripe stage negatively affected the expression of both oxidases, except polyphenol oxidase from 'Madame Moutot' that showed the highest residual activity (68% of the maximum). Peroxidase from both cultivars was much more thermolable as compared with PPO, losing over 60% of relative activity already after 60 min of incubation at 40Co. The POD activation energy was much lower than the PPO activation energy (delta E = 97.5 and 57.8 kJ mol(-1) for 'Elsanta' and 'Madame Moutot', respectively). Results obtained from D-glucose and D-fructose inhibition tests evidenced a decreasing course of PPO and POD activities from both cultivars as the sugar concentration in the assay medium increased. Changes in CIE L*, a*, b*, chroma, and hue angle values were taken as a browning index of the samples during storage at 4 degrees C. A decrease in L* was evident in both cultivars but more marked in 'Elsanta'. PPO and POD activities from cv.

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'Elsanta' were very well-correlated with the parameter L* (r(2) = 0.86 and 0.89, respectively) and hue angle (r(2) = 0.85 and 0.93, respectively). According to these results, the browning of the fruit seemed to be related to both oxidase activities. Mislovicova et al; (2007) studied four immobilized forms of glucose oxidase (GOD) used for biotransformation and removed of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin, A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC, exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 Co) in comparison with those of free GOD. The decrease of V-max values after immobilization was observed, from 256.8 +/- 7.0 mu mol min (-1) mg(GOD)(-1) for free enzyme to 63.8 +/- 4.2 mu mol min(-1) mg(GOD)(-1) for GOD-ConA-TBC and 45 +/- 2.7 mu mol min(1) mg(GOD)(-1) for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.815.6% of its initial amount. The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose). Ricci et al; (2007) developed glucose biosensors based on the use of planar screenprinted electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 Co) with an operative stability of ca. 72 h,

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thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system. Park et al; (2007) demonstrated a new colorimetric method for determining the isomerization activity of sucrose isomerase .This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity. Shan et al; (2007) proposed that calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties.This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 mM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mA cm-2 M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein. Hadas et al; (2007) reported the contribution of gluconic acid secretion to the colonization of apple tissue by Penicillium expansum and was analyzed by modulation (increase or decrease) of gluconic acid accumulation at the infection court .P. expansum

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isolates that express the most GOX2 transcripts and concomitant glucose oxidase (GOX) activity and that secrete the most gluconic acid cause disease of apple at the fastest rate. Cultures grown under reduced oxygen concentration generated fewer GOX2 transcripts, produced less gluconic acid, and led to a 15% reduction in disease. Furthermore, the detection of significantly high levels of transcripts of GOX2 and GOX activity at the edge of the decaying tissue emphasize the involvement of GOX in tissue acidification of the decaying tissue. Taken together, these results emphasize the importance of GOX in the production of the gluconic acid that leads, in turn, to host tissue acidification. This acidification enhanced the expression of pectolytic enzymes and the establishment of conditions for necrotrophic development of P. expansum. According to Worthington (1988) since glucose oxidase discovery as an "antibiotic" (shown subsequently to be due to peroxide formation) there is an increasing interest in it, chiefly because of its utility in glucose estimation. Glucose "kits" and "dip-sticks" become available for screening of blood/urine glucose (qualitative estimation) by coupling the reaction to peroxidase and a chromogen.

2.4

Enzyme sources

GOX is produced by a wide variety of micro organisms including fungi, bacteria and yeasts. Microbial degradation of biological wastes is a natural process that has occurred since the on set of life on earth. In biodegradation system, microorganisms utilize wastes as potential energy source for synthesis of very useful fermentation products such as enzymes, hormones, organic acids, liquid fuels, single cell proteins etc. Acquisition of metabolizable energy through biomass recycling very attractive proposition particularly for country like Pakistan having agro-based economy. Rajoka and Malik (1994). Fungi are potent source of glucose oxidase enzyme. There are about 70,000 species of fungi, most of which are saprophytes (Onions et al; 1981). These saprophytic funji produce a wide range of hydrolytic enzymes to degrade the complex organic substrates on which they grow. Lipolytic fungi can grow on fat while non-lipolytic fungi cannot. There are several fungal species which have both lipolytic and non-lipolytic strains. Fungi A. niger species have been reported to be lipolytic in nature. (Mukherjee, 1951).

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The fungi constitute a most fascinating group of organism exhibiting great diversity in form structure, habitat, life history, mode of nutritional and mycelia tropic stage. Adequately distinguishing the fungi from other groups has provided universal acceptance of fungi as a separate kingdom (Hawksworth et al; 1983). The use of filamentous fungi may have a number of advantages over yeast and bacteria, the most important of which being their capability to be propagated on a wide variety of substrates generally discarded as wastes, it involves simple harvesting and manufacturing operations (Sinskey 1978). The success of A. niger for industrial production of biotechnological products is largely due to metabolic versatility of this strain. A. niger is well known to produce a lot of organic acids, enzymes, plant growth regulators, mycotoxins and antibiotics. The industrial importance of A. niger is not limited to its more than 35 native products but also to the development and commercialization of the new products which are derived by modern molecular biology techniques (Davies, 1991). GOX has been produced from various organisms like Penicillium notatum (Bentley, 1959).P. chrysosporium, Botrytis cinerea (Liu et al; 1998) Fusarium lini, P. reticulosum Tauber, (1949) and Saccharomyces cerevisiae (Baetselier et al;, 1991). According to Worthington (1988), the crude form of A. niger enzyme is satisfactory for the most clinical work. GOX was also found in Penicillium glaucum (Muller, 1936). Coulthard et al; (1942) discovered this enzyme from Penicillium notatum and it was also found in Penicillium amagasakiense by Kusai et al; (1960). But GOX is commercially prepared from two fungal sources. A. niger and Penicillium amaga sakience (Rogalski et al; 1988). Fiedurck et al; (1986) found after screening different fungi that A. niger is the best source for the production of this enzyme. Willis (1966) applied A. niger to submerged culture fermentation for GOX production in a mineral medium containing a source organic nitrogen and carbohydrate. GOX was present in the filtrate and purified by means of ammonium sulphate precipitation technique and DEAE-Cellulose chromatography. Lu et al; (1996) showed that waste mycelium of A. niger from the production of sodium, calcium and magnesium gluconate can be used to produce glucose oxidase.

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Finkel Stein (1987) showed that some fungal strains that are used to produce enzymes in an industrial process are capable to secrete large amounts of the respective products e.g. A. niger produces glucoamylase at 0.5g/L, as a result of mutation as well as medium development and optimization of fermentation condition, the yield increased 40 fold reaching about 20gl-1. Jeens et al; (1991) reported that the filamentous fungi typically, are saprophytic microorganisms, which secrete a wide array of enzymes involved in the breakdown and recycling of complex biopolymers from both plant and animal tissues. Now a days the production of enzymes is an important and well growing sector of the fermentation industries. Several of these enzymes have been developed for a variety of commercial uses; e.g. in textile processing, leather manufacturing, paper and pulp processing, detergent production and in food processing. Fiedurck and Ilezuk (1992) reported 1,486 mould strains isolated from natural sources (screened for extracellular GOX) only 119 (Aspergillus and Penicillium) showed GOX enzyme activity. As the best GOX producer, A. niger 0-1 was isolated from decaying tree. The dynamics of GOX synthesis in A. niger 0-1 during its culture by submerged method show that the intracellular activity of this enzyme is 10-times higher than its extracellular level. Fiedurck et al; (1994) used a new method for GOX production by A. niger conidium's immobilized on seeds was also studied by the adsorption of A. niger spores on wheat seeds are very simple and inexpensive method of immobilization and continued production immobilization and continued production of GOX was observed for 8 repeated batches. Fiedurck and Gramada (1997) studied a strain of A. niger (G-4-10) that was effective for simultaneous production of catalase and glucose oxidase, it was selected out of molds belonging to 15 different species by the method of test-tube micro culture. According to Willis (1966), Nathan and Kaplan (1957) GOX from A. niger is an intracellular enzyme present in the mycelium of organisms. Fiedurck and Gromada (1996) investigated the production of GOX from A. niger in the submerged culture with continuous shaking waste mycelium of A. niger are used to produce the GOX.

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Liu et al; (1999) reported the synthesis of GOX and catalase by A. niger using a resting cell culture system without growth being established. Wosten et al; (1991) explained the protein secretion in A. niger using immunocytochemical methods, it was shown that glucoamylase secretion in A. niger occurred at the growing hyphal tip. While Peberdy, (1994) presumed that the apical vesicles are the final step of the intracellular secondary pathway that begins at the endoplasmic reticultum (ER) and proceeds via a golgi system generally in other eukaryotic cell. According to (High 1992), in Eukaryotic cells, the desired proteins for secretion are synthesized on ribosomes of the endoplasmic reticulum. The secondary process is then initiated by the sequencation of the nascent extracellular portion into the lumen of the rough endoplasmic reticulum. This process is determined by the information of the signal sequence attached to the protein molecule. In general, signal sequence of different organisms shares the common features. They comprise 13-30 amino acids with a basic N-terminal reigion and more polar Cterminal region, which is the cleavage site. Three changes may occur to a protein molecules before secretion: (1) Proteolytic cleavage to remove signal sequence and other peptide sequence, if present. (2) A folding process involving disculphide bonds formation to develop tertiary and quarternary structure of the protein where this bond stabilizes the molecule. (3) Glycosylation.

Farkas (1985) studied the relationship between fungal cell wall and protein secretion. According to him the cell wall of filamentus fungi fulfills the following functions. (1) Formation of rigid barriers on the surface of protoplast to determine the cell shape. (2) Acting as the site of various extracellular enzymes engaged in the exchange of nutrients and products of metabolism as well as hydrolysis of cell wall components during the cell growth. (3) (4) Acting as a reservoir of carbohydrates. Acting as a carrier of specific antigens.

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(5) (6)

Protection from osmotic stress on the protoplast. The cell wall plays an important role as a biobarrier for neutrient uptake and excretion process, (Peberdy, 1994) which are highly dependent on the porosity of the cell wall.

(7)

The external/internal ratio of enzyme was found to be strain dependent in accordance with the differentiation in cell wall composition e.g. Invertase is excreted by both A. niger and A. nidulans. In A. niger, the distribution of the enzyme is 70% cell bound and 30% excreted, but in A. nidulans, the enzyme is distributed more equally. In all cases, 70% of the cell bound enzymes are external to the plasma membrane (Peberdy, 1994).

In submerged cultivation the filamentous fungi morphology can vary from compact pellets of hyphae to homogeneous suspension of dispersed mycelia. The filamentous form of mycelial hyphae easily causes entanglement and the cultivation broth becomes viscous while the pellets form can be an attractive growth form for cultivation of fungi, it decrease the viscosity of the cultivation broth (Van Suij dam et al; 1980) If the pellets consist of more filamentous mycelium agitation of the culture allows neutrients and oxygen to reach all hyphae and support the growth of the entire biomass. Pellets are classified into three types i.e. coagulative, non coagulative and hyphae element agglomeration type investigated by Nielson (1996). The coagulative type is characterized by the coagulation of spores while germination gives rise to a net of intertwined hyphae. A. niger pellets were found to be a good example of this type. Following are the factors which usually affect the microbial pellet formation of filamentous fungi.

(1)

· · · · ·

Medium composition

Carbon source Nitrogen source Addition of complex organic material Addition of polymer Addition of surfactants

That includes

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· · ·

Presence of solid particles Divalent cations Antifoam.

(2)

· · · · · ·

Strain dependent factors

Strain used Type of inoculum Type of aggregate Cell wall composition Degree of aggregation Amount of inoculum.

That includes

(3)

· · · · · · ·

Cultivation conditions

pH Temperature Oxygen supply Shear force Dilution rate Type of bioreactor. Shape of cultivation vessel.

That includes

Medium composition i.e. nutritional dependent factors of A. niger like C-source, Nsource, addition of polymer, addition of surfactants and presence of solid particles (Hermers dor fer et al; 1987, Elmayergi 1975, Metz and Kossen 1977). Strain dependent factors of A. niger like type of inoculum and type of aggregate were explained by Van suijdam et al; (1980) and Olsvik et al; (1993). Cultivation conditions like pH, temperature, oxygen supply, shear force of A. niger were investigated by (Braun and Vecht-Lifshitz, 1991), (Zetelaki and Vas, 1968) and (Metz et al; (1981). (Hermerdofer et al; 1987).

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Isolation of GOX producing bacteria and fungi has been reported by different techniques. The principle behind all these techniques is the selection on a growth medium having some sugars as the prime carbon and energy source. Turner (1929), used agar medium containing dispersed fat and Nile blue sulfate for isolation of lipolytic colonies. Fryer et al; (1967) developed a double layer technique for the screening of lipolytic organisms. The organisms were grown on nutrient agar overlying the tributyrin agar. Kundu and Pal (1970) reported a new method for the isolation of lipolytic fungi from the soil on oil-mineral medium spread on silica gel plates. In most of the cases isolated fungal cultures were maintained on malt-agar and potato dextrose agar media reported by Espinosa et al; (1990). However some workers have chosen other media for culture maintenance. Pal et al; (1978) maintained A. niger on olive oil sucrose-agar medium. Valero et al; (1988) used malt extract peptone-agar medium for the maintenance of Candida rugosa. Guimaraes et al; (2006) demonstrated that many enzymes produced by fungi have relevant biotechnological applications in several industrial areas .The purpose of his study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the Sao Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose- oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose- oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C-8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var.

rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light

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microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of Sao Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti- microbial or anti- parasitic activities. Ramachandran et al; (2006) reported that spores of A. niger were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found to be retained in the spores even after repeated washings. Average rate of reaction was 1·5 g l

1 1 1 1

h

with 102 g l

of gluconic acid produced out of 100 g l

glucose

consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide.

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Chapter No.3

EXPERIMENTAL WORK

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MATERIALS AND METHODS

The study was divided into two parts. (A) (B) Production of glucose oxidase from Aspergillus niger. Commercial application of glucose oxidase.

3.1

(A) Production of glucose oxidase from Aspergillus niger

Glucose oxidase was produced from Aspergillus niger by the submerged fermentation following the methods of Fidurek and Gromada (1996) with some changes. The study of this part includes following aspects: 3.1.1 Screening of sources for the microbial strain producing highest GOX activity along with optimizing pH and carbon source. 3.1.2 Optimization of other fermentation conditions for improved GOX production like fermentation period, MgSO4, KH2PO4, and urea. 3.1.3 Enzyme kinetics 3.1.4 Enhanced GOX production by UV mutation 3.1.5 Purification of GOX. 3.1.1 Screening for the fungal strain producing the highest GOX activity along with optimizing carbon source and pH For commercial production of enzyme first step is the screening of different native strains for the optimal enzyme production. So in this section different strains of A. niger were examined along with optimization of two growth conditions pH and carbon source, (i.e. glucose concentration) for the optimal production of glucose oxidase. This will help the commercial production of glucose oxidase in Pakistan. 3.1.1.1 Microorganism

The strains of A. niger were isolated from five different sources (bread, potato, grapes, pickle, sugar beet) identified from the fungal culture bank (FCB) at Punjab University Lahore. They were grown on malt extract agar medium at pH 5.5 and with following composition (gl-1).

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1. 2. 3.

Malt extract Peptone Agar

20 g 2.5 g 10 g.

The medium was prepared by weighing accurately 1-3 and dissolving them in 500 ml of distilled water. The medium was boiled, with constant stirring for 15 minutes and was poured into clean test tubes. The tubes were plugged with sterilized cotton and autoclaved at 20 lbs PSI at 100°C for 15 minutes. After autoclaving the tubes were placed in slanting position for 24 hours. Transfer of strains on fresh medium continued after every two weeks. These pure and identified colonies were kept in the refrigerator at 4°C for storage. 3.1.1.2 Substrate (carbon source)

Glucose obtained from biochemistry Lab. Chemistry Department, GC University Lahore was used as carbon source for the growth and production of glucose oxidase.

3.1.1.3

Fermentation

The enzyme was produced by submerged fermentation of A. niger in 250 ml shake flask. The fermentation medium with the following composition was used. Table 3.1 No. 1. 2. 3. 4. 5. 6. Composition of fermentation medium. Compounds NaNO3 Urea MgSO4.7H2O FeSO4.7H2O KH2PO4 CaCO3 Amount g/100 ml 12.5 0.2 1.25 0.025 2.5 1.75/25 ml H2O

(Petruccioli and Federici, 1993) with some amendments i.e. urea used instead peptone and no KCl was added in the medium.

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Table 3.2

Composition of components used in the 250 ml flask. No. 1. 2. 3. 4. 5. 6. 7. 8. Ingredients CaCO3 NaNO3 Urea MgSO4.7H2O FeSO4.7H2O KH2PO4 Glucose Spore suspension Amounts 1.75 g/25 ml 2 ml 2 ml 2 ml 2 ml 2 ml 10 ml 5 ml

(i)

Substrate level (carbon source):

The required glucose amount (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0%(w/v) was dissolved in 100 ml water. After sterilization, 10 ml of above different concentrations of glucose solution was added to the flasks containing 35 ml of the sterile fermentation medium containing the compounds mention in table 3.2 (2ml each) and were inoculated with 5 ml spore suspension (107­108 spore/ml) (Collins et al; 1984). Now the flask contained sterilized growth media of about 50 ml (shown in table 3.2) which was already autoclaved at 20 lbs. PSI at 100°C for 15 minutes.

(ii)

pH of media

The required pH of the medium (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) was adjusted by 1M HCl and 1M NaOH. These flasks were then incubated in shaking incubator at 100 rpm and 30°C for 48 hours (Willis, 1966). The fungal spores were also counted by Thoma Counting Chamber (Collins et al; 1984). All the experiments were carried out in triplicate.

3.1.1.4

Enzyme extraction and partial purification

After 48 hours of incubation in shaking incubator at 30°C, the mycelia were collected by filtration and washed with distilled water. The mycelia collected were weighed. The washed mycelia were crushed in sodium citrate buffer (0.1 M pH = 5) In Mortar and Pestle for 30 minutes and centrifuged at 10,000 rpm for 10 minutes mycelium debris was

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separated and kept for dry mass estimation. The filtrate was precipitated with (NH4)2SO4 and kept at 4°C overnight. The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes. Each sample were dissolved in 2 ml of distilled water and refrigerated.

3.1.1.5

Assay of GOX

The glucose oxidase activity was determined by the fast spectrophotometric method by following the enzymatic reduction of benzoquinone to hydroquinone at 290 nm using glucose as substrate (Ciucu and Patroescu, 1984, Dr. Abdul Hameed and Khawar) One unit (u) of enzyme activity was defined as the amount of enzyme producing one micromole of hydrogen peroxide per minute at 30°C. It was also defined as the amount of enzyme catalyzing the decomposition of one micromole of hydrogen-peroxide per minute at 30°C or "One unit catalysis the oxidation of 1 µ mole glucose to gluconic acid per minute at 25°C pH 5 coupled with peroxide and 1-4 benzoquinone". Glucose oxidase oxidize glucose to gluconic acid and H2O2 produced in above reaction reduces benzoquinone to hydroquinone. Both spectrometer cell (A and B) were filled up with 2 ml of 1 M glucose solution which was prepared a day before at room temperature so that and anomers formed at equilibrium. In cell A, 1.0 ml of 0.1% benzoquinone solution (0.01 g in 10 ml distilled water) and 1.0 ml of buffer solution (sodium citrate buffer of 0.1 M, pH = 5) were added. This cell was taken as standard. In cell B, 1.0 ml of 0.1% benzoquinone solution and 0.9 ml of sodium citrate buffer were added. The mixtures of both cell (A and B) were allowed to equilibrate at 25°C for 5 minutes. In cell B, at time zero 0.05 ml of glucose oxidase solution was added and mixture was stirred. An increase in absorbance at 290 nm was recorded for 1 ­ 2 minutes.

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(UV-Cecil CE 7200 Spectrophotometer)

This rate was used to calculate the enzyme activity as follows: Enzyme activity at 25°C Where V µ A290 I A Above equation become. Enzyme activity = = Specific activity = 17.316 x A µ moles HQ/min/ml

17.316 × VA = mgprotein/ml

µ mole HQ/min/mg Protein.

mM

=

VA × V I × A 290 mM × u

µ moles HQ/min/ml

= = =

4.0 ml, volume of reaction mixture 0.05 ml, GOX solution 2.31, molecular absorption co-efficient of HQ (hydroquinone) at = 290 nm

= =

2 cm, length of cell. the actual absorbance of sample after sub starting the control.

3.1.1.6 3.1.1.6.1

Solution A Solution B

Protein estimation Reagents

= = 40 gm sodium carbonate in 500 ml distilled water. 0.38 gm CuSO4.5H2O and 0.6 gm potassium-sodium tartarate in 500 ml distilled water.

Proteins were estimated according to the method described by Moss and Bond (1957).

Solution C

=

1 part Folin reagent (Folin ciocaltem's phenol reagent BDH) and 2 parts of H2O (prepared just before use)

Solution D

=

1N NaOH.

3.1.1.6.2

Standard bovine serum albumin solution (500 µg ml-1)

5.0 mg of Bovine Serum Albumin (BSA) was dissolved in 10 ml of distilled water. The serum albumin dissolved very slowly. The standard was made two days before use and kept at 4°C.

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3.1.1.6.3

Procedure

0.5 ml NaOH 1N (sol. D) was added in each tube containing protein sample (250-750 µg) and were shaken in water bath (37°C) for 30 minutes. After cooling 2.6 ml of solution A and 2.5 ml of solution B was added in each sample as well as in standard tube. The solution in each tube was thoroughly mixed and incubated at 37°C for 30 minutes. Then 0.5 ml of solution C was added, mixed well and kept for 20 minutes at room temperature. BSA (0.05 ­ 1.5 ml) was simultaneously used in experiment. The optical density was measured at 661nm on spectrophotometer (UV-Cecil CE 7200) and a standard calibration curve was drawn and with its help the proteins were estimated.

0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0.00

Absorbance(OD)

0.05

0.10

0.15

0.20

0.25

0.30

Concentrations (mg/mL)

Fig-3.1 Standard curve for bovine serum albumin for protein estimation

3.1.2

Optimization of fermentation conditions for max. GOX production

The conditions for glucose oxidase production by A. niger were also optimized other than substrate level (carbon source i.e. glucose) and pH optimization was carried out along with screening of fungal strains in the previous section (3.1).

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The medium containing glucose was cultured with A. niger isolated from potato (screened in previous section) for different incubation periods with varying concentrations of urea, CaCO3, MgSO4.7H2O and KH2PO4 at 30C° in shake flask.

3.1.2.1

Submerged fermentation

Triplicate flasks of 250 ml containing 50 ml of sterilized growth media along with 5 ml of spore suspension were incubated at 30° on shaking incubator (100 rpm) for each condition to be optimized.

3.1.2.2

Sample preparation

After 48 hours incubation experimental flasks (in each experimental process) were harvested. The process of enzyme extraction is described in section 3.1.

3.1.2.3

Glucose oxidase assay

GOX activity was measured by method of which was Ciucu and Patroescu (1984), described in section 3.1.

3.1.2.4

Fermentation period (incubation time)

The growth media containing 10% (w/v) glucose as substrate at pH = 5.5 (both conditions optimized in previous section) contained in 10 flasks were sterilized, inoculated and were incubated at 30C° under continuous shaking condition (100 rpm). Triplicate growth media were incubated for 12, 24, 36, 48 and 60 hours.

3.1.2.5

CaCO3

The effect of different concentrations of CaCO3 (i.e. 1.5, 2.5, 3.5 and 4.5%) in the medium were studied for maximum production of GOX.

3.1.2.6

Nitrogen source (urea)

The concentrations of urea (nitrogen source i.e. 0.1, 0.2, 0.3, 0.4 and 0.5%) have a considerable influence on GOX production. Different concentrations of urea were added into the fermentation medium having 10% glucose at pH 5.5.

3.1.2.7

KH2PO4

The effect of different concentrations of KH2PO4 (i.e. 0.2, 0.4, 0.6, 0.8 and 1.0%) were studied on GOX production in the medium containing optimum concentration of glucose (10%), Urea (0.2%), and CaCO3 (3.5%) at pH 5.5.

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3.1.2.8

MgSO4.7H2O

Different concentrations of MgSO4.7H2O i.e. 0.01, 0.02, 0.03 and 0.04%were examined for GOX activity in pre-optimized culture medium with 10% glucose, 0.4% KH2PO4, 0.2% Urea, 3.5% CaCO3 at pH 5.5 and 30°C.

3.1.3 Enzyme kinetics

The effect of temperature on the enzyme activity was also investigated. Effect of aeration on GOX production in shake flask (oxygen supply) was also investigated. The effect of aeration rate on GOX production by A. niger (source potato) was studied by changing the shaking speed as well as the volume of the medium in the flasks. Different volumes of the medium in 250 ml conical flasks (such as 20, 30, 40, 50, 60 and 80 ml) and different speeds of the shaker such as (50, 100 and 150 rpm) were tested (Table 4.17).

3.1.4 Enhanced GOX production by UV mutation

A mutation is a permanent change in the DNA sequence of gene. Mutation is a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene. How does this happen? The DNA sequence of each gene determines the amino acid sequence for the protein it encodes. The DNA sequence is interpreted in groups of three nucleotide bases, called codons. Each codon specifies a single amino acid in the polypeptide chain.

3.1.4.1

Microorganism

The fungal strain of A. niger was isolated from potato source and identified from FCB. Punjab University Lahore and was grown on malt extract agar medium at pH 5.5 as described earlier in the section (3.1.1.1). The cultures were stored in the refrigerator at 4°C for further studies.

3.1.4.2

Preparation for mutagenesis

One ml of prepared conidial inoculums was transferred to 100 ml of sterilized water. This conidial suspension was further diluted up to 10-30 by serial dilution method.

3.1.4.3

Ultraviolet treatment

Diluted conidial suspension (10 ml) was transferred in the sterilized petri plates. The Petri plates were placed under the UV lamp (emitting the energy of 2.6 x106 J/m2/S) in the laminar flow for 5- 60 minutes. After different time intervals 0.5 ml of the irradiated

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conidial suspension was transferred to the petri plates containing malt extract agar with 2Deoxy D- glucose medium. The Petri plates were then placed in the incubator at 30oC for 3-4 days. The young colonies of Aspergillus niger on the petri plates were picked up and transferred to the malt extract slants

3.1.4.4

Fermentation technique

Submerged fermentation technique was employed process is same as described in previous section (3.1.1.3).

3.1.4.5

(3.1.1.4)

Enzyme extraction:

The enzyme extraction was carried out using the method explained in previous section

3.1.4.6

Enzyme assay

The process details have been described in previous section (3.1.1.5).

3.1.5

· · · ·

Purification of glucose oxidase

Filtration (NH4)2SO4 Precipitation Dialysis Gel filtration using Sephadex G-75

Following steps are involved for the purification of glucose oxidase from A. niger.

3.1.5.1

Filtration

After 48 hours of incubation at 30oC and 120 rmp the culture medium was filtered through nylon gauze to remove the mycelia. The mycelia were washed with distilled water and the whole solution then filtered through filter paper. The pH of filtrate was adjusted at 5.0 by using citrate buffer and NaOH and filtrate was placed at 4oC for 60 minutes and enzyme activity was calculated.

3.1.5.2

(NH4)2SO4 Precipitation

Ammonium sulphate precipitation (salting out) is a technique used to precipitate proteins from solution by increasing the ionic strength of the solution. The technique is reliant on the hydrophobic nature of proteins, since they contain hydrophilic and hydrophobic groups. When the proteins are dissolved, water is forced into contact with the protein's hydrophobic groups and in the process becomes ordered around the proteins. Increasing

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the concentration of salt ions by the addition of ammonium sulphate causes the water to be removed from around the protein exposing the hydrophobic portions of the proteins. Precipitation of the proteins will then occur due to the aggregation of proteins via the exposed hydrophobic portions. This technique is used to fractionate the proteins from solution since proteins with larger or more hydrophobic portions will aggregate and precipitate before those with smaller or fewer proteins of hydrophobic groups.

Procedure

Solid ammonium sulphate is added to 100ml of solution of glucose oxidase at the concentration of 30% (w/v). The suspension was stirred for half an hour at 4oC. After sufficient shaking the precipitates were collected by centrifugation at 10,000 rpm for 30 minutes. Enzyme activity was determined for each concentration and precipitates were collected for further purification. Enzyme solution was then treated with 40, 50, 60, 70 and finally 80% (w/v) (NH4)2SO4.

3.1.5.3

Dialysis

The precipitates obtained after ammonium sulphate precipitation procedure, were suspended in small volume of Citrate buffer (pH 5) and dialyzed by using 12,000 d molecular weight cut off dialysis bag , which was placed in 2 liters of Citrate buffer (pH 5) for 24 hours at 4oC against three changes. GOX activity of the dialyzed material was determined (section 3.1.1.5).

3.1.5.4

Gel filtration using Sephadex G-75

The principle of gel filtration is based on elution of proteins on the basis of size and shape of the molecules. Molecules of smaller size pass through the beads of gel and the remaining larger sized pass through spaces between the gel beads. Larger molecules are eluted earlier and smaller which travel inside the gel beads eluted later.

Reagents

0.1 M Citrate buffer (pH 5.0) Dextrin blue (molecular weight 2x106) solution (0.5% w/v) Sephadex-G-75

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Procedure

Sephadex-G-75 was soaked in 500 ml of 0.1 M citrate buffer (pH 5.0) containing 0.1 g of sodium azide and incubated at room temperature for 24 hours. After soaking, the gel was deaerated by direct drive rotary vacuum pump and then poured in a 0.9 x 60 cm column. The packed column was washed with 0.1 M citrate buffer (pH5.0). Dextran blue (0.5% w/v) was used for the determination of its void volume. Dialyzed enzyme extract was applied on the column and fractions each of 3 ml were collected. Each fraction was then assayed for enzyme activity and the amount of the protein present. Fractions containing enzyme activity were pooled and lyophilized (Jakoby 1971).

3.2

Commercial applications of glucose oxidase

In the present research projects two commercial applications of GOX were investigated; 3.2.1 Estimation of glucose by standardization of conditions using GOX. 3.2.2 The production of calcium gluconate, gluconic acid and its derivatives using glucose oxidase.

3.2.1

Estimation of glucose by standardization of conditions using GOX

The disease involved in the elevation of blood glucose level is known as diabetes mellitus. It is a very common disease now a days. It is a metabolic problem and is prevalent in many parts of the world. One fundamental aspect of diabetes is an abnormality of glucose metabolism due to in sufficient action of insulin, owing either to its absence or to resist in action (Murray et al 2001). Blood glucose level in diabetes becomes so elevated that the glucose "spills over" into urine, providing a convenient diagnostic test for the disease (Voct et al 1999). In the process of determination of glucose level there are involved three enzymes i.e. mutarotase, glucose oxidase and peroxidase. The overall reaction mechanism is as follows: -D-glucose -D-glucose 2H2O2

Mutarotase Glucose Oxidase

-D-glucose -D-gluconolactone + H2O2

Peroxidase

2H2O + O2.

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The enzymes and their kits for glucose estimation are being imported at high cost and a lot of foreign exchange is required. Economy of our country can not afford such high cost kits so there is a need to optimize the conditions for such methods. So this part of the project was designed to develop the technology to optimize the conditions for glucose estimation using these 3 enzymes and to prepare low priced local kits as compared to imported kits. The three enzymes glucose oxidase, mutarotase and peroxidase were produced/extracted and purified for the preparation and optimization of glucose estimation kit.

3.2.1.1

Glucose oxidase

Glucose Oxidase was produced by submerged fermentation using glucose as substrate and Aspergillus niger (source potato) as the fermentative organism. GOX extraction, purification and assays were described earlier in the section (3.1)

3.2.1.2

Mutarotase

Bovine kidney cortex was used to extract the enzyme mutarotase. It was purified by ammonium sulfate precipitation, dialysis and gel filtration chromatography. 3.1.2.2.1 Enzyme extraction

The method of Bentley (1962) and (Zia M. A.) was applied to prepare extracts. A bovine kidney cortex of about 100g was mixed with 100 ml of 0.1M phosphate buffer of pH 5.8 and 50 ml of chloroform and was homogenized in a blender for 2 minutes. This extract was centrifuged at 8,000 rpm for 15 minutes and the supernatants were dialyzed for 2 hours against 0.1 M phosphate buffer (pH 5.8) with continuous stirring. 3.2.1.2.2 Enzyme assay

The activity of mutarotase was determined by following the procedure of Calzyme Laboratory Manual (1998). i) Determination of spontaneous mutarotation At time zero, 100 mg -D-glucose was dissolved in 10 ml of 5mM sodium EDTA and transferred to polarimeter. Rotation was recorded at 1 minute intervals for the first 10 minutes. After 10 minutes rotation was recorded every 5 minutes intervals. After 30 minutes the rotation was noted at 15 minutes intervals. Until the rotation become constant.

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ii)

Graph rotation vs time

The Initial rotation corresponds to an -D-glucose concentration of 555µ mole. The data obtained were extra plotted to zero to obtain the initial rotation. iii) Blank rotation graph

Graph rotation was determined against time in 1 minute intervals, up to 5 minutes. iv) Standard curve Graph rotation Vs µ mole of -D-glucose using data from spontaneous mutarotation was plotted. It was between 555 µ mole (initial and 195 µ mole (Final rotation). v) Determination of test rotation

At time zero, 0.1 ml of enzyme sample was added to 9.9 ml of 5 mM sodium EDTA solution and dissolved in 100 mg -D-glucose. The rotation was determined at 30 second intervals for 10 minutes. vi) a b c d Calculation = = = = Initial rotation from spontaneous rotation graph. Rotation for 5 minutes from graph (ii) Blank rotation for 5 minutes (c = a-b) Conversion of c to µ moles of -D glucose from standard curve graph. e f g h i j = = = = = = 555 µ mole -D glucose at initial rotation. Spontaneous rate in micromoles/minute (f = e-d/5 minutes) Test rotation per 5 minutes from test graph. Test rotation after 5 minutes (h = a-g) Conversion of `h' to micromole from standard curve. Test rate in µ mole/minute i.e. [j = (e-i)/5 min ­ f].

Units/mg protein = j/mg protein/10.0 ml reaction mixture. 3.2.1.2.3 Protein estimation

Protein were estimated according to the method of Moss and Bond (1957) as mentioned earlier in section (3.1). The optical density was measured at 661 nm wavelength and a standard curve was plotted between concentration (mg/ml) and absorbance. Total protein concentrations were estimated from that curve.

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3.2.1.2.4

Partial purification of mutarotase by (NH4)2SO4 precipitation technique

The enzyme extract was precipitated by ammonium sulfate for partial purification as described by the method of Bentley (1962). Solid ammonium sulphate was added to 100 ml of solution of glucose oxidase at the concentration of 30% (w/v). The suspension was stirred for half an hour at 4oC. After sufficient shaking the precipitates were collected by centrifugation at 10,000 rmp for 30 minutes. Enzyme activity was determined for each concentration and precipitates were collected for further purification. Enzyme solution was then treated with 40, 50, 60, 70 and finally 80% (w/v) (NH4)2SO4.

3.2.1.2.5

Dialysis

The precipitates obtained by ammonium sulphate precipitation were measured in 0.1 m phosphate (pH 5.8) buffer and dialyzed in dialysis bag with constant stirring for 2 hours and then were subjected to enzyme assay (3.2.1.2.2) and protein estimation (3.2.1.2.3).

3.2.1.2.6 Reagents

Gel filtration using sephadex G-75

0.1 M Phosphate (pH 5.8) Dextran blue (molecular weight 2x106) solution (0.5% w/v) Sephadex-G-75

Procedure

Sephadex-G-75 was soaked in 500 ml of 0.1 M phosphate (pH 5.8) containing 0.1 g of sodium azide and incubated at room temperature for 24 hours. After soaking the gel was deaerated by direct drive rotary vacuum pump and then poured in a 0.9 x 60 cm column. The packed column was washed with 0.1 M phosphate (pH 5.8). Dextrin blue (0.5% w/v) was used for the determination of its void volume. Dialyzed enzyme extract was applied on the column and fractions each of 3 ml were collected. Each fraction was then assayed for enzyme activity and the amount of the protein present (Jakoby 1971).

3.2.1.3

Peroxidase

Peroxidase isolated from horse Radish was purified by ammonium sulfate precipitation technique, Dialysis and gel filtration chromatography for utilization in glucose estimation kit.

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3.2.1.3.1

Enzyme extracts preparation

About 100g horse Radish, was thoroughly washed with water, cut into small pieces and homogenized in a blender with 500 ml distilled water. It was centrifuged at 10,000 rpm for 15 minutes at 4°C and filtered (Civello et al 1995) and (Zia M.A.). The filtrate was heated in water bath at 65°C for 3 minute to inactivate catalase and cooled quickly in ice cold water. Enzyme assays were performed and protein was estimated as explained in section (3.2.1.2.3). 3.2.1.3.2 Enzyme assay

The activity of peroxidase enzyme was determined by the method as described by Civello et al (1995). Buffered substrate solution was prepared as follow: Phosphate buffer (pH 6.5) H2O2 (30%) Guaiacol = = = 46.6 ml 0.32 ml 1.00 ml

The reagents were mixed in agitator and completely covered till the whole day. In the blank solution preparation, H2O2 was not added only Guaiacol (1 ml) and phosphate buffer (46.6 ml) were mixed in agitator and used as blank spectrophotometer was set to zero at 470 nm wavelengths after in setting blank solution in it. Then in UV cell 1 ml of buffered substrate solution was taken along with 0.02 ml of enzyme extract and kept in spectrophotometer and absorbance was recorded after 3 minutes which is directly proportional to the enzyme activity. 3.2.1.3.3 Partial Purification of Peroxidase by (NH4)2SO4 precipitation techniques

The enzyme extract was precipitated by ammonium sulfate for partial purification as described by the method of Bentley (1962). Solid ammonium sulphate was added to 100 ml of solution of glucose oxidase at the concentration of 30% (w/v). The suspension was stirred for half an hour at 4oC. After sufficient shaking the precipitates were collected by centrifugation at 10,000 rpm for 30minutes. Enzyme activity was determined for each concentration and precipitates were collected for further purification. Enzyme solution was then treated with 40, 50, 60, 70 and finally 80% (w/v) (NH4)2SO4.

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3.2.1.3.4

Dialysis

The precipitate obtained by (NH4)2SO4 were dialyzed in the dialysis bag with 0.2 M phosphate buffer (pH 6.5) with constant stirring for 2 hours and then the sample was subjected to enzyme assay (3.2.1.3.2) and protein estimation (3.2.1.2.3). 3.2.1.3.5 Reagents 0.2 M Phosphate (pH 6.5) Dextran blue (molecular weight 2x106) solution (0.5% w/v) Sephadex-G-75 Procedure Sephadex-G-75 was soaked in 500 ml of 0.2 M phosphate (pH 6.5) containing 0.1 g of sodium azide and incubated at room temperature for 24 hours. After soaking the gel was deaerated by direct drive rotary vacuum pump and then poured in a 0.9 x 60 cm column. The packed column was washed with 0.2 M phosphate (pH 6.5). Dextrin blue (0.5% w/v) was used for the determination of its void volume. Dialyzed enzyme extract was applied on the column and fractions of three ml were collected. Each fraction was then assayed for enzyme activity and the amount of the protein present (Jakoby 1971). Gel filtration using Sephadex G-75

3.2.1.4

Optimization of conditions for glucose estimation

Glucose oxidase, mutarotase and peroxidase enzymes were used to estimate glucose level. The conditions of these partially purified enzymes were optimized. The standard solution of -D-glucose 100 mg dL-1 was prepared in distilled water. 2 ml of this solution was used in all the parameters to be optimized and the three enzymes added at the same time. Following parameters were optimized. 3.2.1.4.1 Guaiacol time

Timing for the addition of Guaiacol as chromogen was before adding peroxidase. 3.2.1.4.2 Enzyme concentrations

Different concentrations were optimized for three sets of the enzymes as follow: Set A Set B Set C Mutarotase (5 µL) + GOX (15µL) + Peroxidase (10µL) Mutarotase (10 µL) + GOX (30µL) + Peroxidase (20µL) Mutarotase (20 µL) + GOX (60µL) + Peroxidase (40µL)

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3.2.1.4.3

Wavelength

Different wavelengths were optimized at spectrophotometer i.e. i) ii) iii) 3.2.1.4.4 470 nm wavelength 290 nm wavelength 546 nm wavelength Incubation period

Two different incubation periods were investigated at 37°C for best results i.e. i) ii) 3.2.1.4.5 10 minutes 20 minutes Sensitivity of glucose estimation kit

Different concentration of glucose was used to determine the sensitivity of glucose estimation kit. The different concentration were used as: 200, 170, 140, 110, 80 and , 50 mg dL-1 and their absorbance were calculated with the help of standard curve and the sensitivity was measured as shown in Table 4.39 and Fig. 4.67. 3.2.1.4.6 Comparison with standard kit

All the optimized conditions (mentioned above) were used for preparation of glucose estimation kit and then this kit was compared with standard estimation kit. The blood samples of seven diabetic patients were investigated with both kits and comparison was made as shown in Table 4.38 and Fig. 4.66

3.2.2

Production of calcium gluconate, gluconic acid and its derivatives by GOX method

In the present research project gluconic acid, and its derivatives (metal salts) such as sodium, magnesium, copper, nickel and cobalt gluconates were synthesized from calcium gluconate which were earlier obtained by A. niger.

3.2.2.1

Production of calcium gluconate

Calcium gluconate was produced by submerged fermentation.

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3.2.2.1.1

Microorganism

Aspergillus niger strain (source potato) was obtained and identified from fungal Cultural Bank Punjab University Lahore, Pakistan and was used in the present study. The fungal culture was grown on malt extract agar medium at pH 5.5 as described in previous section in (3.1). 3.2.2.1.2 Preparation of growth media

The growth medium for A. niger was prepared by mixing the following quantities of ingredients in each 250 ml Erlenmeyer flask.

Table 3.3 INGREDIENTS MgSO4.7H2O FeSO4.7H2O KH2PO4 Glucose Urea NaNO3 Table 3.4 Ingredients CaCO3 KH2PO4 NaNO3 MgSO4.7H2O FeSO4.7H2O Urea Glucose Spore suspension

Composition of growth medium AMOUNT (gl-1) 0.5 0.01 1.0 100.0 0.2 5 Composition of each 250 ml flask Amounts 1.75 g/25 ml 2 ml 2 ml 2 ml 2 ml 2 ml 10 ml 5 ml (107 ­ 108 spores/ml)

The above quantities were mixed in the flasks except CaCO3 and spore suspension, autoclaved at 121°C for 15 minutes and 15 PSI. CaCO3 was autoclaved separately and mixed with the media under sterile condition, the flasks were then plugged after the

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addition of inoculums (5 ml).The pH of medium was adjusted at 6.0 by using 1M NaOH or 1M HCl. 3.2.2.1.3 Fermentation technique

The flasks containing 50 ml of fermentation medium were placed on a shaker for incubation at 30°C with speed of 100 rpm for 48 hours. All experiments were carried out in triplicates. The samples were drawn at the end of fermentation and the contents of flasks were filtered and filtrate was centrifuged at 5000 rpm for 5 minutes. The supernatant was used for the estimation of calcium gluconate and glucose. The dry cell mass was obtained by drying the wet mycelial mass in oven at 105°C for 24 hours according to method of Haq and Daud (1995). 3.2.2.1.4 (i) Assay methods

Estimation of glucose

Glucose was estimated by DNS method. (Tasun et al 1970) DNS solution was prepared as: Distilled water NaOH 3,5 dinitrosalicyclic acid = = = 1500 ml 20 g 10 g

Above ingredients were dissolved in water and heated in water bath at 80°C until clear solution was obtained. The following chemicals were than added. Rochelle salt Sod. metabisulphate Phenol (melted at 60°C) = = = 300 g 10 g 5 ml

The solution was filtered and stored at room temperature.

Standard curve of glucose

One gram of glucose was dissolved in small quantity of distilled water and the volume was raised to 100 ml this stock solution of glucose contained 10 mg/ml glucose. Five dilutions (0.2 ­ 1.2 mg ml-1) were made. From the stock solution 2 ml of each dilution and 2 ml of DNS solution was transferred in each test tube. Blank was also run in parallel with 2 ml DNS and 2 ml of distilled water. Test tubes were placed in boiling water for 5

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minutes and cooled at room temperature and finally diluted with distilled water upto 20 ml. The absorbance was measured at 546 nm by U.V. spectrophotometer. The standard curve was made by using each absorbance at respective range of glucose concentration

0.6 0.5 Absorbance 0.4 0.3 0.2 0.1 0 1 2 3 Concentration 4 5

Fig. 3.2

Standard curve of glucose

To determine glucose concentration (mg ml-1) the optical density was measured at 546nm on spectrophotometer. (ii) Calcium gluconate estimation

The analysis of calcium gluconate was made by the method of Pharmacopoeia (1990) UK. In 1.0 ml of sample, 2.0 ml of 1M HCl was added and water was added upto 200 ml while stirring. Approximately 20 ml of 0.05M EDTA was added from burette. Then 20 ml of 1M NaOH and 300 mg of Hydroxy naphthol blue indicator was added upto blue end point. Each ml of 0.05 M EDTA is equivalent to 2.004 mg of calcium gluconate. 1 ml of 0.05 M EDTA = 2.004 mg of calcium gluconate AND

Percentage yield of calcium gluconate =

Ca. gluconate Produced x 100 Glucose added

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3.2.2.1.5

Optimization of conditions to enhance calcium gluconate production

The following parameters were studied: (i) Effect of Incubation period on calcium gluconate production

The effect of incubation period on calcium gluconate production was studied at an interval of every 12 hours upto 96 hours. (ii) Effect of different pH on calcium gluconate production

The effect of pH on ca. gluconate production was studied within the range of 4.0 ­ 7.0 pH. The pH was adjusted by 1M HCl or 1M NaOH. (iii) Effect of glucose concentration on calcium gluconate production

To examine the effect of glucose concentration, different concentrations of glucose were added to fermentation medium (6-22%). (iv) Effect of different types of carbonates and CaCO3 concentration

Submerged fermentation was carried out using different metal carbonate such as MgCO3, CaCO3, ZnCO3 and FeCO3 in the medium. The various levels of CaCO3 (0 ­ 7 %) were further optimized. (v) Effect of different nitrogen sources and urea concentration Different nitrogen sources were used at a level of 2-5 gl-1 in the medium for improving the expression of calcium gluconate. Different sources are peptone, Urea, NH4NO3, NH4Cl and NaNO3. The various levels of urea (0.1-0.4 gl-1) were further optimized. (vi) Effect of different PO4 source

To study the effect of different phosphates like K2HPO4 and KH2PO4 on Ca. gluconate production, different concentration of respective phosphates in fermentation medium i.e. 0.15, 0.20, 0.25, 0.30 were added. (vii) The effect of temperature

Effect of temperature on calcium gluconate production was also evaluated. The influence of temperature was studied over the range of 15 to 50°C.

3.2.2.2

Preparation of gluconic acid from calcium gluconate

Gluconic acid was prepared from calcium gluconate. Following methods were employed for its preparation (Prescott and Dunn 1959) and (M. Iqbal and Maqbool).

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3.2.2.2.1

Oxalic acid

Exactly 43.0 gm calcium gluconate was dissolved in 180 ml boiling water. 12.6 gm crystallized oxalic acid was dissolved in minimum quantity of water. Both the solutions were mixed at 60°C with constant stirring. It was filtered to remove calcium oxalate. Gluconic acid was crystallized under vacuum at 30 - 40°C on a rotary evaporator. Reaction

CH2OH (CHOH)4 COOH COO COO (CHOH)4 CH2OH Ca

+

COOH COOH . 2H2O

2 (CHOH)4 CH2OH

+

COO COO Ca

+ 2H2O

Calcium gluconate 3.2.2.2.2

Oxalic acid

Gluconic acid

Calcium oxalate

Sulphuric acid method

Sulphuric acid was employed in place of oxalic acid to remove calcium as calcium sulphate and thus releasing the gluconic acid. 215.0 gm calcium gluconate was dissolved in 900 ml boiling water. This solution was placed in an ice bath and 33.4 ml 30 N sulphuric acid was added to it drop wise. It was stirred constantly for about five minutes and was filtered through a Buckner funnel to remove precipitated calcium sulphate. Gluconic acid solution was obtained as filtrate. This solution was used for subsequent preparations of gluconates.

Reaction:

CH2OH (CHOH)4 COO COO (CHOH)4 CH2OH Ca COOH

+

H2SO 4

2 (CHOH)4 CH2OH

+

CaSO 4

Calcium gluconate

Sulphuric acid

Gluconic acid

Calcium sulphate

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3.2.2.2.3

Identification of gluconic acid

About 5 ml of a warm aqueous solution of gluconic acid (9.03%) were added to 1 ml of freshly distilled phenylhydrazine. The mixture was taken in a test tube and heated on a water bath for 30 minutes. After cooling, the inner surface of the tube was scratched with a glass rod. Crystals of gluconic acid phenylhydrazide were formed (Eric and Cook, 1956). 3.2.2.3 Preparation of gluconic acid derivatives

The derivatives of gluconic acid such as magnesium gluconate, sodium gluconate, etc were prepared either from the calcium gluconate or directly from the gluconic acid. Calcium gluconate on treatment with the metal sulphate under suitable conditions gave the desired gluconate. Metal carbonates while treated with gluconic acid gave the metal gluconate with the evolution of carbon dioxide (Baronnet R. 1948). The appropriate conditions and detailed procedures are given as follows: 3.2.2.3.1 (i) Sodium gluconate

Double decomposition method

Sodium gluconate was prepared by the double decomposition of sodium sulphate and calcium gluconate as given below:

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CH 2 OH (CHOH) COO COO (CHOH) CH 2 OH

4 4

CH 2 OH (CHOH) COONa Ca

4

+

Na 2 SO 4

+

COONa (CHOH) CH 2 OH

4

+

CaSO

4

Calcium gluconate

Sodium sulphate

Sodium gluconate

Calcium sulphate

The 68.8 gm of calcium gluconate was added to 250 ml boiling water and stirred to dissolve it. The solution of calcium gluconate was treated with sodium sulphate (51.52 gm). This solution was heated to boiling. The precipitate of calcium sulphate was removed by filtration while hot. Sodium gluconate solution was concentrated under vacuum at 30°C on a rotary vacuum evaporator. Ethanol was added to crystallize sodium gluconate. It was filtered and the crystals were dried in a desicator over anhydrous calcium chloride. (ii) Gluconic acid method

Sodium gluconate was prepared by the interaction of sodium carbonate and gluconic acid. The reaction is given by the following equation:

COOH 2(CHOH)4 CH2OH COONa

+

Na2CO 3

2 (CHOH)4 CH2OH

+ CO2 +

Carbon dioxide

H2O

Gluconic acid

Sodium carbonate

Sodium gluconate

Water

Sodium carbonate (21.2 gm) was added to 50% gluconic acid (156.8 ml corresponding to 78.5 gm). The solution was heated to expel carbon dioxide. Sodium gluconate solution was concentrated under vacuum at 30°C. Ethanol was added to precipitate the salt. It was recrystallized from water solution and the crystals were dried in a desicator over anhydrous calcium chloride.

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3.2.2.3.2 (i)

Magnesium gluconate

Procedure

Calcium gluconate (86.0 g) was dissolved in minimum quantity of boiling water. Magnesium sulphate (49.2 g) was added to the gluconate solution and it was boiled, filtered, while hot, to remove calcium sulphate. Magnesium gluconate solution was concentrated at 40 - 50°C and the salt was precipitated from the concentrated solution with alcohol. Reaction

CH2OH (CHOH)4 COO COO (CHOH)4 CH2OH Ca CH2OH (CHOH)4

+

MgSO 4

COO COO (CHOH)4 CH2OH Mg

+

CaSO 4

Calcium gluconate (ii) Procedure

Magnesium sulphate

Magnesium gluconate

Calcium sulphate

Magnesium carbonate (84.0 g) was added to 50% gluconic acid solution (784.0 ml corresponding to 392.0 g). The solution was heated to expel carbon dioxide and concentrated under vacuum. Magnesium gluconate was precipitated with ethanol. Reaction

CH2OH COOH 2(CHOH)4 CH2OH (CHOH)4

+

MgCO 3

COO COO (CHOH)4 CH2OH Mg

+

CO 2

+

H2O

Gluconic Acid

Magnesium gluconate

Magnesium gluconate

Carbon dioxide

Water

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3.2.2.3.3 (i)

Copper gluconate

Procedure:

Solution of calcium gluconate (86.0 g) was made in minimum quantity of boiling water. This solution while boiling was treated with copper sulphate (49.8g) and filtered to separate calcium sulphate. Copper gluconate solution was concentrated to crystallization and the crystals were dried in a desicator over anhydrous calcium chloride. Reaction

CH2OH (CHOH)4 COO COO (CHOH)4 CH2OH Ca CH2OH (CHOH)4

+

CuSO 4

COO Cu COO (CHOH)4 CH2OH

+

CaSO 4

Calcium gluconate (ii) Procedure

Copper sulphate

Copper gluconate

Calcium sulphate

A 50% Gluconic acid solution (78.4 ml equivalent to 39.2 g) was treated with cuprous carbonate (22.1 g). Carbon dioxide was removed by heating and the resulting cuprous gluconate solution was concentrated under vacuum. Cuprous gluconate was precipitated with alcohol.

CH2OH COOH 2(CHOH)4 CH2OH (CHOH)4

+

COO CuCO 3 Cu COO (CHOH)4 CH2OH

+

CO 2

+

H2O

Gluconic acid

Copper carbonate

Copper gluconate

Carbon dioxide

Water

3.2.2.3.4

Nickel gluconate

56.2 g Nickel sulphate was treated with 86.0 g calcium gluconate previously dissolved in minimum quantity of water. The solution was heated, filtered to remove calcium

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sulphate. Nickel gluconate solution was concentrated and precipitated with alcohol. The precipitate of nickel gluconate was dried by placing it in a desicator. Reaction

CH2OH (CHOH)4 COO Ca COO (CHOH)4 CH2OH CH2OH (CHOH)4 COO

+

NiSO 4

Ni COO (CHOH)4 CH2OH

+

CaSO 4

Calcium gluconate (ii) Procedure

Nickel sulphate

Nickel gluconate

Calcium sulphate

The 12.6 g of nickel carbonate was reacted with 78.4 ml (equivalent to 39.2 g) 50% gluconic acid solution. It was heated to release carbon dioxide and concentrated. Nickel gluconate was crystallized from the concentrated solution with ethanol. Reaction

CH2OH (CHOH)4 COOH 2(CHOH)4 CH2OH COO

+

NiCO 3

Ni COO (CHOH)4 CH2OH

+

CO 2

+

H2O

Gluconic acid Percentage Yield Formula used

Nickel carbonate

Nickel gluconate

Carbon dioxide.

Water

%ageYield =

Actualyield x 100 Theoretical Yield

These salts of gluconic acid have been prepared and used in view of their comparatively high water solubility.

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Chapter No. 4

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSIONS

4.1 Production of glucose oxidase from A. niger

The present studies describe the production of glucose oxidase by A. niger using submerged fermentation. To study the production of GOX, first of all screening of fungal sources producing highest GOX activity was done, along with optimization of pH and carbon source. Different fermentation parameters like fermentation period, levels of Urea, MgSO4.7H2O, KH2PO4 and CaCO3 were also optimized. Glucose was used as the carbon source for GOX production. Effects of aeration were measured and enzyme kinetics were also investigated. The GOX production was enhanced by UV mutation and it was purified also. The results thus obtained have been discussed here under.

4.1.2 Screening for microbial strain producing the highest GOX activity along with optimizing pH and carbon source

Different experiments were conducted for screening of fungal strain. Five strains of A.

niger isolated from bread, potato, grapes, pickle and sugarbeet were optimized for GOX production. Different concentrations of glucose (carbon source) were used in fermentation media in which fungi were grown. In this way the carbon source level was optimized for GOX production for all five strain of A. niger. pH values of the media were also optimized for GOX production for five strains. Mycelial mass, enzyme activities, total protein and specific enzyme activities were also calculated for different pH and glucose concentration of five strains. Crude enzyme extract was obtained by breaking down the mycelia after 48 hours incubation (Willis, 1966) in shaking incubator at 30°C with 100 rpm. In this study enzyme was found to be intracellular, by breaking down the mycelia and no activity was found in filterate after filtration of mycelia. So no extracellular GOX activity was found for all of our selected strains. Similarly Markwell et al (1989), Mischak et al (1985) and Doppner & Hartmeir (1984) reported the enzyme to be intracellular. Enzyme activity was measured by the method of Ciucu and Patroescu (1984). GOX activity was determined by enzymatic reduction of benzoquinone to hydroquinone and then by the measurement of increase of hydroquinone absorbance at 290 nm. The enzymatic activity is deducted from the initial rate and expressed in µ mole

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of hydroquinone per minute per ml. Like other methods this method requires no other coupled enzymatic reaction.

4.1.1.1

Screening

For commercial production of the enzyme the first step is the screening of different native strains for the optimal production of enzyme. So in this study different strains of A. niger were examined. Five strains of A. niger isolated from grapes, potato, bread, pickle and sugar beet were screened and optimized for GOX production. Enzyme activity of A. niger isolated from potato was found to be maximum (1.59 and 1.56 µ mole HQ min-1 ml-1) in medium containing 10% (w/v) glucose and at pH 5.5. This strain showed also the maximum specific enzyme activity (2.44 and 2.51 µ mole/HQ/min/mg protein). These values are presented in Tables (4.2 and 4.7) and Figs. (4.8 and 4.33). Mycelial mass and total amount of protein were also calculated along with enzyme activities and specific enzyme activities for all five strains shown in Tables (4.1 to 4.10) and Figs. (4.1 to 4.50). This strain of A. niger (potato source) selected in this study can be used for commercial production of GOX in Pakistan.

4.1.1.2

Substrate (carbon source)

Glucose was used as carbon source for the production of glucose oxidase. In the fermentation media different concentrations of glucose (4% to 13%) were used to examine its effect on GOX production. The results are shown in Tables (4.1 to 4.5) for five strains. In Table 4.1 Aspergillus niger strain isolated from bread showed maximum enzyme activity (1.23 µ mole HQ min-1ml-1) at 10% glucose mycelial mass, specific enzyme activities and total amount of protein were also calculated and shown in Table 4.1 and Figs (4.1 to 5.5). In table 4.2, the effect of glucose concentration on enzyme activity, mycelial mass, amount of protein and specific enzyme activity of A. niger isolated from potato is shown. It also gave the maximum enzyme activity (1.59 µ mole HQ min-1 ml-1) at 10% glucose. This activity was found to be the highest activity among the five strains. Mycelial mass, protein amount and specific enzyme activity were also estimated and shown in table (4.2) and fig. (4.6 to 4.10). In table 4.3 A. niger isolated from grapes source showed maximum GOX activity (1.38 µ mole HQ min-1 ml-1) also at 10% (w/v) glucose. It showed a maximum specific activity (2.07 µ mole HQ min-1 ml-1 protein) also

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at 10% (w/v) glucose concentration. Mycelial mass and protein amount were also estimated and shown in Table (4.3) and Figs. 4.11 to 4.15. Table 4.4 shows the effect of glucose concentrations of A. niger (source pickle) on GOX activity specific activity, mycelial mass and amount of protein. It showed the maximum enzyme activity (1.38 µ mol HQ min-1 ml-1) in media containing 8% glucose concentration. The values are shown in table 4.4 and in figs (4.16 to 4.20). Aspergillus niger strain isolated from sugar beet showed the maximum enzyme activity (1.23 µ mole HQ min-1 ml-1) in media containing 9.0% (w/v) glucose concentration. While amount of protein, mycelial mass and specific enzyme activity also calculated and shown in Table 4.5 and Figs (4.21 to 4.25). Above results indicate that GOX production is greatly affected by the glucose concentration in fermentation medium GOX production is directly influenced by glucose concentration. Aspergillus niger strain isolated from bread, potato, grape, pickle and sugar beet showed maximum enzyme activities in media containing, 10.0%, 10.0%, 10%, 8.0% and 9.0% glucose concentration respectively. These results are in agreement with the results of Petruccioli and Federici (1993) and Rogalski et al (1988) obtained highest GOX activity at 8% glucose. Ray and Banik (1999) concluded that 15% glucose induced GOX synthesis. In this study maximum enzyme activity was produced at 10% glucose concentration. A further increase in substrate concentration resulted in decrease in enzyme production.

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4.1

Effect of glucose concentration on glucose oxidase production by A. niger (source bread) Enzyme Activity (u moles HQ /min/ml) 0.37 ± 0.12 0.71 ± 0.4 0.73 ± 0.01 0.89 ± 0.02 0.97 ± 0.12 1.10 ± 0.25 1.23 ± .35 1.20 ± 0.41 0.84 ± 0.02 0.63 ± 0.1 0.21 Specific Enzyme activity ( moles HQ/min/mg protein) 0.77 ± 0.1 1.19 ± 0.02 1.15 ± 0.3 1.33 ± 0.01 1.36 ± 0.02 1.51 ± 0.03 1.74 ± 0.01 1.71 ± 0.23 1.37 ± 0.54 1.08 ± 0.21 0.14

Glucose concent ration % 4 5 6 7 8 9 10 11 12 13 LSD

Net weight of mycelium (g/100ml) 4.05 ± 0.15 7.1 ± 0.02 7.35 ± 0.21 8.15 ± 0.07 8.6 ± 0.17 8.35 ± 0.07 7.95 ± 0.1 7.1 ± 0.02 6.05 ± 0.01 4.65 ± 0.32 0.63

Mycelium mass (dry) (mg/ml) 2.58 ± 0.1 5.1 ± 0.07 5.2 ± 0.10 5.7 ± 0.04 6.03 ± 0.15 5.84 ± 0.23 5.54 ± 0.05 5.14 ± 0.09 4.06 ± 0.21 2.86 ± 0.04 0.43

Total Protein (mg/ml) 0.48 ± 0.21 0.59 ± 0.02 0.64 ± 0.015 0.67 ± 0.17 0.71 ± 0.31 0.73 ± 0.2 0.70 ± 0.07 0.70 ± 0.02 0.61 ± 0.01 0.58 ± 0.02 0.39

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Mycelium Mass Production with glucose cocentration 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose concentration Glucose concentration % (w/v)

Fig. 4.1

Mycelium mass

Effect of glucose concentration on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.2

Effect of glucose concentration on mycelium mass (Dry)

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Enzyme Activity

1.6 Enzyme Activity (u moles HQ/min/ml) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.3

Effect of glucose concentration on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v)

Fig. 4.4

Effect of glucose concentration on total protein

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Specific Enzyme Activity

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v) Specific Enzyme Activity (u moles HQ/min/mg)

Fig. 4.5

Effect of glucose concentration on specific enzyme activity

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Table 4.2

Effect of glucose concentration on glucose oxidase production by A. niger (source potato ) Specific Enzyme activity (u moles HQ/min/mg protein 0.66 ± 0.12 1.44 ± 0.13 1.52 ± 0.2 1.59 ± 0.13 1.43 ± 0.15 1.65 ± 0.1 2.44 ± 0.12 2.15 ± 0.14 1.3 ± 0.23 0.94 ± 0.49 0.52

Glucose concent ration % 4 5 6 7 8 9 10 11 12 13 LSD

Net weight of mycelium (g/100ml) 4.15 ± 0.01 6.7 ± 0.03 7.15 ± 0.04 8.55 ± 0.21 9.15 ± 0.14 8.3 ± 0.09 8.15 ± 0.09 7.15 ± 0.04 5.55 ± 0.03 4.05 ± 0.1 0.19

Mycelium mass (dry) (mg/ml) 2.90 ± 0.02 4.67 ± 0.19 5.00 ± 0.07 5.50 ± 0.04 6.40 ± 0.03 5.81 ± 0.02 5.70 ± 0.01 5.00 ± 0.1 3.85 ± 0.03 3.00 ± 0.04 0.24

Enzyme Activity (u moles HQ /min/ml) 0.30 ± 0.07 0.82 ± 0.02 0.89 ± 0.29 1.03 ± 0.15 1.03 ± 0.007 1.20 ± 0.01 1.59 ± 0.12 1.34 ± 0.2 0.75 ± 0.01 0.48 ± 0.01 0.39

Total Protein (mg/ml) 0.46 ± 0.03 0.57 ± 0.02 0.59 ± 0.01 0.64 ± 0.04 0.72 ± 0.03 0.73 ± 0.03 0.65 ± 0.02 0.62 ± 0.1 0.58 ± 0.2 0.51 ± 0.4 0.16

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Mycelium Mass Production with glucose cocentration 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose concentration Glucose concentration % (w/v)

Fig. 4.6

Mycelium mass

Effect of glucose concentration on mycelium mass

Mycelium Dry Mass production 7.00 Mycelium Mass (Dry) 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.7

Effect of glucose concentration on mycelium mass (dry)

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Enzyme Activity

1.8 Enzyme Activity (u moles HQ/min/ml) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.8

Effect of glucose concentration on enzyme activity

Proteins production Protein concentration

0.800 Total Protein (mg/ml) 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000 0 2 4 6 8 10 12 14

Glucose concentration % (w/v) Glucose Concentration (%) Fig. 4.9 Effect of glucose concentration on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 3 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14

Glucose concentration % (w/v) Glucose Concentration (%) Fig. 4.10 Effect of glucose concentration on specific enzyme activity

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Table 4.3

Effect of glucose concentration on glucose oxidase production by A. niger (source grapes) Specific Enzyme activity (u moles HQ/min/mg protein 0.98 ± 0.12 1.17 ± 0.09 1.26 ± 0.06 1.5 ± 0.03 1.53 ± 0.02 1.61 ± 0.01 2.07 ± 0.02 1.67 ± 0.07 1.5 ± 0.05 1.09 ± 0.01 0.23

Glucose concentrati on % 4 5 6 7 8 9 10 11 12 13 LSD

Net weight of mycelium (g/100ml) 3.95 ± 0.1 4.9 ± 0.19 6.75 ± 0.05 7.7 ± 0.06 7.95 ± 0.13 8.35 ± 0.09 8.05 ± 0.03 7.85 ± 0.21 6.65 ± 0.09 4.05 ± 0.1 0.43

Mycelium mass (dry) (mg/ml) 3 ± 0.21 3.55 ± 0.19 4.7 ± 0.02 5.36 ± 0.09 5.85 ± 0.05 5.66 ± 0.07 5.68 ± 0.09 5.4 ± 0.1 4.61 ± 0.2 3.05 ± 0.1 0.03

Enzyme Activity (u moles HQ /min/ml) 0.41 ± 0.03 0.72 ± 0.1 0.79 ± 0.07 0.96 ± 0.04 1.09 ± 0.3 1.17 ± 0.1 1.38 ± 0.2 1.10 ± 0.3 0.89 ± 0.29 0.58 ± 0.31 0.12

Total Protein (mg/ml) 0.41 ± 0.15 0.62 ± 0.29 0.62 ± 0.01 0.63 ± 0.03 0.71 ± 0.01 0.72 ± 0.02 0.66 ± 0.05 0.66 ± 0.07 0.59 ± 0.3 0.53 ± 0.14 0.32

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Mycelium Mass Production with glucose cocentration 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14

Mycelium mass

GlucoseGlucose concentration concentration % (w/v) Fig. 4.11 Effect of glucose concentration on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14

GlucoseGlucose Concentration concentration % (w/v) Fig. 4.12 Effect of Glucose concentration on Mycelium mass (Dry)

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Enzyme Activity

1.6 Enzyme Activity (u moles HQ/min/ml) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14

Glucose concentration % (w/v) Glucose Concentration

Fig. 4.13

Effect of glucose concentration on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v)

Table: 4.14

Effect of glucose concentration on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v)

Fig. 4.15

Effect of glucose concentration on specific enzyme activity

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Table 4.4

Effect of glucose concentration on glucose oxidase production by A. niger (source pickle) Specific Enzyme activity (u moles HQ/min/mg protein 0.7 ± 0.1 0.88 ± 0.09 1.21 ± 0.1 1.68 ± 0.03 2.18 ± 0.06 1.94 ± 0.04 1.63 ± 0.1 1.38 ± 0.17 1.37 ± 0.09 1.23 ± 0.07 0.01

Glucose concentra tion

% 4 5 6 7 8 9 10 11 12 13 LSD

Net weight of mycelium (g/100ml) 4.6 ± 0.01 6.55 ± 0.03 7.8 ± 0.04 8.5 ± 0.05 8.2 ± 0.1 8.05 ± 0.23 7.4 ± 0.05 6.8 ± 0.02 6.05 ± 0.09 5 ± 0.06 0.02

Mycelium mass (dry) (mg/ml) 3.02 ± 0.1 4.55 ± 0.2 5.2 ± 6 ± 0.09 6.05 ± 0.1 5.96 ± 0.1 5.31 ± 0.15 4.53 ± 0.12 4.02 ± 0.13 3.05 ± 0.14 0.32

Enzyme Activity (u moles HQ /min/ml) 0.29 ± 0.09 0.53 ± 0.02 0.81 ± 0.03 1.19 ± 0.02 1.38 ± 0.24 1.20 ± 0.19 0.97 ± 0.07 0.73 ± 0.01 0.72 ± 0.1 0.63 ± 0.15 0.43

Total Protein (mg/ml) 0.41 ± 0.25 0.61 ± 0.09 0.67 ± 0.03 0.70 ± 0.01 0.63 ± 0.1 0.61 ± 0.09 0.59 ± 0.03 0.53 ± 0.02 0.52 ± 0.01 0.51 ± 0.21 0.06

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Mycelium Mass Production with glucose cocentration 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose concentration Glucose concentration % (w/v)

Fig. 4.16

Mycelium mass

Effect of glucose concentration on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.17

Effect of glucose concentration on mycelium mass (dry)

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Enzyme Activity

1.6 Enzyme Activity (u moles HQ/min/ml) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.18

Effect of glucose concentration on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 8 10 12 14

GlucoseGlucose Concentration (%) concentration % (w/v) Fig. 4.19 Effect of Glucose concentration on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v)

Fig. 4.20

Effect of glucose concentration on specific enzyme activity

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Table 4.5

Effect of glucose concentration on glucose oxidase production by A. niger (source sugar beet)

Glucose concentration %

Net weight of mycelium (g/100ml) 5.15 ± 0.01 6.2 ± 0.02 6.45 ± 0.12 7.5 ± 0.19 7.55 ± 01 7.95 ± 0.1 8.05 ± 0.1 7.15 ± 0.07 5.7 ± 0.03 5.4 ± 0.04 0.25

Mycelium mass (dry) (mg/ml )

Enzyme Activity (u moles HQ /min/ml) 0.28 ± 0.04 0.43 ± 0.02 0.49 ± 0.09 0.68 ± 0.1 0.99 ± 0.3 1.23 ± 0.4 1.10 ± 0.15 0.71 ± 0.12 0.49 ± 0.027 0.44 ± 0.04 0.24

Total Protein (mg/ml)

Specific Enzyme activity (u moles HQ/min/mg protein 0.70 ± 0.01 1.02 ± 0.05 1.08 ± 0.07 1.19 ± 0.03 1.67 ± 0.02 1.97 ± 0.05 1.70 ± 0.09 1.15 ± 0.04 0.95 ± 0.09 0.92 ± 0.02 0.22

4 5 6 7 8 9 10 11 12 13 LSD

3.05 ± 0.09 4.01 ± 0.1 4.05 ± 0.12 5.1 ± 0.13 5.1 ± 0.21 5.37 ± 0.27 5.45 ± 0.19 4.8 ± 0.07 3.85 ± 0.04 3.6 ± 0.23 0.32

0.40 ± 0.03 0.42 ± 0.09 0.45 ± 0.1 0.57 ± 0.16 0.59 ± 0.13 0.62 ± 0.14 0.64 ± 0.13 0.61 ± 0.05 0.52 ± 0.1 0.48 ± 0.3 0.08

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Mycelium Mass Production with glucose cocentration 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose concentration Glucose concentration % (w/v)

Fig. 4.21

Mycelium mass

Effect of glucose concentration on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.22

Effect of glucose concentration on mycelium mass (dry)

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Enzyme Activity

1.4 Enzyme Activity (u moles HQ/min/ml) 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 Glucose Concentration Glucose concentration % (w/v)

Fig. 4.23

Effect of glucose concentration on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 8 10 12 14 Glucose Concentration (%) Glucose concentration % (w/v)

Fig. 4.24

Effect of glucose concentration on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 2.50 2.00 1.50 1.00 0.50 0.00 0 2 4 6 8 10 12 14

Glucose concentration % (w/v) Glucose Concentration (%) Fig. 4.25 Effect of glucose concentration on specific enzyme activity

4.1.1.3

pH

The pH has great effect on enzyme production. In this experiment fungal strains were checked on pH ranging from 4.0 to 8.0. The effect of pH on GOX activity, specific GOX activity, Mycelial mass and amount of proteins were investigated. The results are shown in Tables (4.6 to 4.10). In Table 4.6 Aspergillus niger strain isolated from bread showed maximum GOX activity (1.23 µ moles HQ/min/ml) and maximum specific activity (1.76 µ moles HQ/min/mg protein) in medium at pH 5.5. While the maximum mycelial mass and highest amount of proteins were noted at pH 5.0 also shown in Figs (4.26 to 4.30). In Table 4.7 the effect of pH on GOX activity, specific activity, mycelial mass and amount of protein of A. niger isolated from potato is shown. The maximum GOX activity (1.56 µ moles HQ min-1ml-1) at pH 5.5. The values of specific enzyme activities mycelial mass and protein amount were also calculated and shown in Table (4.7) and Fig (4.31 to 4.35). This strain shows the highest activity among the others at pH 5.5 Table 4.8 shows the effect of pH of A. niger (source grapes) on GOX activities, specific activities mycelial mass and protein amount. It showed the maximum enzyme activity (1.38 µ moles HQ min-1 ml-1) in the fermentation medium at pH 6.0. Mycelial mass, protein amount and specific. Activities were also determined at different pH and their values represented in Table 4.8 and Figs (4.36 to 4.40). Aspergillus niger isolated from pickle showed maximum GOX activity (1.37 µ moles HQ min-1 ml-1) and maximum specific enzyme

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activity at pH 5.0, while highest mycelial yield and highest protein amount of pH 5.5. Their values are presented in Table 4.9 and Figs (4.41 to 4.45). Table 4.10 shows the pH optimization of A. niger isolated from sugar beet for maximum GOX production. This strain gave maximum enzyme activity (1.23 µ moles HQ min-1 ml-1) in medium at pH 5.0. The values of other parameters are presented in Table 4.10 and Figs 4.46 to 4.50. It is concluded from the above results that pH range from 5-6.0 is best for GOX production and at pH 5.5 highest GOX activity observed. Our findings are in conformity with the Fiedurck and Gromada (2000) who reported the increased GOX and catalase production at pH 5.0. The enzyme activity was negligible at pH 7.0, 7.5 and 8.0. In this study A. niger strain isolated from potato was found to be the best for GOX production. This strain showed the maximum enzyme activity in medium containing 10% glucose and at pH 5.5

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Table 4.6

Effect of pH on glucose oxidase production by A. niger (source bread) Net weight of mycelium (g/100ml) 7.35 ± 0.1 7.8 ± 0.13 8.4 ± 0.19 8 ± 0.12 7.6 ± 0.16 5.15 ± 013 2.55 ± 0.11 0.65 ± 0.1 0.25 ± 0.09 0.09 Enzyme Activity (u moles HQ /min/ml) 0.77 ± 0.23 0.89 ± 0.19 1.10 ± 0.16 1.23 ± 0.14 0.98 ± 0.19 0.42 ± 0.16 0.17 ± 0.15 0 ± 0.00 0 ± 0.00 0.31 Specific Enzyme activity (u moles HQ/min/mg protein 1.2 ± 0.1 1.24 ± 0.13 1.5 ± 0.12 1.76 ± 0.14 1.54 ± 0.12 0.9 ± 0.11 0.89 ± 0.1 0 ± 0.00 0 ± 0.00 0.34

pH

Mycelium mass (dry) (mg/ml) 5.15 ± 0.13 5.36 ± 0.10 5.69 ± 0.07 5.55 ± 0.04 4.94 ± 0.03 3.33 ± 0.06 1.51 ± 0.09 0.45 ± 0.1 0.14 ± 0.15 0.09

Total Protein (mg/ml) 0.64 ± 0.03 0.70 ± 0.04 0.73 ± 0.25 0.69 ± 0.2 0.63 ± 0.19 0.46 ± 0.16 0.19 ± 0.12 0.12 ± 0.19 0 ± 0.00 0.29

4 4.5 5 5.5 6 6.5 7 7.5 8 LSD

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Mycelium Mass Production with pH Changes 9 8 7 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.26

Mycelium mass

Effect of pH on mycelium mass

Mycelium Dry Mass production 6 Mycelium Mass (Dry) 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.27

Effect of pH on mycelium mass (dry)

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Enzyme Activity

1.4 Enzyme Activity (u moles HQ/min/ml) 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.28

Effect of pH on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.29

Effect of pH on total protein

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Specific Enzyme Activity

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 4

Specific Enzyme Activity (u moles HQ/min/mg)

4.5

5

5.5

6 pH

6.5

7

7.5

8

8.5

Fig. 4.30

Effect of pH on specific enzyme activity

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4.7

Effect of pH on glucose oxidase production by A. niger (source potato) Specific Enzyme activity (u moles HQ/min/mg protein 1.64 ± 0.12 1.6 ± 0.13 1.85 ± 0.1 2.51 ± 0.09 1.48 ± 0.07 0.92 ± 0.06 0.64 ± 0.04 0 ± 0.00 0 ± 0.00 0.43

pH

Net weight of mycelium (g/100ml) 7.15 ± 0.19 7.8 ± 0.16 8.6 ± 0.14 8.45 ± 0.09 7.85 ± 0.07 5.4 ± 0.04 2.15 ± 0.03 0.7 ± 0.12 0.3 ± 0.09 0.12

Mycelium mass (dry) (mg/ml) 5 ± 0.3 5.35 ± 0.4 5.9 ± 0.9 5.6 ± 0.1 5.45 ± 0.13 3.55 ± 0.1 1.45 ± 0.2 0.45 ± 0.4 0.15 ± 0.7 0.09

Enzyme Activity (u moles HQ /min/ml) 0.76 ± 0.21 0.92 ± 0.19 1.22 ± 0.23 1.56 ± 0.11 1.01 ± 0.12 0.38 ± 0.09 0.18 ± 0.03 0 ± 0.00 0 ± 0.00 0.31

Total Protein (mg/ml) 0.46 ± 0.07 0.58 ± 0.09 0.65 ± 0.19 0.62 ± 0.13 0.68 ± 0.11 0.41 ± 0.12 0.28 ± 0.2 0.17 ± 0.1 0 ± 0.00 0.29

4 4.5 5 5.5 6 6.5 7 7.5 8 LSD

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Mycelium Mass Production with pH Changes 10 9 8 7 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.31

Mycelium mass

Effect of pH on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.32

Effect of pH on mycelium mass (dry)

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Enzyme Activity

1.8 Enzyme Activity (u moles HQ/min/ml) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.33

Effect of pH on enzyme activity

Proteins production Protein concentration

0.8 Total Protein (mg/ml) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.34

Effect of pH on total protein

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Specific Enzyme Activity

3 Specific Enzyme Activity (u moles HQ/min/mg) 2.5 2 1.5 1 0.5 0 -0.5 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.35

Effect of pH on specific enzyme activity

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Table 4.8

Effect of pH on glucose oxidase production by A. niger (source grapes)

pH

Net weight of mycelium (g/100ml) 5.15 ± 0.09 5.55 ± 0.02 7.95 ± 0.07 8.25 ± 0.1 8.15 ± 0.12 6.15 ± 0.07 2.65 ± 0.06 1.3 ± 0.05 0.5 ± 0.02 0.19

Mycelium mass (dry) (mg/ml)

Enzyme Activity (u moles HQ /min/ml) 0.38 ± 0.21 0.62 ± 0.19 0.99 ± 0.17 1.12 ± 0.15 1.38 ± 0.22 0.48 ± 0.23 0.17 ± 0.07 0.11 ± 0.13 0 ± 0.00 0.29

Total Protein (mg/ml)

Specific Enzyme activity (u moles HQ/min/mg protein 0.902 ± 0.12 1.05 ± 0.12 1.55 ± 0.09 1.73 ± 0.04 2.07 ± 0.19 0.93 ± 0.21 0.76 ± 0.17 0.57 ± 0.10 0 ± 0.00 0.12

4 4.5 5 5.5 6 6.5 7 7.5 8 LSD

3.56 ± 0.1 3.9 ± 0.19 5.36 ± 0.12 5.7 ± 0.13 5.67 ± 0.14 4.16 ± 0.10 1.65 ± 0.11 0.53 ± 0.13 0.21 ± 0.17 0.43

0.422 ± 0.149 0.591 ± 0.13 0.638 ± 0.07 0.649 ± 0.04 0.665 ± 0.09 0.512 ± 0.113 0.226 ± 0.01 0.193 ± 0.1 0.166 ± 0.19 0.31

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Mycelium Mass Production with pH Changes 9 8 7 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.36

Mycelium mass

Effect of pH on mycelium mass

Mycelium Dry Mass production 6 Mycelium Mass (Dry) 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.37

Effect of pH on mycelium mass (dry)

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Enzyme Activity

1.6 Enzyme Activity (u moles HQ/min/ml) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 4 5 6 7 pH 8 9 10 11

Fig. 4.38

Effect of pH on enzyme activity

Proteins production Protein concentration

0.7 Total Protein (mg/ml) 0.6 0.5 0.4 0.3 0.2 0.1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.39

Effect of pH on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 2.5 2 1.5 1 0.5 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.40

Effect of pH on specific enzyme activity

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Table 4.9

Effect of pH on glucose oxidase production by A. niger (source pickle)

pH

Net weight of mycelium (g/100ml) 6.7 ± 0.01 7.9 ± 0.07 8.3 ± 0.01 8.55 ± 0.09 7.45 ± 0.04 4.85 ± 0.05 1.9 ± 0.1 0.55 ± 0.21 0.25 ± 0.19 0.13

Mycelium mass (dry) (mg/ml) 4.6 ± 0.12 5.2 ± 0.13 6.05 ± 0.09 6.15 ± 0.17 5.1 ± 0.16 2.95 ± 0.11 0.6 ± 0.16 0.35 ± 0.09 0.1 ± 0.01 0.09

Enzyme Activity (u moles HQ /min/ml) 0.73 ± 0.13 1.1 ± 0.1 1.37 ± 0.09 1.22 ± 0.07 0.96 ± 0.04 0.41 ± 0.01 0.20 ± 0.03 0 ± 0.00 0 ± 0.00 0.24

Total Protein (mg/ml) 0.52 ± 0.13 0.57 ± 0.12 0.62 ± 0.10 0.68 ± 0.02 0.59 ± 0.01 0.49 ± 0.03 0.31 ± 0.04 0.20 ± 0.05 0.14 ± 0.02 0.11

Specific Enzyme activity (u moles HQ/min/mg protein 1.4 ± 0.01 1.97 ± 0.1 2.19 ± 0.17 1.79 ± 0.14 1.62 ± 0.10 0.84 ± 0.09 0.64 ± 0.04 0 ± 0.00 0 ± 0.00 0.32

4 4.5 5 5.5 6 6.5 7 7.5 8 LSD

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Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.41

Effect of pH on mycelium mass

Mycelium Dry Mass production 7 Mycelium Mass (Dry) 6 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.42

Effect of pH on mycelium mass (dry)

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Enzyme Activity

1.6 Enzyme Activity (u moles HQ/min/ml) 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.43

Effect of pH on enzyme activity

Proteins production Protein concentration

0.8 0.7 Total Protein (mg/ml) 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.44

Effect of pH on total protein

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Specific Enzyme Activity

2.5 Specific Enzyme Activity (u moles HQ/min/mg) 2 1.5 1 0.5 0 -0.5 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.45

Effect of pH on specific enzyme activity

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Table4.10

Effect of pH on glucose oxidase production by A. niger (source sugar beet) Enzyme Activity (u moles HQ /min/ml) 0.63 ± 0.07 1.10 ± 0.04 1.23 ± 0.01 1.09 ± 0.03 0.97 ± 0.05 0.38 ± 0.01 0.10 ± 0.3 0.10 ± 0.01 0 ± 0.00 0.41 Specific Enzyme activity (u moles HQ/min/mg protein 1.52 ± 0.03 1.9 ± 0.1 1.97 ± 0.07 1.78 ± 0.04 1.67 ± 0.04 1.05 ± 0.2 0.51 ± 0.4 0.5 ± 0.5 0 ± 0.00 0.10

pH

Net weight of mycelium (g/100ml) 7.05 ± 0.09 7.7 ± 0.04 8 ± 0.03 7.85 ± 0.02 6.05 ± 0.03 4.9 ± 0.01 2.1 ± 0.1 1 ± 0.01 0.55 ± 0.12 0.19

Mycelium mass (dry) (mg/ml) 4.67 ± 0.11 5.23 ± 0.09 5.4 ± 0.04 5.31 ± 0.01 3.98 ± 0.04 2.83 ± 0.01 1.38 ± 0.09 0.45 ± 0.1 0.35 ± 0.09 0.21

Total Protein (mg/ml) 0.41 ± 0.01 0.58 ± 0.03 0.62 ± 0.01 0.61 ± 0.02 0.58 ± 0.01 0.36 ± 0.03 0.21 ± 0.09 0.2 ± 0.07 0.16 ± 0.02 0.09

4 4.5 5 5.5 6 6.5 7 7.5 8 LSD

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Mycelium Dry Mass production 6 Mycelium Mass (Dry) 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.46

Effect of pH on mycelium mass

Mycelium Dry Mass production 6 Mycelium Mass (Dry) 5 4 3 2 1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.47

Effect of pH on mycelium mass (dry)

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1.4 Enzyme Activity (u moles HQ/min/ml) 1.2 1 0.8 0.6 0.4 0.2 0 4 4.5 5

Enzyme Activity

5.5

6 pH

6.5

7

7.5

8

8.5

Fig. 4.48

Effect of pH on enzyme activity

Proteins production Protein concentration

0.7 Total Protein (mg/ml) 0.6 0.5 0.4 0.3 0.2 0.1 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.49

Effect of pH on total protein

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Specific Enzyme Activity

Specific Enzyme Activity (u moles HQ/min/mg) 2.5 2 1.5 1 0.5 0 4 4.5 5 5.5 6 pH 6.5 7 7.5 8 8.5

Fig. 4.50

Effect of pH on specific enzyme activity

4.1.2 Optimization of other fermentation conditions for maximum GOX production

Fermentation conditions like fermentation period, level of CaCO3, Urea, KH2PO4 and MgSO4.7H2O were optimized other than glucose level and pH which were optimized alongwith screening of fungal strain described earlier in the previous section (4.1.1). The growth medium was fermented with A. niger strain isolated from potato (screened earlier) for different fermentation parameters. The results obtained have been discussed here under.

4.1.2.1

Fermentation period

The media containing 10% glucose in duplicate flasks were incubated for 12, 24, 36, 48 and 60 hours. The maximum GOX activity was obtained after 48 hours of fermentation at pH 5.5 and 30°C and decreased there after (Table 4.11 and Fig 4.51). Similar finding has been reported by Willis (1966), he obtained highest GOX yield after 48 hours of fermentation.

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Table 4.11

Effect of fermentation period on GOX production GOX activity (µ moles HQ/min/ml) 1.021± 0.07 1.64 ± 0.03 2.59 ± 0.12 2.45 ± 0.23 1.83 ± 0.07 0.291

Fermentation period (hrs) 24 36 48 60 72 LSD

Gox activity (u moles HQ/min/ml

3 2.5 2 1.5 1 0.5 0 24 36 48 60 72 Fermentation Period (Hours)

Fig 4.51

Effect of fermentation period on GOX production

4.1.2.2

Effect of CaCO3

GOX production was enhanced by using CaCO3. Different concentration of CaCO3 was used in triplicate flasks i.e. 1.5%, 2.5%, 3.5% and 4.5. The results have been shown in Table 4.12 and Fig. 4.52. It was observed that maximum GOX production (2.337 µ mole HQ/min/ml) was obtained with 3.5% CaCO3 in the medium. Fiedurck and Szezodark (1995) also obtained maximum GOX production with 3.5% CaCO3 (35 g/L).

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Table 4.12

Effect of CaCO3 concentration on GOX production GOX activity (µ moles HQ/min/ml) 2.01 ± 0.03 2.13 ± 0.02 2.25 ± 0.1 2.33 ± 0.09 2.23 ± 0.03 0.39

CaCO3 (%) Control 1.5 2.5 3.5 4.5 LSD

Gox activity (u moles HQ/min/ml)

2.4 2.3 2.2 2.1 2 1.9 1.8 0 1.5 2.5

CaCO3 % (w/v) CaCO3 (%)

3.5

4.5

Fig 4.52

Effect of CaCO3 concentration on GOX production.

4.1.2.3

Effect of urea

According to results the maximum GOX activity was (2.00 µ moles HQ/min/ml) obtained with 0.2% Urea in the medium. Further increase in Urea resulted in decrease in enzyme activities. Results are shown in (Table 4.13 and Fig 4.53). This result is in agreement with the work of Ray and Banik (1999). They added urea in 0.14% concentration for maximum yield of enzyme. While Zubair et al obtained highest GOX activity at 0.3% urea.

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Table 4.13

Effect of urea concentration of GOX production Enzyme activity (µ moles HQ/min/ml) 1.66 ± 0.06 2.00 ± 0.01 1.59 ± 0.07 1.53 ± 0.09 1.47 ± 0.1 0.09

Urea Concentration (%) 0.1 0.2 0.3 0.4 0.5 LSD

Enzyme activity (u moles HQ/min/ml)

2.5 2 1.5 1 0.5 0 0.1 0.2 0.3 0.4 0.5 Urea Concentration (%)

Fig. 4.53

Effect of urea concentration of GOX production.

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4.1.2.4

Effect of KH2PO4

Different concentrations of KH2PO4 were examined for GOX production in pre optimized culture medium. Our finding revealed that maximum GOX production was recorded (2.96 µ mole HQ/min/ml) with 0.4% KH2PO4 in the medium and then decreased thereafter (Table 4.14 and Fig. 4.54). The results are in agreement with the work of Petrucciolit Federici (1993) and Zubair et al.

Table 4.14

Effect of KH2PO4 concentration on GOX production Enzyme activity (µ moles HQ/min/ml) 2.58 ± 0.05 2.96 ± 0.01 2.59 ± 0.02 2.44 ± 0.12 2.25 ± 0.09 0.16

KH2PO4 (%) 0.2 0.4 0.6 0.8 1.0 LSD

Enzyme activity (u moles HQ/min/ml)

3.5 3 2.5 2 1.5 1 0.5 0 0.2 0.4 0.6 KH2PO4 (%) KH2PO2(%) 0.8 1

Fig. 4.54

Effect of KH2PO4 concentration on GOX Production.

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4.1.2.5

Effect of MgSO4.7H2O

Different concentrations of MgSO4.7H2O were examined for GOX production in preoptimized culture medium (Table 4.15 and Fig. 4.55). It was observed that with the increase of MgSO4.7H2O concentration GOX activity decreases gradually. It should not be added into the fermentation medium for intracellular GOX production by A. niger. These results are in line with the work of Fiedurck and Szezoderk (1995). They demonstrated that addition of Mg+2 in the medium strongly inhibited the production of GOX. Table 4.15 Effect of MgSO4.7H2O concentration on GOX production MgSO4.7H2O (%) 0.01 0.02 0.03 0.04 LSD Enzyme activity (µ mole HQ/min/ml) 1.92 ± 0.01 1.74 ± 0.05 1.47 ± 0.02 1.34 ± 0.09 0.32

Enzyme activity (u mole HQ/min/ml)

2.5 2 1.5 1 0.5 0 0.01 0.02

4 2

0.03

0.04

MgSO4. 7H2O (%) MgSO .7H O (%)

Fig. 4.55 Effect of MgSO4.7H2O concentration on GOX production

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4.1.3

Enzyme kinetics

Kinetic studies explained the effect of pH, temperature and aeration (oxygen supply) on GOX production. These parameters were investigated and the results are as under.

4.1.3.1

Effect of pH

The pH has great influence on the activity of GOX as reported earlier in section (4.1.1.3). It was found that the enzyme activity reached maximum at pH 5.5 and decreased thereafter.

4.1.3.2

Effect of temperature

Different temperature i.e. 20°C, 30°C, 40°C, 50°C, and 70°C were checked for the GOX activity. It was found that GOX worked properly within the range 20-30°C. The activity of enzyme increased from 20-30°C and reached its maximum at 30°C and after 40°C a great decrease in the enzyme activity was observed (Table 4.16 and 4.56 Fig). Sidney and Northon (1955) investigated that GOX is lost on heating at temperature greater than 39°C. Petruceioli et al (1994) obtained highest GOX activity at 28°C.

Table 4.16

Effect of temperature on GOX production Temperature °C 20 30 40 50 60 70 LSD Enzyme activity (µ moles HQ/min/ml) 2.77 ± 0.01 2.94 ± 0.07 2.59 ± 0.03 2.07 ± 0.05 1.21 ± 0.1 0.10 ± 0.02 0.23

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Enzyme activity (u moles HQ/min/ml)

3.5 3 2.5 2 1.5 1 0.5 0 20 30 40 50 60 70 Temperature C

Fig. 4.56

Effect of temperature on GOX production.

4.1.3.3

Effect of aeration on GOX production

Effect of aeration rate on GOX production by A. niger in shake flask was studied by changing the speed of shaker as well as the volume of the fermentation medium in shake flasks. Different volumes of the fermentation medium were taken in 250 ml conical flask (20, 30, 40, 50, 60, 70 and 80 ml) and different shaker speed (50, 100 and 150 rpm) were optimized. The maximum amount of GOX was produced in the shake flasks which contained 50 ml of the fermentation medium at 100 rpm. At lower speeds of shaker or more medium per flask, the fungal growth and GOX formation were limited. The fungal growth and GOX formation were reduced in such cases, because dissolved oxygen becomes the limiting nutrient (Giuseppon 1984). Chander et al (1980) obtained similar results after working on Aspergillus wentii and Penicillium chrysogenum. He found that a shaking speed of 200 rpm was the best for enzyme reduction in 250 ml flasks containing 50 ml fermentation medium.

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Table 4.17 Volu me per flask (cm)3 20 30 40 50 60 70 80 LSD

Effect of aeration on GOX production 50 rpm Fungal Biomass (g/dm3) Gox activity (µ moles HQ/min/ml 2.44 ± 0.05 2.45 ± 0.06 2.08 ± 0.12 1.84 ± 0.13 1.43 ± 0.14 0.97 ± 0.12 0.45 ± 0.09 0.21 Shaking Speed 100 rpm Gox Fungal activity (µ Biomass moles (g/dm3 HQ/min/ml 11.9 ± 0.1 2.58 ± 0.02 12.0 ± 0.09 12.5 ± 0.06 12.5 ± 0.03 11.8 ± 0.01 10.0 ± 0.1 6.0 ± 0.2 0.13 2.60 ± 0.03 2.74 ± 0.09 2.74 ± 0.01 2.51 ± 0.02 2.30 ± 0.07 1.92 ± 0.04 0.45 150 rpm Fungal Biomass (g/dm3) 8.0 ± 0.01 9.2 ± 0.03 10.5 ± 0.07 11.3 0.09± 11.4 0.1 ± 11.0 ± 0.14 11.0 0.09± 0.35 Gox activity (µ moles HQ/min/ml) 2.06 ± 0.01 2.45 ± 0.17 2.62 ± 0.15 2.62 ± 0.10 2.52 ± 0.07 2.30 ± 0.04 1.99 ± 0.03 0.28

10.2 ±0.01 9.9 ± 0.02 8.8 ± 0.01 7.6 ± 0.01 6.0 ± 0.05 4.8 ± 0.02 3.8 ± 0.03 0.23

4.1.4

Enhanced GOX production by UV mutation

Mutagenesis is the source of all genetic variation. UV radiations were used to produce mutations in the genome of Aspergillus niger. In these studies the purpose of mutagenesis was to select the colonies of Aspergillus niger with improved expression of glucose oxidase enzyme. UV mutation was carried out to obtain enhanced production of glucose oxidase activity.

4.1.4.1

Mutant selection

The dilutions were made of the spore suspension after mutagenesis in such a way that 0.1 ml of it was plated on Malt extract agar media. The number of colonies was restricted to 20 or less than this by using oxgall as colony restrictor.

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4.1.4.2

Restriction of colony

Colonies were restricted to small size on selection medium, with colony restrictors. With the help of colony restrictor we can observe small colonies very clearly for their selection (table 4.18). (Wood et al; 1987)

Table 4.18

Different colony restrictor used in A. niger

Colony restrictor Rose Bengal Oxgall Sorbose

Colony size Large Small Medium

The use of Ox gall was found to be best for colony restriction and clearance.

4.1.4.3

Aspergillus niger mutant using 2-Deoxy-2-glucose

The UV treated spores were spread on Malt extract agar plates with 2-Deoxy-2-glucose. A few colonies were selected based on large clearance zones than wild type micro organisms. Some of the mutant colonies showed variations in appearance on malt extract agar plates. Moreover some colonies showed considerably smaller size of colonies as compared to other darker colonies with much larger size. The colonies were cultured on malt extract agar slants for preservation.

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4.1.4.4

Production of enzyme in shake flask

All the selected mutants along with wild type Aspergillus niger were used for the production of enzyme (GOX) in shake flask fermentation. Shake flasks containing 100 ml fermentation medium were inoculated with 5 ml of vegetative inoculums from the different mutant strains along with wild type strain. These cultures were incubated at 30oC for 48 hours at 120 rpm.

4.1.4.5

Glucose oxidase activity

The Intracellular glucose oxidase activity from wild type and mutant strains was measured and results are (Tables 4.19). It is reported that mutant-9 gives the maximum production of GOX for intra cellular GOX activity. Literature revealed the possibility of obtaining high effectiveness of GOX in A. niger by induction with various mutagens ( Markwell et al 1989 and Petruecioli et al 1997). Our results can be compared with Gromada and Fiedruck (1997) who improved GOX production over 125% after mutagenesis with UV of A. niger. It was noted that no extra cellular GOX activity was found in mutant as well as in wild type strains. Table 4.19 Sr. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Intracellular glucose oxidase activity in shake flask cultures Absorbance (OD) 0.91 ± 0.03 0.60 ± 0.01 0.87 ± 0.02 0.67 ± 0.01 0.51 ± 0.1 0.62 ± 0.12 0.80 ± 0.1 0.41 ± 0.03 0.93 ± 0.02 0.69 ± 0.07 0.89 ± 0.03 0.80 ± 0.01 0.68 ± 0.04 0.26 Enzyme activity (µ moles HQ/min/ml) 15.75 ± 0.01 10.39 ± 0.02 15.06 ± 0.13 11.68 ± 0.04 8.83 ± 0.07 10.74 ± 0.04 13.93 ± 0.02 7.09 ± 0.03 16.19 ± 0.01 12.03 ± 0.02 15.50 ± 0.04 13.93 ± 0.01 11.86 ± 0.05 0.09

Strains Type Mutant-1 Mutant-2 Mutant-3 Mutant-4 Mutant-5 Mutant-6 Mutant-7 Mutant-8 Mutant-9 Mutant-10 Mutant-11 Mutant-12 Wild Type LSD

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Enzyme activity

20 15 10 5 0 Mut Mut Mut Mut Mut Mut Mut Mut Mut Mut Mut Mut Wild

Specific Activity (µ mole HQ/min/mg Protein) 7.60

1. 2. 3. 4. 5. 6. 7. 8. 9. Mutant type

Fig 4.57

10. 11. 12. 13.

Intracellular glucose oxidase activity in shake flask culture

4.1.5

Purification of GOX

Purification of protein was done by ammonium sulphate dialysis and gel chromatography.

4.1.5.1

GOX activity in crude extract

Glucose oxidase was produced by fermentation using Aspergillus niger as test organism. Glucose was used as substrate and culture was incubated at 30oC for 36 hours. The crude extract was subjected to analysis for GOX activity (Table 4.20). Table 4.20 GOX activity in crude extracts Enzyme activity (µ moles HQ/min/ml) 11.96 Protein contents (mg/ml) 1.56

Enzyme fraction Crude Enzyme

Absorbance (OD) 0.685

4.1.5.2

Precipitation by (NH4)2SO4

Precipitation of proteins from cell free supernatant by the addition of different concentrations of (NH4)2SO4 is shown in Table 4.21 and Fig 4.58. Only 73% of the total proteins could be precipitated by the addition of 80% (w/v) (NH4)2SO4. Hence, 80gm (NH4)2SO4 was found suitable for the precipitation of GOX proteins.

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Table 4.21 Sr. No. 1. 2 3 4 5 6 7

Precipitation of protein by ammonium sulphate (NH4)2SO4 Conc. (%) 30 40 50 60 70 80 90 LSD Enzyme activity (µ moles HQ/min/ml) 8.76 ± 0.012 11.42 ± 0.12 11.82 ± 0.09 12.31 ± 0.04 13.33 ± 0.01 14.90 ± 0.1 14.80 ± 0.11 0.32

20 Gox µ/ml 15 10 5 0 30 40 50 60 70 80 90 (NH4)2SO4 (NH4)2SO4

Fig 4.58 Precipitation of protein by ammonium sulphate

Supernatant of crude extract was subjected to ammonium sulfate precipitation. (Table 4.22). Table 4.22 GOX purification by ammonium sulphate precipitation technique Enzyme activity (µ moles HQ/min/ml) 14.90 Protein contents (mg/ml) 1.13 Specific Activity (µ mole HQ/min/mg Protein) 13.19

Enzyme fraction Ammonium sulphate precipitation

Absorbance (OD)

0.861

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4.1.5.3

Dialysis

Dialysis was performed after ammonium sulphate precipitation. The detailed findings are as follows (Table 4.23). Table 4.23 GOX purification by dialysis Enzyme activity (µ moles HQ/min/ml) 18.45 Protein contents (mg/ml) 0.824 Specific Activity (µ mole HQ/min/mg Protein) 22.5

Enzyme fraction Dialysis

Absorbance (OD) 1.071

4.1.5.4

Purification through gel filtration (Sephadex G-75)

Samples concentrated by (NH4)2SO4 precipitation and dialysis were added to an equal volume of 0.1 M Citrate buffer (pH 5.0). This enzyme preparation was then loaded on sephadex G-75 column and 20 fractions each of 3 ml were collected. Each fraction was assayed for GOX activity. Results of Gel-filtration showed that increase in specific activity indicated removal of considerable amounts of non-GOX proteins (Table 4.24 and Fig. 4.59). Purification of 11.55 folds to that of crude enzyme was achieved through gelfiltration. A total of 20 fractions, No. 8 has the maximum activity of 37.24 µ/ml.

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Table 4.24 Enzyme fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 LSD

Analysis of gel filtration chromatography for GOX Enzyme activity (µ moles HQ/min/ml) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.12 0.10 ± 0.09 8.88 ± 0.02 24.50 ± 0.03 37.24 ± 0.12 37.10 ± 0.14 23.63 ± 0.12 14.82 ± 0.09 3.84 ± 0.01 1.90 ± 0.02 0.39 ± 0.3 0.31 ± 0.01 0.27 ± 0.03 0.17 ± 0.02 0.03 ± 0.01 0.00 ± 0.02 0.01 ± 0.09 0.42 Protein contents (mg/ml) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.01 0.14 ± 0.03 0.22 ± 0.1 0.31±0.0.3 0.42 ± 0.04 0.56 ± 0.02 0.42 ± 0.01 0.41 ± 0.09 0.16 ± 0.07 0.12 ± 0.06 0.02 ± 0.04 0.02 ± 0.03 0.03 ± 0.04 0.02 ± 0.03 0.02 ± 0.05 0.00 ± 0.01 0.00 ± 0.03 0.09 Specific Activity (µ mole HQ/min/mg Protein) 0.000 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.01 0.71 ± 0.03 40.36 ± 0.02 79.03 ± 0.07 88.66 ± 0.09 66.25 ± 0.08 56.26 ± 0.06 36.14 ± 0.12 24.00 ± 0.15 15.83 ± 0.19 19.50 ± 0.14 15.50 ± 0.09 9.00 ± 0.07 8.50 ± 0.04 1.50 ± 0.03 0.00 ± 0.01 0.00 ± 0.1 0.32

Absorbance (OD) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.01 0.00 ± 0.02 0.51 ± 0.12 1.41 ± 0.01 2.15 ± 0.03 2.14 ± 0.01 1.36 ± 0.07 0.85 ± 0.02 0.22 ± 0.01 0.11 ± 0.01 0.02 ± 0.1 0.01 ± 0.03 0.01 ± 0.02 0.01 ± 0.04 0.00 ± 0.05 0.00 ± 0.01 0.00 ± 0.02 0.32

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Gox Activity (u/ml)

40 30 20 10 0 1 3 5 7 9 11 13 15 17 19 Enzyme Fraction

Fig: 4.59

Analysis of gel filtration chromatography for GOX

Protein contents (mg/ml)

0.6 0.5 0.4 0.3 0.2 0.1 0 1 3 5 7 9 11 13 15 17 19 Enzyme Fraction

Fig: 4.60

Analysis of gel filtration chromatography for GOX

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Specific Activity (u/ml)

100 80 60 40 20 0 1 3 5 7 9 11 13 15 17 19 Enzyme Fraction

Fig: 4.61 Table 4.25

Analysis of gel filtration chromatography for GOX Summary of GOX purification Enzyme activity (µ moles HQ/min/ml 11.86 ± 0.01 14.90 ± 0.03 18.54 ± 0.09 37.24 ± 0.12 0.23 Protein contents (mg/ml) 1.56 ± 0.09 1.13 ± 0.12 0.82 ± 0.09 0.424 ± 0.02 0.19 Specific activity (µ moles HQ/min/mg Protein 7.60 ± 0.12 13.19 ± 0.09 22.5 ± 0.04 87.84 ± 0.01 0.24

Samples

Fold purification 1.00 ± 0.03 1.73 ± 0.09 2.96± 0.04 11.55 ± 0.19 0.13

Crude (NH4)2SO4 Dialysis Gel filtration LSD

4.2

Commercial applications of glucose oxidase

In the present research project two commercial applications of GOX were investigated; 4.2.1: Estimation of glucose by optimization of conditions 4.2.2: The production of calcium gluconate, gluconic acid and its derivatives

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4.2.1 Estimation of glucose by optimization of conditions

The objective of the present research projects was to develop and optimize the condition for the glucose estimation, depending on our own sources and in view of the economy of our country. The three enzymes i.e. mutarotase, glucose oxidase and peroxidase in this concern were produced, purified and the results are arranged here in this chapter under specified sections.

4.2.1.1

Glucose oxidase

Glucose oxidase produced by fermentation and purified in the previous part of project was used in this part for the estimation of glucose. The summary of result are shown in Table 4.26. Table 4.26 Summary of GOX purification Enzyme activity (µ moles HQ/min/ml 11.86 ± 0.01 14.90 ± 0.03 18.54 ± 0.09 37.24 ± 0.12 0.236 Protein contents (mg/ml) 1.56 ± 0.09 1.13 ± 0.12 0.82 ± 0.09 0.424 ± 0.02 0.193 Specific activity (µ moles HQ/min/mg Protein 7.60 ± 0.12 13.19 ± 0.09 22.5 ± 0.04 87.84 ± 0.01 0.245

Samples

Fold purification 1.00 ± 0.03 1.73 ± 0.09 2.96± 0.04 11.55 ± 0.19 0.136

Crude (NH4)2SO4 Dialysis Gel filtration LSD

4.2.1.2

4.2.1.2.1

Mutarotase

Protein estimation

Protein contents were determined by following the method of Moss and Bond, (1957). Standard curve was prepared (Fig 3.1) using bovine serum albumin, with various dilutions.

4.2.1.2.3

Standard curve of -D-glucose for mutarotase analysis

The activity of different purified enzyme samples was calculated by using this curve which is shown in Fig. 4.62.

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140 120 100 80 60 40 20 0 195 255 315 375 435 495 555 Alpha-D-glucose concentrations (µ mol)

Specific rotational (Alpha)

Fig 4.62

Standard curve of -D-glucose for mutarotase analysis

4.2.1.1.4

Mutarotase activity in crude extract

Bovine kidney cortex extract was prepared to isolate and purify the mutarotase enzyme. After extraction, the crude sample was subjected to analysis for the determination of enzyme activity (Table 4.27). Table: 4.27 Enzyme fraction Crude Enzyme Mutarotase activity in crude extract -Glucose conc. (µM) 422 Activity (/mL) Protein contents (mg/mL) 2.120 Specific activity ( /mg) 0.226

Specific rotation [] 88

0.480

4.2.1.1.5

Ammonium sulfate precipitation for mutarotase purification

The addition of different concentrations of (NH4)2SO4 were used for precipitation of proteins. (Table 4.28 and Fig. 4.63). The maximum amount of the total proteins could be precipitated by the addition of 60% (NH4)2SO4. Hence, 60% (NH4)2SO4 was found to be suitable for the precipitation of mutarotase. Crude extract was applied to 60% ammonium sulfate. The maximum activity (1.22 µ ml1

) was observed in 60% ammonium sulphate concentration (Table 4.28 and Fig. 4.63).

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Table 4.28 Ammonium sulphate coc. (%) 20% 40% 60% 80%

Mutarotase purification by (NH4)2SO4 precipitation technique Specific rotation [] 76 95 69 80 LSD -Glucose conc. (µM) 345 449 311 367 Activity (u/mL) 1.07 ± 0.01 0.72 ± 0.09 1.22 ± 0.03 0.98 ± 0.01 0.89 Protein contents (mg/mL) 2.57 ± 0.08 0.97 ± 0.08 1.86 ± 0.12 1.41 ± 0.09 0.46 Specific activity ( /mg) 0.41 ± 0.02 0.74 ± 0.03 0.65 ± 0.12 0.69 ± 0.09 0.36

Activity of mutarotase (u/ml)

1.4 1.2 1 0.8 0.6 0.4 0.2 0 20% 40% 60% 80%

Ammonium sulphate conc. (%) Ammonium slphate conc. ((%)

Fig 4.63

Mutarotase purification by (NH4)2SO4 precipitation technique

4.2.1.1.6

4.29.

Dialysis

Dialysis was done after ammonium sulphate precipitation. The result are shown in Table

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Table 4.29

Mutarotase purification by dialysis Specific rotation [] 65 -Glucose conc. (µM) 283 Activity ( /mL) 1.32 Protein contents (mg/mL) 1.69 Specific activity ( /mg) 0.78

Enzyme fraction

Dialysis

4.2.1.1.7

Gel filtration chromatography of mutarotase

The enzyme fraction having the maximum activity 1.32 µ mL-1 after dialysis was applied to Sephadex G-75 column of 1 x 16 cm for gel filtration chromatography. A total of 20 fractions of 3 ml each were collected and it was observed that the fraction 6 has the maximum activity of 1.71 µ mL-1. (Table 4.30 and Fig. 4.64) Table 4.30

Enzyme fraction

Analysis of gel filtration chromatography mutarotase

Specific rotation [] -glucose conc. (µM) Activity ( /mL) Protein contents (mg/mL) Specific activity ( /mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

104 95 92 61 57 54 62 76 95 95 98 99 100 102 100 102 103 103 106 106 LSD

510 450 442 256 234 217 264 345 459 459 476 481 486 498 486 498 504 504 522 522

0.17 ± 0.003 0.39 ± 0.005 0.46 ± 0.004 1.55 ± 0.005 1.62 ± 0.006 1.71 ± 0.006 1.47 ± 0.006 1.07 ± 0.10 0.40 ± 0.006 0.40 ± 0.007 0.339 ± 0.004 0.324 ± 0.005 0.309 ± 0.006 0.240 ± 0.005 0.309 ± 0.006 0.240 ± 0.004 0.206 ± 0.005 0.206 ± 0.004 0.103 ± 0.001 0.103 ± 0.005 0.032

0.000 ± 0.00 0.734 ± 0.006 0.744 ± 0.008 0.741 ± 0.008 0.545 ± 0.008 0.570 ± 0.006 0.609 ± 0.004 0.571 ± 0.006 0.372 ± 0.005 0.376 ± 0.004 0.391 ± 0.005 0.443 ± 0.006 0.607 ± 0.004 0.777 ± 0.005 0.476 ± 0.004 0.478 ± 0.005 0.411 ± 0.006 0.424 ± 0.005 0.295 ± 0.008 0.262 ± 0.003 0.054

0.000 ± 0.00 0.531 ± 0.003 0.618 ± 0.004 2.099 ± 0.002 2.977 ± 0.001 3.00 ± 0.003 2.413 ± 0.008 1.877 ± 0.003 1.082 ± 0.003 1.071 ± 0.008 0.867 ± 0.007 0.731 ± 0.005 0.509 ± 0.003 0.308 ± 0.005 0.649 ± 0.005 0.502 ± 0.006 0.501 ± 0.007 0.485 ± 0.008 0.349 ±0.004 0.393 ± 0.005 0.034

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3.5 3 2.5 2 1.5 1 0.5 0 1 3 5 7 9 11 13 15 17 19

Activity (U/mL)

Protein contents (mg/mL)

Specific Activity (U/mg)

Fig. 4.64: Analysis of gel filtration chromatography mutarotase

Table 4.31

Summary of mutarotase purification Activity ( /ml) 0.480 ± 0.02 1.220 ± 0.09 1.325 ± 0.07 1.712 ± 0.12 0.189 Protein contents (mg/ml) 2.120 ± 0.12 1.860 ± 0.14 1.691 ± 0.11 0.570 ± 0.15 0.236 Specific activity ( /mg) 0.226 ± 0.16 0.655 ± 0.09 0.783 ± 0.07 3.003 ± 0.06 0.59 Fold purification 1.00 ± 0.02 2.89 ± 0.12 3.46 ± 0.19 13.28 ± 0.36 0.236

Enzyme samples Crude (NH4)2SO4 Ppt. Dialysis After gel filtration chromatography LSD

Mutarotase accelerate the anomeric inter conversion of D-glucose and related sugars. Purification of mutarotase has been reported from kidneys of various mammals as hog, sheep, rat and human (Toyada et al; 1983). The crude extract was applied to ammonium sulfate precipitation which is most commonly used reagent for salting out of proteins because of its high solubility with high ionic strength (Voet et al., 1999). The activity was increased to 1.220 µ mL-1 and 0.655 µ mg-1 (specific activity). Desalting was carried out by dialysis against buffer.

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The protein contents of crud extract were decreased from 2.120 mg mL-1 to 1.691 mg mL-1 which, indicate that unwanted proteins have been removed. Toyoda et al; (1983) stated 2.45 mg mL-1 of protein contents increased. Dialyzed fraction was applied to sephadex G-150 column for gel filtration chromatography. It was observed that maximum activity 1.712 µ/mL obtained in 6th fraction during experiment with 0.570 mg/mL protein contents and 3.003U/mg of specific activity. Now, the enzyme obtained was 13.287 fold purified after this treatment.

4.2.1.3

Peroxidase

4.2.1.3.1

Peroxidase activity in crude extract

Horseradish extract was prepared to purify the enzyme peroxidase. After extraction, it was subjected to analyze the activity. The findings are arranged in table 4.32.

Table 4.32

Analysis of peroxidase in crude extract

Enzyme fraction Crude enzyme

Absorbance

Activity (µ/mL) 16.792

1.340

Protein contents (mg/mL) 7.339

Specific activity (µ/mg) 2.288

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4.2.1.3.2

Purification of peroxidase by ammonium sulfate precipitation technique

Precipitation of proteins by the addition of different concentrations of (NH4)2SO4 is shown in Table 4.33. The maximum amount of the total proteins could be precipitated by the addition of 80% (NH4)2SO4. Hence, 60% (NH4)2SO4 was found suitable for the precipitation of mutarotase. Crude extract was applied to ammonium sulfate precipitation. The activity was maximum (17.860 µ/ml) in 80% ammonium sulphate concentration which indicates the presence of enzyme (Table 4.33). Table 4.33 Summary of peroxidase purification by (NH4)2SO4 precipitation technique Activity (µ mL-1) 12.656 ± 0.56 15.213 ± 0.49 16.541 ± 0.36 17.860 ± 0.26 0.206 Protein contents (mg mL-1) 0.737 ± 0.29 1.480 ± 0.31 0.350 ± 0.26 0.524 ± 0.25 0.342 Specific Activity (µ/mg-1) 16.718 ± 0.06 10.279 ± 0.05 47.260 ± 0.04 34.062 ± 0.03 0.294

Ammonium sulphate conc. (%) 20% 40% 60% 80% LSD

Absorbance 1.010 1.214 1.320 1.425

4.2.1.3.3

4.34. Table 4.34

Dialysis

Dialysis was done after ammonium sulphate precipitation. The result is shown in table

Peroxidase purification by dialysis

Absorbance 1.543 Activity (µ/mL) 21.137 Protein contents (mg/mL) 0.445 Specific activity (µ/mg) 47.498

Enzyme fraction Dialysis

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4.2.1.3.4

Gel filtration chromatography of peroxidase

The enzyme fraction having the maximum activity of 21.137 µ/ml and 47.498 µ/mg specific activities due to ion exchange chromatography was subjected to sephadex G-75 column for gel filtration chromatography. From the total of 20 fractions, number 4 has the maximum activity 26.529 µ/mL. (Table 4.35 and Fig.4.65) Table 4.35

Enzyme fractions

Analysis of gel filtration chromatography for peroxidase

Absorbance Activity (µ/mL) Protein Contents (mg/mL) Specific activity (µ/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 LSD

0.000 ± 0.00 0.000 ± 0.00 1.543 ± 0.015 2.117 ± 0.017 2.042 ± 0.20 0.496 ± 0.009 0.175 ± 0.008 0.117 ± 0.007 0.086 ± 0.006 0.042 ± 0.001 0.038 ± 0.007 0.035 ± 0.008 0.033 ± 0.003 0.022 ± 0.030 0.019 ± 0.004 0.015 ± 0.001 0.010 ± 0.001 0.005 ± 0.001 0.003 ± 0.001 0.001 ± 0.00 0.053

0.000 ± 0.00 0.000 ± 0.00 19.335 ± 0.050 26.529 ± 0.20 25.59 ± 0.13 6.215 ± 0.007 2.193 ± 0.008 1.470 ± 0.008 1.078 ± 0.003 0.526 ± 0.005 0.475 ± 0.003 0.439 ± 0.008 0.414 ± 0.006 0.276 ± 0.003 0.188 ± 0.004 0.187 ± 0.003 0.125 ± 0.005 0.063 ± 0.005 0.037 ± 0.003 0.013 ± 0.003 0.041

0.008 ± 0.001 0.015 ± 0.003 0.279 ± 0.004 0.253 ± 0.001 0.255 ± 0.006 0.109 ± 0.007 0.094 ± 0.005 0.078 ± 0.003 0.079 ± 0.004 0.044 ± 0.006 0.064 ± 0.007 0.061 ± 0.005 0.061 ±0.007 0.046 ± 0.007 0.038 ± 0.005 0.035 ± 0.003 0.045 ± 0.005 0.031 ± 0.004 0.056 ± 0.003 0.016 ± 0.004 0.039

0.000 ± 0.00 0.000 ± 0.00 69.301 ± 0.032 104.857 ± 0.054 100.352 ± 0.050 57.018 ± 0.003 23.33 ± 0.041 18.846 ± 0.008 13.645 ± 0.009 11.954 ± 0.015 7.421 ± 0.013 7.196 ± 0.007 6.000 ± 0.009 4.947 ± 0.051 5.342 ± 0.031 2.777 ± 0.009 2.032 ± 0.010 0.650 ± 0.019 0.812 ± 0.009 0.715 ± 0.27 0.057

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120 100 80 60 40 20 0 1 3 5

Activity (U/mL)

7

9

11

13

15

17

19

Protein Contents (mg/mL)

Specific Activity (U/mg)

Fig. 4.65

Analysis of gel filtration chromatography for peroxidase

Table 4.36 Enzyme samples Crude Ammonium sulphate PPt. Dialysis

Summary of peroxidase purification Activity (µ/ml) 16.792 ± 0.12 17.860 ± 0.19 21.137 ± 0.65 26.529 ± .63 0.421 Protein contents (mg/ml) 7.339 ± 0.86 0.524 ± 0.36 0.445 ± 0.12 0.253 ± 0.3 0.521 Specific activity (µ/mg) 2.288 ± 0.21 34.062 ± 0.12 47.508 ± 0.13 104.857 ± 0.04 0.431 Fold purification 1.00 ± 0.01 14.88 ± 0.19 20.76 ± 0.29 45.82 ± 0.29 0.289

After gel filtration chromatography LSD

Peroxidase is one of those enzymes that have much wide application in health and clinical sciences as a diagnostic tool. In the present project, peroxidase was extracted by blending the fresh horse Radish for 15 minutes. The activity and specific activity of the crude enzyme was 16.792 µmL-1 and 2.288 µ mg-1 having 7.339 mg mL-1 of protein contents. To purify the desired enzyme,

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extract was subjected to 20-80% saturation with ammonium sulfate.

The activity

obtained 17.860 µ mL-1 and 34.062 µ mg-1 was specific activity. The activity of dialysed enzyme was 21.137 µ mL-1 and it was greater from 17.860 /mL of ammonium sulphate precipitation. Gel filtration chromatography was applied by sephadex G-75 which proved to be very efficient as activity of dialysed fraction was 26.529 µ mL-1 while 104.857 U/mg was specific activity with 45.829 folds purification as compared to crude. Such good value of specific activity appreciates this method and proves the high degree of purity.

4.2.1.4

Optimization of conditions for glucose estimation

The concentrations of mutarotase, glucose oxidase and peroxidase enzymes, incubation period for reaction and wavelength were optimized to standardize the glucose level determination. The results of these parameters are (Table 4.37) indicating that 5µl

mutarotase, 15µL glucose oxidase and 10 µl of peroxidase, added at same time utilizing 50 µL of guaiacol before peroxidase addition proved to be best than other trails. It was observed that the incubation period of 10 minutes at 30oC is sufficient for the reaction and wavelength of 470 nm is more suitable than other two. The maximum period for recording the observations of tests was estimated to be 30 minutes.

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Table 4.37

Analysis of results for glucose estimation to standardize the conditions (addition of enzymes at same time)

WAVELENGTH

Observa tions 1 Set A Set B Set C LSD x x x

290 nm 2 0.572 ± 0.008 0.600 ± 0.009 0.510 ± 0.012 0.069 1

546 nm 2 0.778 ± 0.009 0.838 ± 0.018 0.899 ± 0.009 0.296 1

470nm 2 0.747 ± 0.008 0.885 ± 0.020 1.320 ± 0.005 0.068

0.485 ± 0.008 0.711 ± 0.005 0.726 ± 0.008 0.087

0.848 ± 0.006 0.727 ± 0.015 0.759 ± 0.017 0.0235

0.912 ± 0.013 0.752 ± 0.013 0.759 ± 0.005 0.078

A= Mutarotase: 5µL + Glucose oxidase: 15µL + Peroxidase: 10µL B= Mutarotase: 10µL + Glucose oxidase: 30µL+ Peroxidase: 20µL C= Mutarotase: 20µL + Glucose oxidase: 60µL + Peroxidase: 40µL x= 1= 2= Guaiacol before peroxidase 10 min incubation 20 min incubation

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4.2.1.4.1

Comparison with standard kit

The optimized value of all the parameters (given above) was utilized to compare with standard kit (Medisense Abbott UK) on serum of diabetic patients. The results of both the kits have been shown (Table 4.38 and Fig. 4.66), which are comparable in this concern. Table 4.38 Comparison with standard kit (Medisense Abbott UK) Patients Absorbance of sample 1.132 ± 0.012 1.224 ± 0.015 1.017 ± 0.009 1.102 ± 0.008 0.656 ± 0.012 0.747 ± 0.005 1.015 ± 0.008 0.121 Our local kit glucose level (mg/dL) 199 ± 2 215 ± 3 178 ± 4 193 ± 1 115 ± 1 131 ± 5 178 ± 6 0.698 Standard kit glucose level (mg/dL) 117 ± 2 174 ± 2 99 ± 3 269 ± 2 91 ± 2 116 ± 3 136 ± 4 0.592

1 2 3 4 5 6 7 LSD

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Fig. 4.66

Comparison with standard kit.

4.2.1.4.2

Determination of sensitivity our local kit

It was observed that our prepared and standardized kit (Medisense abbott UK) is very much sensitive as it can determine the glucose level as low as 50 mg/dL. (Table 4.39). Table 4.39 Determination of Sensitivity of our local kit. Absorbance

1.105 ± 0.005 0.913 ± 0.006 0.812 ± 0.009 0.658 ± 0.007 0.597 ± 0.004 0.455 ± 0.003 0.400 ± 0.12 0.092

-D-Glucose Concentration (mg/dL)

200 175 150 125 100 75 50 LSD

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Fig. 4.67

Determination of sensitivity of self prepared kit

Due to marked affinity and specificity for -D-glucose, glucose oxidase is being used to determine the true glucose value of plasma/serum as well as other biological materials (Kaplan, 1957). The production of H2O2 form glucose oxidase on glucose serves as substrate for the peroxidase, which simultaneously converts a colourless compound into a coloured one with the help of chromogen where absorbance is easily measured. It was found that rate of production of coloured complex was proportional to the rate of H2O2 production which in turn was proportional to the activity of glucose oxidase (Hames and Hooper, 2001 Wong et al; 1981). The concentrations of 5 µL mutarotase, 15 µL glucose oxidase and 10µL peroxidase enzymes were proved to be the best for estimation of glucose serum samples. Guaiacol as chromogen was added before peroxidase while the incubation at 30oC for 10 minutes obtained the best results. Many other reagents like benzidine, O-toluidine, O-dianisidine are being used as chromogen in glucose determination which are carcinogenic. But guaiacol is appropriate for this method. According to Wong et al., (1981) glucose can be measured either kinetically or by fixed time assay having a short incubation of 5 minutes.

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Horse Radish peroxidase in presence of H2O2 catalyzes the reaction having a broad spectrum band between 500-600 nm with maximum at 590 nm (Wong et al; 1981). We carried out the trials at 546, 290 and 470 nm wavelength. It was observed that at 470 nm wavelength, the values of 0.912 is highest which indicates that it should be used for the estimation of glucose. In the comparison of the two kits i.e. standard and our prepared, although there is a difference between results but our findings are higher than that of standard kit. Such difference is due to precipitation. Much improved techniques like ion exchange chromatography and FPLC can remove this problem. The sensitivity of our prepared kit was found as 50mg/dL having 0.400 absorbance value when tested upon D-glucose standard solution. All values from 200 mg/dL to 50 mg/dL have gradually a fine decreasing order. Wong et al (1981)calculated the lower limit of 10-100 µg of Dglucose.

4.2.2

The production of calcium gluconate, gluconic acid and its derivatives by GOX method

Submerged fermentation process was used for the production of calcium gluconate, gluconic acid and its derivatives by GOX method. GOX was produced by A. niger (potato source) which convert glucose to gluconic acid by simple oxidation reaction. Due to the presence of CaCO3 in the fermentation broth the gluconic acid produced immediately converted into its salt i.e. calcium gluconate which was the stable product. Later on gluconic acid was obtained from calcium gluconate.

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4.2.2.1

Production of calcium gluconate

Calcium gluconate was produced by submerged fermentation using GOX method. Aspergillus niger strain was isolated from potato source (screened in previous section 4.1 for highest production of GOX) used in the present study for the production of calcium gluconate. Glucose salt medium containing CaCO3 in 250 ml Erlenmeyer flasks was used for the production of calcium gluconate by Aspergillus niger. The shake flask cultures containing 50 ml of fermentation medium were incubated at 30°C for 48 hours. All the experiments were performed in triplicate. Different fermentation conditions such as fermentation period, pH, temperature, glucose concentration, carbonates, phosphates and nitrogen sources etc. were optimized for maximum production of enzyme GOX and the bioconversion of glucose into calcium gluconate or gluconic acid (Prescott and Dunn, 1987 and 1959).

4.2.2.1.1.

Effect of fermentation period on calcium gluconate production

Optimum fermentation period is one of the most important factors in calcium gluconate production (Pons et al 2000). The results indicated that after 12 hours fermentation, the production of calcium gluconate was negligible. However its production was increased after 24 hours. The calcium gluconate production was maximum at 48 hours (Table 4.40 and Fig 4.68). Further incubation did not increase the production of calcium gluconate which might be due to the over growth of mycelium. The mycelial mass and glucose consumption were also calculated for every time period. While Buzzini et al; (1993) investigated maximum yield of calcium gluconate at 72 hours.

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Table 4.40

Effect of fermentation period on calcium gluconate production Wet mycelial weight g/100ml 5.20 ± 0.01 6.10 ± 0.05 7.70 ± 0.03 7.10 ± 0.09 6.90 ± 0.10 0.092 Glucose used g/L 40.0 ± 0.1 55.3 ± 0.5 80.0 ± 0.6 85.1 ± 0.7 90.3 ± 0.3 0.136 Calcium gluconate produced g/L 33.00 ± 1.9 53.3 ± 2.5 85.0 ± 1.6 80.6 ± 0.8 70.0 ± 0.9 0.236 %age yield of calcium gluconate 33 ± 2.1 53.3 ± 1.3 85.0 ± 3.2 80.6 ± 2.5 70.0 ± 2.3 0.684

Fermentation period (hrs) 12 24 48 72 96 LSD pH

= 5.5 Temperature 35°C Glucose added = 100 g/L

Calcium gluconate Produced (g/l)

100 80 60 40 20 0 12 24 48 72 96 Fermentation Period (Hrs)

Fig. 4.68 Effect of fermentation period on calcium gluconate production

4.2.2.1.2

Effect of pH

The pH of fermentation medium is of great significance for microbial production of calcium gluconate (Lee et al (1998). In this experiment calcium gluconate production was investigated within the range of 4.0-7.0 pH (Table 4.41 and Fig 4.69). Results show that the production of calcium gluconate was minimum at pH 4.0 (i.e. 25.0 gl-1) and mycelial mass was also poor at this pH. The production of calcium gluconate increased

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by increasing the pH from 4.0­5.5 of the medium. Calcium gluconate produced was maximum (i.e.80.1 g/L) at pH 5.5. Further increase in pH from 5.5 to 7.0 reduced the gluconate production. While Ray and Banik (1994) found maximum amount of calcium gluconate by mutant A. niger at a pH of 6.5. Table 4.41 pH 4.0 4.5 5.0 5.5 6.0 6.5 7.0 LSD pH Effect of pH on calcium gluconate production Wet mycelial weight g/100ml 6.3 ± 0.1 11.4 ± 0.5 12.3 ± 0.2 15.1 ± 0.2 13.3 ± 0.3 11.5 ± 0.1 11.0 ± 0.1 0.086 Glucose consumed g/L 27.0 ± 2.1 38.6 ± 0.9 53.3 ± 0.8 74.0 ± 1.1 62.5 ± 1.5 60.0 ± 0.8 57.3 ± 0.7 0.435 Calcium gluconate produced g/L 25.0 ± 0.9 37.5 ± 0.3 60.0 ± 0.8 80.1 ± 0.4 66.6 ± 0.5 71.0 ± 0.6 56.0 ± 0.9 0.392 %age yield of calcium gluconate 25.0 ± 0.5 37.5 ± 0.3 60.0 ± 0.4 80.1 ± 0.1 66.6 ± 0.3 71.0 ± 0.4 56.0 ± 0.5 0.542

= 5.5 Fermentation period = 48 hours

Temperature 35°C Glucose added = 100 g

Calcium gluconate Produced (g/l)

100 80 60 40 20 0 4 4.5 5 5.5 pH 6 6.5 7

Fig. 4.69:

Effect of pH on calcium gluconate production.

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4.2.2.1.3

Effect of glucose concentration

Different concentrations of glucose in medium (5 -24%) were used to study the effect of glucose level for calcium gluconate production. The carbon source concentration plays an important role in the conversion of glucose into gluconic acid and its calcium salt. The data is presented in Table 4.42 and Fig. 4.70 Consumption of glucose and mycelial mass were also calculated. The maximum yield of calcium gluconate was found when the glucose concentration was 10% w/v (i.e. 75.9 g/L). It might be due to fact that at this concentration the GOX activity by A. niger was optimum, which resulted in high yield of calcium gluconate. Further increase in glucose concentration reduced the production of calcium gluconate. The decrease in the production may be due to catabolic repression of A. niger (Doneva et al; 1999). Vroomen et al (1999) investigated that 10% glucose is suitable for maximum production of gluconic acid while Ray and Banik (1994) observed higher yield of calcium gluconate at 15% glucose concentration. Table 4.42: Effect of glucose concentration on calcium-gluconate production Wet Mycelial weight g/100ml 5.20 ± 0.05 6.10 ± 0.09 7.70 ± 0.08 8.80 ± 0.09 9.30 ± 0.07 11.75 ± 0.05 12.2 ± 0.06 12.8 ± 0.08 13.2 ± 0.09 0.62 Glucose used g/L 36 ± 0.1 53.3 ± 0.2 73.6 ± 0.4 74.5 ± 0.1 78.7 ± 0.8 79.5 ± 0.7 79.02 ± 0.5 76.0 ± 0.3 65.8 ± 0.5 0.192 Calcium gluconate produced g/L 50.6 ± 0.4 65.25 ± 0.5 75.9 ± 0.4 63.08 ± 0.4 58.2 ± 0.4 49.6 ± 0.5 45.3 ± 0.8 39.5 ± 0.8 28.9 ± 0.6 0.099 %age yield of calcium gluconate 50.6 ± 0.3 65.25 ± 0.2 75.9 ± 0.1 63.08 ± 0.1 58.2 ± 0.1 49.6 ± 0.5 45.3 ± 0.4 39.5 ± 0.3 28.9 ± 0.5 0.108

Concentration of glucose (%) 6 8 10 12 14 16 18 20 22 LSD pH

= 5.5 Fermentation period = 48 hours

Temperature 35°C

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Calcium gluconate produced (g/l)

80 60 40 20 0 6 8 10 12 14 16 18 20 22 Cocentration of glucose (%)

Fig 4.70 pH

Effect of glucose concentration on calcium-gluconate production

= 5.5 Fermentation Period = 48 hours

Temperature 35°C Glucose added = 100 g

4.2.2.1.4

Effect of different carbonates

Different metal carbonates such as MgCO3, CaCO3, MnCO3, ZnCO3, and FeCO3 were used in medium to evaluate the influence on production calcium gluconate (Fig. 4.71) shows the comparison between various carbonate sources for ca-gluconate produced by A. niger. CaCO3 was found to be the best metal carbonate for highest calcium gluconate production and 7% calcium carbonate concentration gave maximum percentage yield of calcium gluconate (Table 4.43). Table 4.43 Effect of different carbonates on calcium gluconate production Glucose used g/L 71.7 75.3 48.3 73.0 45.0 Wet mycelial mass weight g/100 ml. 6.31 ± 0.05 7.23 ± 0.08 5.33 ± 0.04 6.21 ± 0.06 4.91 ± 0.02 0.082

Metal Calcium %age yield of carbonate gluconate calcium (1.75 g/25ml) produced g/L gluconate MgCO3 70.0 70 CaCO3 80.0 80.0 ZnCO3 55.0 55.0 FeCO3 75.0 75.0 MnCO3 50.0 50.0 LSD pH = 5.5 Fermentation period = 48 hours Temperature 35°C Glucose added = 100 g

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Calcium gluconate Produced (g/l)

100 80 60 40 20 0 MgCO3 CaCO3 ZnCO3 FeCO3 MnCO3 Metal Carbonate (1.75 g/25ml)

Fig. 4.71

Effect of different carbonates on calcium gluconate production

Table 4.44

pH

Effect of different concentration of calcium carbonate on calcium gluconate production Calcium carbonate conc. % age yield of Calcium Sr. No. (%) gluconate 1. 0 21 ± 2 2. 1 38 ± 3 3 2 49 ± 2 4 3 61 ± 2 5 4 76 ± 4 6 5 90 ± 5 7 6 90 ± 6 8 7 98 ± 2 0.683 LSD = 5.5 Fermentation period = 48 hours

Temperature 35°C Glucose added = 100 g While Elnaghy and Megalla (1975) reported higher yield of calcium gluconate in the presence of 3% CaCO3 and 15% glucose and fermentative materials.

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%age Yield of Calcium gluconate

120 100 80 60 40 20 0 0 1 2 3 4 5 6 7 Calcium carbonate conc. (%)

Fig 4.72

Effect of different concentration of calcium carbonate on calcium gluconate production

4.2.2.1.5

Effect of different nitrogen sources

Different sources such as peptone, urea, NH4NO3, NH4Cl, and NaNO3 were added in culture media to investigate the effect of nitrogen sources on mycelial mass and Cagluconate production and glucose consumption (Table 4.45and Fig. 4.73). The most suitable nitrogen source for the production of calcium gluconate by A. niger was urea (i.e. 84.4 gl-1). However, sodium nitrate resulted in minimum yield of Ca. gluconate (i.e.52.1 gl-1. While Elanghy and Megalla (1995) reported that the peptone was best sources for the production of calcium gluconate. Ray (1999) and Banik (1994) added urea in 0.14% concentration for enhanced yield of calcium gluconate. The various concentrations of urea (0.1-0.4 gl-1) were further studied and presented in (Table 4.46). The optimum level of urea was 0.2 gl-1. The yield of Ca-gluconate was also decreased with further increase of level of urea. The glucose consumption and mycelial mass were also calculated because these are also related to maximum gluconate production.

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Table 4.45

Effect of different nitrogen sources on calcium gluconate production Wet mycelial weight g/100ml 6.21 ± 0.09 8.21 ± 0.06 9.74 ± 0.08 9.00 ± 0.01 10.99 ± 0.06 0.138 Glucose used g/L 72.7 ± 0.3 74.3 ± 0.2 47.2 ± 0.5 69.0 ± 0.4 62.0 ± 0.5 0.294 Calcium gluconate produced g/L 70.5 ± 0.3 84.4 ± 0.2 45.9 ± 0.5 67.0 ± 0.4 52.1 ± 0.5 0.386 %age yield of calcium gluconate 70.5 ± 0.3 84.4 ± 0.4 45.9 ± 0.1 67.1 ± 0.2 52.1 ± 0.5 0.429

Nitrogen sources (3g/L) NH4NO3 Urea Peptone NH4Cl NaNO3 LSD pH

= 5.5 Fermentation period = 48 hours

Temperature 35°C Glucose added = 100 g nitrogen Source added = 3 g/L

Calcium gluconate produced (g/l)

100 80 60 40 20 0 NH4NO3 Urea Peptone NH4Cl NaNO3 Nitrogen Sources (3g/l)

Fig. 4.73

Effect of different nitrogen source on calcium gluconate production.

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Fig. 4.46

Effect of urea concentration on calcium gluconate production

Urea concentration (g/L) 0.1 0.15 0.2 0.25 0.3 0.35 0.4 LSD pH

Wet mycelial mass weight (g/100ml) 6.35 7.30 8.25 8.81 8.90 9.01 8.95

Glucose used (g/L) 60.6 70.0 85.0 82.0 74.0 61.3 64.6

Calcium gluconate produced (g/L) 58.8 ± 0.3 71.3 ± 0.5 88.1 ± 0.3 73.3 ± 0.2 63.2 ± 0.5 46.0 ± 0.4 41.5 ± 0.1 0.214

%age yield of calcium gluconate 58.8 ± 0.3 71.3 ± 0.4 88.1 ± 0.3 73.3 ± 0.4 63.2 ± 0.4 46.0 ± 0.8 41.5 ± 0.6 0.346

= 5.5 Fermentation period = 48 hours

Temperature 35°C Glucose added = 100 g

Calcium gluconate produced (g/l)

100 80 60 40 20 0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 Urea concentration (g/l)

Fig 4.74

Effect of urea concentration on calcium gluconate production

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4.2.2.1.5

Effect of different Phosphate sources

Table 4.47 and Figure 4.75 shows the effect of addition of different phosphate (KH2PO4 and K2HPO4) in different concentration ranging from 0.10 to 0.30% w/v on Ca-gluconate fermentation by A. niger (Table 4.48). The addition of KH2PO4 in the fermentation broth resulted in maximum calcium gluconate yield (i.e. 62.0 gl-1) in concentration 0.15%. The production of Calcium gluconate was gradually decreased. Therefore 0.15% KH2PO4 concentration was found to be optimum. Trager et al; (1992) used 1 gl-1 KH2PO4 in fermentation medium for optimum GOX production.

Table 4.47

Effect of different phosphate sources on calcium gluconate production Wet mycelial weight g/100ml 12.0 9.01 Glucose used g/L 60.0 58.6 Calcium gluconate produced g/L 62.0 54.6 %age yield of calcium gluconate 62.0 54.6

Phosphate sources 1.5g/L KH2PO4 K2HPO4 pH

= 5.5 Fermentation period = 48 hours

Temperature 35°C Glucose added = 100 g Phosphate source added 1.5 gl-1

64 62 60 58 56 54 52 50 KH2PO4 K2HPO4 Phosphate Sources 1.5 g/l

Fig: 4.75

Calcium gluconate produced (g/l)

Effect of different phosphate sources on calcium gluconate production:

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Table 4.48

Effect of different concentration of KH2PO4 on the production of calcium gluconate Calcium gluconate produced g/L 70.7 ± 0.3 77.6 ± 0.2 72.0 ± 0.1 55.1 ± 0.5 42.6 ± 0.4 0.382 %age yield of calcium gluconate 70.7 ± 0.4 77.6 ± 0.4 72.0 ± 0.6 55.1 ± 0.7 42.6 ± 0.4 0.524

KH2PO4 % (w/v) 0.10 0.15 0.12 0.25 0.30 LSD pH

Wet mycelial weight g/100ml 7.89 10.98 8.30 7.30 6.99

Glucose used g/L 71.3 ± 0.3 76.0 ± 0.4 72.6 ± 0.1 60.4 ± 0.2 55.4 ± 0.1 0.186

= 5.5 Fermentation period = 48 hour s

Temperature 35°C Glucose added = 100 g

Calcium gluconate produced (g/l)

100 80 60 40 20 0 0.1 0.15 0.12 0.25 0.3

KH2KH2PO4 PO4 % (w/v)

Fig. 4.76

Effect of different concentration of KH2PO4 on the production of calcium gluconate Effect of temperature

4.2.2.1.6

The influence of temperature on Ca-gluconate production was studied over the range of 20 to 45°C by measuring the initial rates of synthesis. Maximum initial rates were obtained at 35°C. It is similar to the work of Tomotani et al; (2005), they found that

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when GOX was used as catalyst for oxidizing glucose into gluconic acid utilizing 10 ml Bioengineering enzyme membrane reactor at 30°C, a yield of about 75% was attained. Similarly Savas Anastassiadis et al; (2005) have observed that the temperature of new fermentation process for continuous gluconic acid production by the isolated yeast like strain Aureobasidium pullulams was between 29 and 31°C.

Calcium gluconate (g/l)

120 100 80 60 40 20 0 15 20 25 30 35 40 45 50 Temperature (C)

Fig 4.77

Effect of temperature on calcium gluconate production.

4.3

Production of gluconic acid and its derivatives

Yield of gluconic acid from calcium gluconate Methods Oxalic acid Sulphuric acid Yields 80% 90%

Table: 4.49

Sr. No. 1. 2.

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Table: 4.50

Yields of metal gluconates

Percentage yields Sr. No. Derivatives Double decomposition method 1. 2. 3. 4. Sodium gluconate Magnesium gluconate Copper gluconate Nickel gluconate 80% 81% 83% 84% Gluconic acid method 92% 94% 91% 88%

This project describe the production of the gluconic acid and its derivatives on the laboratory scale. Gluconic acid and its metal salts such as sodium, ferrous, magnesium, copper, nickel and cobalt gluconates were synthesized from calcium gluconate which is produced by fermentation. The % age yield of gluconic acid is given in (Table 4.49.) The gluconic acid was produced by the action of oxalic acid and sulphuric acid on calcium gluconate. Sulphuric acid gave better yield i.e. 90% as compared to oxalic acid i.e. 80%. It may be due to the difficulty in cooling the reaction mixture of calcium gluconate and oxalic acid. Sulphuric acid is cheap and readily available in the local market and gave higher yields. Table 4.50 shows the yields of metal gluconates which were produced by double decomposition method and gluconic acid method respectively. It is clear from the table that gluconic acid method gave better yield in comparison with the other method used. The reason was the equilibrium between the reactants and the products. In metal sulphates the equilibrium obtained by the reactants and the products was equal on both sides. The result was that no further reaction was occurred and hence the yield was less. The metal carbonates gave maximum yield because their direct action with gluconic acid and removal of carbon dioxide helped the reactants to give more and more products. Forward reaction is maximum over here and stops the chances of backward reaction which was clear in case of metal sulphates. (Prescott and Dunn 1959).

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The metal gluconates were isolated, crystallized and all these seemed to be pure. Also showed that the derivatives produced by double decomposition method with less yield. But in the case of gluconic acid method, the derivatives were obtained with greater yield.

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SUMMARY

The main project was planned for the optimum production of glucose oxidase (EC 1.1.3.4) by Aspergillus niger and its utilization for estimation of glucose and for the production of calcium, gluconate, gluconic acid and its derivatives. The project was divided into two parts: (A) (B) Production of glucose oxidase from Aspergillus niger. Commercial applications of glucose oxidase.

(A)

Production of Glucose Oxidase from Aspergillus niger

Here the aim was to improve GOX production using mutagenesis of A. niger, to optimize the conditions of fermentation; to screen fungal strains producing highest GOX activity, and to medium composition. Mutagenesis was carried out on several strains at different time intervals. GOX enzyme was purified by (NH4)2SO4 precipitation technique, dialysis and gel filtration chromatography. The enzyme was found to be intracellular. It was extracted by breaking the mycelia and no activity was found in culture filtrate obtained after the separation of mycelium from growth medium. Five strains of A. niger isolated from grapes, bread, potato, pickle and sugar beet sources were screened for maximum GOX production. The fungi were grown in mineral broth media with different concentrations of glucose and pH values. Enzyme activities, mycelial masses, amount of total protein and specific enzyme activities were calculated by keeping one factor at optimal condition while obtaining the optimal condition for other factor by using a range of conditions. It is clear from our results that the A. niger strain isolated from potato was best for GOX production. This strain showed the maximum enzyme activity in medium containing 10% (w/v glucose and at pH 5.5. In Pakistan there are few fungal sources with improve production of glucose oxidase. Hence this project will help the commercial production of GOX. Different conditions like the fermentation period, varying concentrations of urea, MgSO4.7H2O, CaCO3 and KH2PO4 were optimized by conducting different experiments. The maximum activity of

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glucose oxidase was recorded after 48 hours of continuous shaking fermentation of optimum growth medium containing 3.5% (w/v) CaCO3, 0.2% (w/v) Urea, 0.4% (w/v) KH2PO4 and 0.01% (w/v) MgSO4.7H2O. It was observed that addition of Urea, CaCO3, and KH2PO4 in the medium enhanced the GOX production whereas addition of MgSO4.7H2O decreased the GOX production. The GOX was found to be quite active upto 60oC with optimum temperature at 30oC. The batch fermentation volume of 50 ml at 100 rpm speed shaker was found to be the optimum for GOX production. Among the mutants both improved GOX production and reduced GOX mutants were found. The values of enzyme activities of mutants showed that there was an increase in enzyme activity as compared to wild type strain However, some mutants showed negative growth results due to the effect of UV radiations on the genetic make up of culture. It was found that mutant (9) owe maximum activity and growth. The UV induced mutation gave a stable and viable culture for hyper production of GOX as the production was enhanced. This may be due to gene copy number or improvement in gene expression or both. Then the enzyme was purified by (NH4)2SO4 precipitation technique, Dialysis and Gel filtration chromatography. It was observed that enzyme activity was increased by increasing (NH4)2SO4 concentration. Enzyme activity also increased by Dialysis and Gel filtration chromatography from 11.90 to 37.24 µ/ml purification was 11.55 folds than simple precipitation at this final step.

(B)

(1) (2)

Commercial applications of glucose oxidase

Estimation of glucose by standardization of conditions using GOX. The production of calcium gluconate, gluconic acid and its derivatives using GOX.

In this part of project two commercial applications of GOX were investigated i.e.

(1)

Estimation of glucose by Standardization of Conditions using GOX

Diabetes mellitus is a metabolic problem in which elevated blood and urine glucose, provide a convenient diagnostic test for the disease. The three enzymes GOX, mutarotase (EC # 5.1.3.3) and peroxidase (EC # 1.11.1.) were produced, extracted and purified for the preparation and optimization of glucose estimation kit. GOX; which was produced, extracted and purified in the previous section,

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was used. Mutarotase, accelerated the anomeric interconversion of D-glucose and related sugars. Mutarotase was isolated and purified from bovine kidney cortex by (NH4)2SO4 precipitation technique, dialysis and gel filtration chromatography. Mutarotase increased activity from 0.480 to 1.712 µ/ml by purification. It has 13.28 folds purifications at this final step. Peroxidase was isolated and purified from horse radish and was subjected to (NH4)2SO4 precipitation technique, Dialysis and Gel filtration chromatography. Peroxidase gained activity from 16.792 to 26.592 µ/ml. It was 45.82 fold purified by this stage. The enzyme concentrations of 5 µL mutarotase, 15 µL glucose oxidase and 10 µL of peroxidase with Guaiacol added before peroxidase, proved to be best for estimations of glucose in blood samples. The sensitivity of the local kit was as low as 50 mg/dL glucose. The wavelength of 470 nm was best for the test. The results were comparable with standard kit of Medisense Abbott (UK).

(2)

The Production of Ca-gluconate, gluconic acid and its derivatives:

The production of calcium gluconate and gluconic acid by glucose oxidase from Aspergillus niger is a challenge in fermentation technology. The present study describes the production of calcium gluconate and gluconic acid by GOX. The time course during fermentation showed that the calcium gluconate production was maximum at 48 hours after conidial inoculation. The cultural conditions optimized for maximum calcium gluconate production were, glucose concentration 10% (w/v), pH 5.5, 7% (w/v) CaCO3, 0.2% (w/v) urea 0.15% (w/v) KH2PO4 concentration at 35oC. Different nitrogen, phosphate and metal carbonate sources were also optimized. In view of the increasing importance of calcium gluconate as the vehicle for administering metals in pharmaceutical industries. Further studies are required for large scale production of calcium gluconate. It is evident that the biosynthesis of calcium gluconate was strongly dependent on the culture conditions employed. The present study also described the production of gluconic acid and its derivatives on the laboratory scale. Gluconic acid and its metal salts such as sodium, magnesium, copper and nickel gluconates were synthesized from calcium gluconate which was produced by fermentation. The gluconic acid was released by the action of oxalic acid and sulphuric

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acid on calcium gluconate. Sulphuric acid gave better yields i.e. (90%) as compared to oxalic acid (80 %.). So the organic acid was obtained by H2SO4 in the present work because it was cheap and readily available in local market. Metal gluconates were also produced by both the double decomposition and gluconic acid methods respectively. It is clear from the study that the gluconic acid method gave greater yields compared to the double decomposition method. The project enhanced GOX production, and shows it could be used for the production of calcium gluconate, gluconic acid and its derivatives. GOX was used develop and standardize a glucose estimation kit made in Pakistan. The final work of this project was to develop and encourage local technology to save substantial amounts of foreign exchange being drained by the import of GOX derives. This project will help in the commercial production of products using GOX in Pakistan.

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RECOMMENDATIONS

In order to achieve better production of GOX for its efficient and commercial use the recommendation are as follows: · Activities of all three enzymes i.e. glucose oxidase, mutarotase and peroxidases can be increased by much improved techniques of purification like ion exchange chromatography or FPLC hence enhanced the production of these enzymes. · Enzyme production can also be enhanced by adopting the latest and modern technology to introduce the mutation in wild type strains i.e. gene cloning. Chemicals mutagens can also be used besides physical treatments. · · · Chemical or electrical method can be used besides fermentation method that would help to achieve higher amount of required product. Low priced local kits should be prepared as compared to imported kits by using present project in Pakistan. The scores of factors that contribute to the overall mismanaged system should be removed some of these factors include: o Lack of chemicals o Lack of apparatus o Lack of instruments o Limited use of purification technologies. o Lack of infra structure facilities (Research lab availabilities). o Lack of local plants availability for preparation of kits and organic salt production from lab to industrial level for commercialization purpose. o Lack of funding agencies and Govt. support. Therefore, there is an urgent need to improve all the above factors by the support of Government of Pakistan.

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FUTURE EXTENSION OF RESEARCH PROJECT

Keeping in mind the general view of the importance of this enzyme (GOX), the results shows reasonable findings. Although in this study short duration data was analyzed, the true picture may emerge by the applications of long term processes. Regarding this aspect and others, the following recommendations are made to extend this research project further. · Adequate methodologies may be developed to carry out financial economic and social benefit-cost analyses of the projects aimed at the commercial applications of GOX in Pakistan. · In future our aim is to: o Encourage the local technology and skill o Disseminate technical know-how for the development of glucose estimation kit. o Improvement of GOX production on glucose by manipulation of growth morphology. o Improvement of cell morphology to the desired form (growth in a small aggregate form, minimizing the mass transfer limitation inside the microbial pellet) with the aim to increase the GOX productivity. Change of cell morphology was done by cultivation under different hydrodynamic stresses conditions and with addition of a soluble biopolymer (xylan). o Develop income generating activities for those who are well qualified and are unemployed. o Save substantial amount of foreign exchange being drained on the import of such items. I want to work on other applications like to. (1) (2) (3) (4) Be used as preservative in different foods. Be used in biofuel cells and bioelectrochemical cells. Remove blackening of potato chips. Be used as Antioxidant

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REFERENCES

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REFERENCES

1) Amberkar, G.R., Thadani, S.B. and Doctor, V.M. (1965). Production of calcium gluconate by penicillum chrysogenum in submerged culture. App. Microbiol. 13: 713-719. 2) Attia, R.M., Rawia, F.G. and Dokhan, A.M. (1987). Physico-chemical properties of glucose oxidase. Egypt. J. Microbiol. 22(1): 35-44. 3) Baajaree, J. and Kannika, C. (1993). Utilization of starch hydrolyzed for gluconic acid production by A. niger G-153. Microb. Util. Renewable Resource: 8: 611616. 4) Baetselier, D.A., Vasavada, A., Dohet, P., Thi, V.H., Beukelaer, M.D., Erpicum, T., Clerck, L.d., Honotier, J. and Rosenberg, S. (1991). Fermentation of a yeast producing A. niger GOX: Scale-up purification and characterization of the recombinant enzyme. Biotechnol. 9(6): 559-561. 5) Bailey, J. M., Fishman P.H., and Pentchew, P.G. (1969). Studies on mutarotases: Isolation and characterization of a mutarotase from bovine 6) 7) Baronnet, R., Ann. Pharm. Franc. 6: 256-259 (1948). Chem. Abstr. 43: 5743. Beltrame, P., Comotti, M., Della, Pina. C., and M. Rossi. (2004). Aerobic oxidation of glucose I. Enzymatic catalysis. J. Catalysis. 228: 282-287. 8) Bentley, R. (1959). Glucose aerodehydrogenase (glucose oxidase). Methods in Enzymology. Vol. 1. Academic press, USA. PP: 340-345. 9) Bentley, R. (1959). Glucose aerodehydrogenase (GOX) Method in Enzymology. Vol. 1. Academic Press, USA. PP: 340-345. 10) Bentley, R., (1962). Mutarotase Methods in Enzymology. Academic Press, USA. PP: 219-225. 11) Bentley, R., Boyer, P., Lardy, H. and Myrback, K. (1963). "The Enzymes" 2nd Ed. Academic press, New York. PP. 567. 12) Bhatti, H.N., Madeeha, M., Asgher, M., Batool, N., (2006). Canadian Journal of Microbiology, Volume 52: pp. 519-524.

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