Mutagenesis of Aspergillus niger for hyperproduction of glucose oxidase to prepare glucose diagnostic kit

By

Muhammad Anjum Zia M.Sc., M.Phil. Biochemistry (UAF)

A Thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

.

In

Biochemistry

Department of Chemistry

Faculty of Sciences University of Agriculture,

Faisalabad, Pakistan.

2007

IN THf: NAME OF ALLAH,

THE MOST MERCIFUL AND 6RACIOUS

, -, OlJl

The Controller of Exa~-,' trol'fs,'

University of Agriculture, Faisalabad,

CJJAIR,MA ~ - Dc:p:;!11!'n~'f1: "rChcmistt University u( Agriculmr Faisulabad ;~-

To

"We, the Supervisory Committee, cenify that the contents and form of thesis submitted by Mr. Muhammad Anjum Zia, Reg. No. 96-ag-831, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of degree".

1. Chairman

unir Ahmad Sheikh)

JA~

Supervisory Committee

2. Member

3.

Member

(Prof. Dr. Iftikhar Ahmad Khan)

.n ... /

DEDICATED

'To

my jlffectionate

(Pjl~w.rS,

my Looinq c5l Carine WIPE c5l

:My Innocent Son

Shahood

[11

ACKNOWLEDGEMENT

All praises and thanks are for one and only, ALMIGHTY ALLAH who is the lord of all universe. Allah created eve,)' thing of the worlds and endowed knowledge to our HOLY PROPHET MUHAMMAD (Peace Be Upon him), for the guidance in every field of life and after life. The blessings of ALLAH and HOLY PROPHET (Peace Be Upon him) are so many but my knowledge and words are limited that could not cover their eve,), aspects.

The completion of this research work would have been a dream ./01' me without the consistent motivation and appreciation by Dr. Khalil-ur-Rahman, Associate Professor of Biochemistry, Department of Chemistry, University of Agriculture, Faisalabad. I will ever acknowledge his dexterous guidance, never-ending moral support and enlightened supervision. Special thanks to Dr. M. Ibrahim Rajoka, TI, Deputy Chief Scientist/Head Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad for providing technical help during my research and thesis.

I would like to express my heartiest gratitude and deep sense of indebtedness to Prof. Dr. Munir A. Sheikh, Professor of Biochemistry: Chairman, Department of Chemistry, University of Agriculture, Faisalabad for his inspiring guidance and extremely kind behavior. I offer my thanks to Prof. Dr. Iftikhar A. Khan, Professor and Chairman, Department of Plant Breeding and Genetics/Director CABB. University of Agriculture, Faisalabad for his nice cooperation and valuable suggestions about my research and thesis.

A great contribution through good wishes from my family members has special appreciations. I offer my feelings of obligations to my great father Prof. Dr. Muhammad Aslam Zia, Professor of Urdu, Govt. College Jhang and kind mother (May Allah grant all my life to them) whose super-moral training and unique impressive life style enabled me to build my personality. This is all because of extreme care and prayers of my parents, grandmother, wire, son, sisters, brothers and in-laws, whose hands are always raisedfor me even at this moment.

May ALLAH grant the above cited personalities with long and happiest life, peace of mind, prosperity, honor and greater heights ..... Aamin!

MUHAMMAD ANJUM ZIA

IV

No.

~L~(

S I/Jutlcro,.

ADVANCED STUD/_~

CON TEN T '\i.l'lIv;t.IY 01 :;:Jilt.,. .;

• '. ,~~ ISAL '8 % if~ r

Title Page l"1f t)~-

e-r- I INTRODUCTION 1
~ II REVIEW OF LITERATURE 8
.--- II! MATERIALS AND METHODS 38
- IV RESULTS AND DISCUSSION 58
"'---V SUl\IlMARY 139
t,i LITERATURE CITED 142 v

TABLE OF CONTENTS

No.

Title

Page

1. Introduction 1

2. Review of literature 8

2.1. Enzyme production by fungi 8

2.2. Glucose oxidase 8

2.3. Glucose diagnostic kit 34

3. Materia1s and Methods 38

3.1. Chemicals and enzyme 38

3.2. Microorganism 38

3.2.1. Counting of spores 39

3.3. Strain improvement techniques 39

3.3.1. Radiation mutagenesis 39

3.3.1.1. Mutagenesis using gamma radiations 39

3.3.1.2. Mutagenesis using UV lamp 40

3.3.2. Chemical mutagenesis 40

3.3.2.1. Mutagenesis using NrNNG 40

3.3.2.2. Mutagenesis using ethidium bromide 41

3.4. Selection of mutant 41

3.4.1. Selection of colony restrictor 41

3.4.2. Selection of3 log kill mutant dose by kill curve 41

3.4.2.l.Calculation of colony forming units (C.F.U. mL-1) 41

3.4.3. Screening procedures 42

3.4.3.1. Plate screening method 42

3.4.3.2. Isolation of mutants by selective marker 42

3.4.4. Identification of mutant 42

3.4.4.1. Enzyme diffusion zone test 42

3.4.4.2. Analytical test 42

3.5. Production of glucose oxidase 42

3.5.1. Preparation of inoculum 42

3.5.2. Enzyme production by liquid-state fermentation 43

3.5.3. Optimization of conditions for glucose oxidase production 43

3.5.4. Sample harvesting 43

3.6. Enzyme assay 44

3.6.1. Preparation of 0.1 M phosphate buffer 44

3.6.2. 1 M nc: 44

3.6.3. I M NaOH 44

3.6.4. 1 % o-Dianisidine 44

3.6.5. 18 % D-glucose solution 44

3.6.6. Peroxidase 44

3.6.7. Dianisidine-buffer mixture 44

3.6.8. Preparation of buffered-substrate solution 45

3.6.9. Preparation of blank solution 45

VI

3.6.10. Procedure 45
3.7. Biomass estimation 45
3.8. Carbohydrate analysis 45
3.8.1. Preparation of ONS reagent 45
3.8.2. Procedure 46
3.9. Estimation of protein contents 46
3.9.1. Preparation of Biuret reagent 46
3.9.2. Procedure 46
3.10. Determination of growth kinetic parameters 46
3.11. Purification of glucose oxidase 48
3.11.1. Ammonium sulfate precipitation technique 48
3.11.1.1. Salting out of other proteins 48
3.11.1.2. Salting out of glucose oxidase 48
3.11.1.3. Desalting of glucose oxidase 48
3.11.2. Purification by ion exchange chromatography 48
3.11.2.1. Preparation of 0.5 N HCI 48
3.11.2.2. Preparation of 0.5 M NaOH 49
3.11.2.3. Preparation of column 49
3.11.2.4. Washing the column with base 49
3.11.2.5. Washing the column with acid 49
3.11.2.6. Equilibration of column 49
3.11.2.7. Application of sample 49
3.11.3. Gel filtration chromatography 50
3.11.3.1. Swelling of the resin 50
3.11.3.2. Filling the column 50
3.11.3.3. Application of sample 50
3.12. Electrophoresis 50
3.12.1. Stock solutions 50
3.12.2. Preparation of resolving gel 51
3.12.3. Preparation of stacking gel 51
3.12.4. Sample buffer 51
3.12.5. Stock electrode buffer 52
3.12.6. Preparation of glucose oxidase for SOS-PAGE 52
3.12.7. Preparation of protein markers ladder for SOS-PAGE 52
3.12.8. Running of PAGE 52
3.12.9. Protein staining of SOS-polyacrylamide gels 52
3.13. Molecular mass determination 53
3.13.1. Native molecular mass 53
3.13.2. Subunit molecular mass 53
3.14. Kinetic and Thermodynamic Studies 54
3.14.1. Optimum pH 54
3.14.2. Optimum Temperature 54
3.14.3. Activation energy (Ea) 54
3.14.4. Determination of Michaelis-Menten constants 54
3.14.5. Irreversible thermal denaturation 54
3.14.6. Activation energy of thermal denaturation 54 Vll

3.14.7. Thermodynamics of irreversible thermal inactivation 55
3.15. Determination of stability 55
3.16. Glucose estimation kit 55
3.16.1. Enzymes concentrations 56
3.16.2. Enzymes schedule 56
3.16.3. Guaiacol concentration 56
3.16.4. Amount of sample 56
3.16.5. Comparison with standard kit 56
3.16.6. Determination of sensitivity of kit 57
3.17. Statistical analysis 57
4. Results and discussion 58
4.1. Kill Curve Determination 59
4.2. Selection and evaluation of mutant 61
4.2.1. Colony Restriction 61
4.2.2. Selection of glucose oxidase hyperproducing 64
Aspergillus niger mutants using 2-deoxy-D-glucose
4.2.3. Selection of specific mutants by enzyme diffusion zone 64
4.2.4. Analytical test 65
4.3. Production of enzyme in shake flask 73
4.3.1. Effect of substrate 73
4.3.2. Effect of fermentation period 74
4.3.3. Effect of pH 75
4.3.4. Effect of temperature 85
4.3.5. Effect of urea 85
4.3.6. Effect of KH2PO" 92
4.3.7. Effect of CaCO:;: 92
4.3.S. Effect of MgS04.7H20 99
4.3.9. Effect of glucose 99
4.3.10. Growth kinetics 106
4.4 Purification of glucose oxidase lOS
4.4.1 Isolation by ammonium sulfate precipitation lOS
4.4.2 Ion exchange chromatography 109
4.4.3 Gel filtration chromatography: 110
4.5 SDS-PAGE 115
4.6 Molecular mass determination 115
4.7 Kinetic and Thermodynamic studies 116
4.7.1 Optimum pH 116
4.7.2 Optimum temperature 1 17
4.7.3 Determination of Michaelis-Menten constants 121
4.7.4 Irreversible thermal denaturation 123
4.7.5 Thermodynamics of irreversible thermal inactivation: 124
4.S Determination of stability 130
4.9 Glucose Estimation Kit 130
4.10 Conclusions 138
5. Summary 139
Literature Cited 142 Vtll

LIST OF TABLES

Table

Page

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

Title

Composition of potato-dextrose-agar medium (PDA) Composition of Vogel's media

Activity of glucose oxidase shown by various mutants by analytical test

Analysis of variance for glucose oxidase production by parental culture at different substrate concentrations

Analysis of variance for glucose oxidase production by mutant culture at different substrate concentrations

Analysis of variance for glucose oxidase production by parental derivative at different fermentation periods

Analysis of variance for glucose oxidase production by mutant derivative at different fermentation periods

Analysis of variance for glucose oxidase production by parent derivative at different pH

Analysis of variance for glucose oxidase production by mutant derivative at different pH

Analysis of variance for glucose oxidase production by parent derivative at different temperatures

Analysis of variance for glucose oxidase production by mutant derivative at different temperatures

Analysis of variance for glucose oxidase production by parent derivative at different urea concentrations

Analysis of variance for glucose oxidase production by mutant derivative at different urea concentrations

Analysis of variance for glucose oxidase production by parent derivative at different levels of KH2P04

Analysis of variance for glucose oxidase production by mutant derivative at different levels of KH2P04

Analysis of variance for glucose oxidase production by parent derivative at different levels of CaCO]

Analysis of variance for glucose oxidase production by mutant derivative at different levels of CaCO]

Analysis of variance for glucose oxidase production by parent derivative at different levels of MgSO.t. 7H20

Analysis of variance for glucose oxidase production by mutant deri vative at different levels of MgSO.t. 7H20

Analysis of variance for glucose oxidase production by parent derivative at different levels of glucose

Analysis of variance for glucose oxidase production by mutant derivative at different levels of glucose

Summary of glucose oxidase production by parent and mutant derived strains

38 39 72

77

78

80

81

83

84

87

88

90

91

94

95

97

98

101

102

104

105

107

ix

4.21

4.22

4.23 4.24 4.25

4.26

4.27

4.28 4.29 4.30

4.31

Data on comparative fermentation kinetic parameters of A. niger and its mutant derivative BCG-4

Comparative fermentation kinetic parameters of A. niger and its mutant derivative BCG-4

Purification summary of glucose oxidase produced by parent strain Purification summary of glucose oxidase produced by mutant strain Kinetic and thermodynamic parameters for irreversible thermal inactivation of glucose oxidase from parental culture of Aspergillus niger

Kinetic and thermodynamic parameters for irreversible thermal inactivation of glucose oxidase from mutant derivative of Aspergillus niger

Analysis of glucose estimation by optimization of enzyme concentrations

Results for glucose estimation after mixing of enzymes (single vial) Results to optimize the amount of guaiacol for glucose estimation Determination of amount of serum glucose in diabetic patient sample

Comparison of glucose estimation through self prepared with standard (Human) kits

107

108

114 114 129

129

134

135 135 136

136

x

LIST OF FIGURES

Figure

Page

Title

1.1

4

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

Enzymatic conversion of glucose to gluconic acid by glucose oxidase

Computer model of glucose oxidase 3D structure

Standard curve of glucose for carbohydrates estimation by DNS method

Standard curve of bovine serum albumin for protein estimation by Biuret method

Aspergillus niger (Mycelial growth) Typical characteristic spore) Effect of y-radiation to formulate the survival curve

Effect 0 f UV -radiation to formulate the survival curve

Effect ofMNNG to formulate the survival curve

Effect of ethidium bromide to formulate the survival curve Screening of glucose oxidase hyperproduced by gamma rays using 2-deoxy-D-glucose resistant mutant derivative

Screening of glucose oxidase hyperproduced by UV -rays induced mutant resistant to 2-deoxy-D-glucose

Screening of glucose oxidase hyperproduced by MNNG induced mutant resistant to 2-deoxy-D-glucose

Screening of glucose oxidase hyperproduced by ethidium bromide induced mutant resistant to 2-deoxy-D-glucose

Selection of glucose oxidase hyperproducing gamma rays induced mutants by enzyme diffusion zone

Selection of glucose oxidase hyperproducing mutants by enzyme diffusion zone

Production of glucose oxidase by parental culture with varying substrate levels

Production of glucose oxidase by mutant culture with varying substrate levels

Effect of fermentation period on enzyme production by parental culture

Effect of fermentation period on enzyme production by mutant culture

Optimization of pH for maximal yield of glucose oxidase by parental culture

Optimization of pH for maximal yield of glucose oxidase by mutant culture

Production of glucose oxidase at varying temperatures by parental culture

Production of glucose oxidase at varying temperatures by mutant culture

Production of glucose oxidase with varying levels of urea by parental culture

6 47

47

59 62 62 63 63 66

67

68

69

70

71

76

76

79

79

82

82

86

86

89

Xl

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

Production of glucose oxidase with varying levels of urea by mutant culture

Optimization of KH2P04 for glucose oxidase production by

parental culture

Optimization of KH2P04 for glucose oxidase production by mutant

culture

Production of glucose oxidase with varying levels of CaCO] by parental culture

Production of glucose oxidase with varying levels of CaCO] by mutant culture

Production of glucose oxidase with varying concentrations of MgS04.7H20 by parental culture

Production of glucose oxidase with varying concentrations of MgS04.7H20 by mutant culture

Effect of glucose on production of glucose oxidase by parental culture

Effect of glucose on production of glucose oxidase by mutant culture

Isolation of glucose oxidase by ammonium sulfate precipitation from parental fungus

Isolation of glucose oxidase by ammonium sulfate precipitation from mutant fungus

Ion exchange chromatography of glucose oxidase from parental A. niger

Ion exchange chromatography of glucose oxidase from mutant derived A. niger BCGA

Gel filtration chromatography of glucose oxidase from parental culture of A. niger

Gel filtration chromatography of glucose oxidase from mutant culture of A. niger BCG-4

Determination of molecular weight of mutant derived glucose oxidase by Sephadex

Effect of pH on parent derived glucose oxidase activity Effect of pH on mutant derived glucose oxidase activity

Effect of temperature on parent derived glucose oxidase activity Effect of temperature on mutant derived glucose oxidase activity Arrhenius plot for activation energy of substrate oxidation for parent derived enzyme

Arrhenius plot for activation energy of substrate oxidation for mutant derived enzyme

Effect of substrate on parent derived glucose oxidase activity

Effect of substrate on mutant deri ved glucose oxidase acti vity Melting temperature of glucose oxidase purified from parental A. niger

Melting temperature of glucose oxidase purified from mutant derivative of A. niger

89

93

93

96

96

100

100

103

103

I 11

I I I

112

112

113

I 13

116

118 118 119 1 19 120

120

122 [22 126

126

XI!

4.47 4.48 4.49

4.50

4.51 4.52 4.53 4.54 4.55

Irreversible thermal denaturation of parent derived glucose oxidase Irreversible thermal denaturation of mutant deri ved glucose oxidase Arrhenius plot for activation energy of thermal denaturation of parent derived enzyme

Arrhenius plot for activation energy of thermal denaturation of mutant derived enzyme

Effect of copper chloride on mutant derived enzyme stability Effect of sodium fluoride on mutant derived enzyme stability Effect of silver sulfate on mutant derived enzyme stability Effect of zinc sulfate on mutant derived enzyme stability Determination of sensitivity of glucose kit

127 127 128

128

131 131 132 132 137

Xlll

LIST OF ABBREVIATION & SYMBOLS

ABTS:

A. niger:

ATP:

DNA:

FAD:

Fig.:

GOxJGOD:

GRAS:

HPLC:

RNA: [J [h-1]:

T [K]:

a:

p:

H202: rpm:

MNNG:

NTG:

EB:

UV:

Co:

ROS:

IR:

NFCCP: mL:

ilL:

CFU:

°C: g:

L:

PDA: ill:

U: mg: mm:

DNS:

Yx;s:

Qs Qp:

YPiS:

YP/X: qs: qp:

2,2' -Azino-di-[3-ethylbenzthiazolin sulfonate] Aspergillus niger

Adenosin triphosphate

Deoxy ribonucleic acid

Flavin adenine dinucleotide

Figure

Glucose oxidase

Generally Regarded As Safe

High Performance Liquid Chromatography Ribonucleic acid

Specific growth rate

Absolute temperature

Alpha

Beta

Hydrogen peroxide

revolutions per minute N-methyl-N-nitro-N-nitrosoguanidine N i tro- N -nitro so guanidine

Ethidiurn bromide

Ultraviolet

Cobalt

Reactive oxygen species Infra red

National Fungal Culture Collection of Pakistan Milli liter

Micro liter

Colony forming units Degree Celsius

Gram

Liter

Potato dextrose agar International unit Unit

NElli gram

Milli meter Dinitro-salicylic acid Growth yield coefficient

Rate of Substrate consumption Volumetric rate of product formation Product yield

Specific product yield

Specific rate of substrate consumption Specific rate of enzyme production

XIV

M:

N: w/v: v/v:

SDS: kD: 0:

PAGE: v:

v;

Vi:

e;

Vmax:

x ; kca!: u;«; Kd:

H: kI3:

R:

N:

T:

I1H*:

I1G*:

I1S*:

EMS:

CSL:

ANOVA:

Molar

Normal Weight/Volume VolumeNolume Sodium dodecyl sulfate Kilo Dalton

Dalton

Polyacrylamide gel electrophoresis

Elution volume/retention time (glucose oxidase or protein marker) Void volume/retention time of blue dextran

Total internal volume/retention time of tyrosine

Activation energy

Maximum velocity

Michaelis-Menten constant

Catalytic constant

Specificity constant

Irreversible thermal denaturation

Planck's co nstant > 6.63 x 10-34 Js Boltzman's constant (R/N) = 1.38 x 10-23 JK-1 Gas constant = 8.314 JK-1 mor'

Avogadro's No. "" 6.02 X 1023 morl

Absolute temperature

Enthalpy of activation of denaturation Free energy of activation of denaturation Entropy of activation of denaturation Ethyl methane sulfonate

Corn steep liquor

Analysis of variance

xv

Chapter 1

INTRODUCTION

Diabetes mellitus is a metabolic problem and is prevalent in many parts of the world. In developed countries, the incidence rate is 5% and an equal number is liable to develop the disease (McKee and McKee, 1996). One fundamental aspect of diabetes is an abnormality of glucose metabolism as due to insufficient action of insulin, owing either to its absence or to resistance in action (Murray et al., 2000). 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).

Three enzymes mutarotase, glucose oxidase and peroxidase are involved in the process of determination of glucose level. The overall mechanism of the reaction is as follows:

a-D-glucose Mutarotase

------------------.

P-D-glucose Glucose oxidase ,.

2H202 Peroxidase

~~~==~-------- ••

P-D-glucose 8-D-gluconolactone + H202 2H20 + O2

The filamentous fungi, typically, are saprophytic microorganisms, which secrete a wide variety of enzymes involved in the breakdown and recycling of complex biopolymerslbiomolecules from both plant and animal tissues. Although the majority of these enzymes are hydrolytic and play an important role in fungal nutrition, releasing carbon and nitrogen locked in insoluble macromolecules obtained from the metabolic activities of other organisms. This makes the filamentous fungi as host for the production of heterologous proteins (Jeenes et al., 1991).

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 a lot of organic acids, enzymes, plant growth regulators, mycotoxins and antibiotics. The industrial importance of A. niger is not only limited to its more than 35 native products but also on the development and commercialization of the new products

which are derived by modern molecular biology techniques (Swoboda and Massey, 1965).

During the past few years numerous studies have been presented on A. niger, presumably the most important fungus for production and secretion of protein. The employment of A. niger as a host organism for production and secretion of homologous and heterologous proteins demonstrates many advantages such as:

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

>- A. niger has a long history of usage within the fermentation industry and is generally regarded as safe (GRAS). This often facilitates the path toward regulatory improvement of the production system.

>- 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 capable of carrying out efficient post-translational modifications of products, e.g, gIycosylation. This is especially important for some proteins derived from higher eukaryotes.

;.. A. niger 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.

>- Mutant stability is relatively high, therefore the threat of revertants 1S less pronounced.

Conversely a number of points can be made against Aspergillus as a potential system for protein production, which includes:

>- The capability of Aspergillus to form the complex morphological, mycelial and pellet structures, which are usually associated with respect to mass and heat

2

transfer limitation, mixing and monitoring the process as the concentration of the biomass increases.

:r Aspergillus secretes significant quantities of other products such as organic acids, which may reduce the pH of the medium. This represents a diversion of the carbon from the desired activity. A second possible difficulty lies in the effect on the protein being secreted into a low-pH environment. This may cause modification or even denaturation of the protein.

};- Aspergillus is also capable of producing extracellular proteases, which not only damage the product directly but also contaminate the final products. So, proteases require an additional purification step.

:r The production of fungal toxins should be also considered when using Aspergillus strains (Bosch et al., 1995).

Several microbial enzymes are known which have the capability of oxidizing glucose. Out of these, glucose oxidase also knO\\'11 as P-D-glucose: oxygenoxidoreductase (EC 1.t.3.4). is of commercial interest, produces D-glucono-I,S-lactone and a reduced acceptor (Crueger and Crueger, 1990). Glucose oxidase is FAD dependent glycoprotein catalyzing the oxidation of P-D-glucose to glucono-I ,S-lactone. It removes hydrogen from glucose and reduces itself, which is then re-oxidized by molecular oxygen. The developed hydrogen peroxide is decomposed by peroxidase or catalase to water and oxygen giving the net reaction as shown in Fig. I (Gibson et al., 1964; Duke et al., 1969).

The reaction catalyzed by glucose oxidase is:

P-D-glucose + Enzyme-FAD .. Enzyme-FADH2 + o-D-gluconolactone

Enzyme-FADH2 + O2

3

H20 + 1/2 Crz t

09-

I

: Colaho;,u I

,

O=l~

H-I---~~ I

f-K) _c_ H 0 + GOO·F.ADH2.

H-~ OH I

H-!

I

HCt-i2C

Q-K) ~b-H I

H_C-~ +GOO·FAD

~b_H I

HO--c-H

I

CH2O-i

P..- D-glucose

D-\1ILlcono.-1 ,5-loctone

! 1120

1-0 0

-C'

I

I-O-C- H

I

H -C-C4--I

r-o-!-H ,

I-O-C-H

~

CH20H

Gluconic Z1c1d

Fig. 1.1.

Enzymatic conversion of glucose to gluconic acid by glucose oxidase

(Rando et al., 1997).

The importance of glucose oxidase comes from its wide applications in many fields in crude/purified form or by using the producer strain for important uses, as some are as follows:

4

};- In food industries, glucose oxidase is used to remove the glucose and oxygen from beverages, powdered eggs and as a source of hydrogen peroxide in food preservation. Moreover, it works as a stabilizer for some food additives such as ascorbic acid and vitamin BI2 (Baldwin 01 al., 1953; Begliomini et 01., 1995; Liu el 0/., 1999; Petruccioli el 01" 1999).

~ Glucose oxidase is used for the production of beer, wine and soft drinks, in which its reaction serves an antioxidant function (Witt et al., 1998; Malherbe et 0/., 2003; Zoldak et 01" 2004), improve their shelf life (Park et al., 2000; Kapat et 01" 2001) and to maintain flavor and color stability (Power, 1998; Vemulapalli et 01., 1998),

~ Glucose oxidase has also a main role in gluconic acid production by A. niger (Petruccioli et 01" 1999).

};- In pharmaceutical and analytical biochemistry, it is used for quantitative determination of glucose in biological fluids. Glucose "dip-sticks" became available for screening of blood/urine glucose by coupling the reaction to peroxidase and a chromogen (Worthington, 1988; Petruccioli et al., 1999). Glucose oxidase use in diagnostic assays accounts for more than 8% of the total yearly budget of the enzymatic kit market worldwide, about US$ 61 million (Ferreira et 01" 2005).

':;- In manufacturing of glucose biosensors (Worthington, 1988; Tsuchiya et al. 2005).

J;;. It has been used an ingredient of toothpaste (Richter, 1983).

);> Glucose oxidase is also used in textile industry and environmental monitoring as discussed by Zoldak el al. (2004),

':;- The production of fructose from sucrose can be achieved by treatment with glucose oxidase after pre-incubation with invertase. The resulting gluconate can be separated more easily from fructose as it would be the case with glucose (Hayashi and Nakamura, 1981).

The enzyme, glucose oxidase, shows a very high degree of specificity for P-Dglucose although 2-deoxy-D-glucose, D-mannose and D-fructose are also oxidized, but at a much reduced rate (Hamid et 01., 2003). Glucose oxidase is highly specific for P-D-

5

glucose while cc-anomer is not acted upon (Bentley, 1955). Glucose oxidase from Aspergillus niger is a highly glycosylated protein, the carbohydrate content is ranged from 10 to 24% of its molecular weight (Pazur et al., 1965; Hayashi and Nakamura, 1981).

Glucose oxidase was first isolated from mycelia of Aspergillus niger and Penicillium glaucum by Muller (1928). Now-a-days, the industrial production of glucose oxidase is carried out using both Aspergillus niger and Penicillium amagasakiense. Beside these two fungi, many other microorganisms were recorded as glucose oxidase producers such as Phanerochaete chrysosporium (Kelley and Reddy, 1986), Talaromyces flavus (Kim et al., 1990), Penicillium variabile (Petruccioli and Federici, 1993), Penicillium expansum, Penicillium italiacum (Petruccioli et al., 1993), Penicillium notatum, Penicillium paxilli (Fiedurek et al., 1986) and Penicillium pinophilum (Rando, et al., 1997).

(a) (b)

Fig. 1.2. Computer model of glucose oxidase 3D structure (Raba and Mottola, 1995).

(a) Overall topology of glucose oxidase holoenzyme

(b) Subunit structure of glucose oxidase showing FAD

6

Traditionally, strain development requires painstaking, lengthy and tedious procedures to identify superior isolates among a mutagen-treated population. Several attempts have been made to improve glucose oxidase production by strain selection using classical screening and mutagenesis techniques. Special environmental conditions, toxic to the majority of the cell type (wild type) but less toxic or non-toxic to a desired minority of cells (mutant), have often been applied to enrich a cell population to obtain desired mutants (Direct selection). The greatest advantage of this screening method is its simplicity that does not require any profound understanding of the molecular biology and physiology of the microorganisms being manipulated (Gromada and Fiedurek, 1997).

Aims and Objectives:

;. To produce the mutant of Aspergillus niger for the hyperproduction of glucose oxidase.

'y Optimization of conditions for the production of glucose oxidase. ;. Thermal Characterization I kinetics of glucose oxidase.

7

chapter 2

REVIEW OF LITERATURE

2.1 Enzyme production by fungi

J eenes et al. (1991) stated that filamentous fungi, typically, are saprophytic microorganisms, which secrete a wide array of enzymes involved in the breakdown and recycling of complex biopolyrners from both plant and animal tissues. Although the majority of these enzymes are hydrolytic and play an important role in fungal nutrition, releasing carbon and nitrogen locked in insoluble macromolecules obtained from the metabolic activities of other organisms. This makes the filamentous fungi as hosts for the production of secreted heterologous proteins.

2.2 Glucose oxidase

Zetelaki and Vas (1968) have investigated the effect of aeration and agitation on glucose oxidase production by A. niger in a 5 liter stirred tank bioreactor. They found that the maximum enzyme production was achieved at 700 rpm. Further increase 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 1.5 fold and the glucose oxidase production was doubled compared to the aerated culture.

O'Malley and Weaver (1972) in their pioneer work on glucose oxidase reported that enzyme from Aspergillus niger is a branched protein containing 16 carbohydrate and two flavin-adenine dinucleotide cofactors per molecule. The results of these studies indicated that glucose oxidase is a subunit enzyme. The native enzyme has a molecular weight of 160,000 and light-scattering measurements in 6 M guanidine hydrochloride indicated the denatured enzyme has the same molecular weight as the native enzyme. However, chemical reduction with p-mercaptoethanol of the enzyme's two disulfide bonds resulted in the formation of molecular species with molecular weights of 80,000. Schlieren patterns taken during sedimentation velocity runs show single sharp

8

sedimentation peaks for both the denatured and the denatured-reduced species. These data are consistent with a model for glucose oxidase in which the enzyme is composed of two polypeptide chains, equal in molecular size, which are covalently linked by disulfide bonds.

Nakamatsu e/ al. (1975) studied the effect of different complex carbon sources as well as different nitrogen sources on glucose oxidase production. Among eight sources of widely differing natural carbon sources, they found that beet molasses was the best carbon source to support growth and glucose oxidase production. On the other hand, nitrate and urea gave a better glucose oxidase yield than ammonium salts.

Hayashi and Nakamura (1976) studied the glucose oxidase from two fungal genera (Aspergillus and Penicillium) chemically, physicochemically and immunologically elucidated the similarities and dissimilarities between these enzymes. Investigation of circular dichroism spectra revealed that these enzyme proteins possess essentially identical conformations. However, differences found in thermal inactivation parameters, catalytic parameters and quantitative immunological reactivities indicated that these enzymes must have some minor but distinct variations in their structures. Interestingly, it was observed that the Penicillium enzyme cross-reacted with the antiserum against the Aspergillus enzyme with an association constant of two orders of magnitude lower than that of the Aspergillus enzyme, and that the precipitin line of the Penicillium enzyme fused together with that of the Aspergillus enzyme in the immunodouble diffusion test. These results lead to the conclusion that these enzymes Eire closely related but not completely identical, and suggest that they might have evolved from a common ancestral precursor.

Musilkova et al. (1978) reported that N-methyl-N-nitro-N-nitrosoguanidine (J\.11\JNG) strongly influences the variability of Aspergillus niger MU-90, particularly on long term treatment. The number of spores capable of growth, decreased with the duration of treatment while morphological and biochemical mutants considerably increased. The high number of mutants with increased organic acid production was obtained after mutagenic treatment when the number of surviving spores decreased significantly.

9

McKee and Lawrence (1979) found that partially different sets of gene functions were required for the production of different kinds of mutations induced by Co6Q rays in Saccharomyces cerevisiae. This observation was very similar to others made previously with respect to UV mutagenesis and confirmed the conclusion that such distinctive patterns of genetic control reflect properties of the test alleles and their genetic locations, rather than the kinds of lesions required to revert them.

Bruce (1985) particularly emphasized to targeted and non-targeted gamma-ray processes that involved the generation of reactive oxygen species (ROS) because of the roles ROS may play in mediating DNA damage, cell cycle related effects and genomic instability. The extent, quality and repair of DNA damage induced by basal oxidative metabolism and low dose/low dose rate IR in the presence and absence of IR-induced increases in cell-mediated ROS production and IR-associated intracellular ROS increases that occur as a bystander effect. elucidated the molecular and biochemical pathways that underlie ROS and cell cycle responses. Moreover it was determined that how the ROS and cell cycle responses in irradiated and bystander cells affect cell growth, radioresistance and genomic stability following subsequent bouts of exposure to JR.

Kelley and Reddy (1986) reported that glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion exchange and molecular sieve chromatography. The enzyme found to be a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme did not appear to be a glycoprotein. It gave optimal activity with D-glucose, which was stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It was inhibited by Ag + (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF or KCN (each 10 mM).

Fiedurek et al. (1987) reported that as a result of mutagenization and passaging on 2-deoxy-D-glucose containing medium, I 0 Aspergillus niger strains resistant to this agent were obtained. These showed (with one exception) an increase in the activity of glucoamylase, the level of which ranged widely in individual cases from several to over 200% in comparison with the parent strain. A weaker rate of glucose accumulation in derepressed strains may account for the fact that the mechanism of their resistance to

10

deoxy-glucose is connected with disturbance of the system of glucose transport. Apart from the biochemical character of the catabolic de-repression, it seems that it can already be successfully utilized to increase the productivity of industrial mould cultures.

Frederick et at. (1990) cloned the gene for Aspergillus niger glucose oxidase from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consisted of 583 amino acids and was preceded by a 22 amino acid pre-sequence. The enzyme contained 3 cysteine residues and 8 potential sites for N-linked glycosylation. Recombinant yeast expression plasmids were constructed containing a hybrid yeast alcohol dehydrogenase 11- glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase pre-sequence and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids directed the synthesis and secretion between 75 and 400 ~g mL-1 of active glucose oxidase. Analysis of the yeastderived enzymes showed that they were of comparable specific activity and had more extensive N-linked glycosylation than the A. niger protein.

Kim et al. (1990) produced Talaromyces flavus glucose oxidase, which may be involved in biocontrol of the fungal plant pathogen, Verticil/fum dahliae. A strain of T. flavus was selected from the wild type population for the production of extracellular glucose oxidase and the enzyme was purified by a combination of acetone precipitation and high performance liquid chromatography. Approximately 12-25 mg of pure protein was obtained from 2 L of culture and the total recovered activity ranged from 5 to lOx I o' ~ mol min". Homogeneity of the purified enzyme was demonstrated by HPLC and by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was 164,000 and that of the subunit was 7 1,000, which indicated that the native enzyme is a dimer. The apparent Km value for D-glucose was 10.9mM.

Fiedurek (1991) showed that the effect of various kinds of starch, as the sole source of organic carbon on the biosynthesis of glucose oxidase by Aspergillus niger GIV-IO (a mutant strain). A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium)

11

was developed that increased 2-3.8 times glucose oxidase yield, as compared to that described earlier.

The production of glucose oxidase was carried out successfully using immobilized cells. Fiedurek and llczuk (1991) studied the glucose oxidase production by A. niger immobilized on sintered glass, rasching rings, pumice stones and polyurethane foam for extracellular glucose oxidase production. Glucose oxidase produced by the immobilized cells was 2.7 times higher than that of free cells.

Fiedurek and Ilczuk (1992) worked out that among 1486 mold strains isolated from natural sources (screened for extracellular glucose oxidase) only 119 (Aspergillus and Penicillium) showed enzyme activity. As the best glucose oxidase producer, A. niger 0-1 was isolated from decaying tree. The dynamics of glucose oxidase synthesis in A. niger 0-1 during its culture by submerged method showed that the intracellular activity of the enzyme was 10 times higher than its extracellular level. Some properties of the crude glucose oxidase preparation, isolated from the postcuIture liquids by lyophilization, were examined.

Hodgkins et af. (l993) studied the glucose oxidase gene from Aspergillus niger, expressed in Hansenula polymorpha. The mutant strain was grown to high cell density by fed-batch fermentation. At the end of fermentation, the culture density was 76 g dry weight L-1 and 108 IV mL-1 glucose oxidase was found in the culture medium. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants, which showed abnormal regulation of glucose oxidase expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g L-1) and produced 445 IU mL-1 extracellular and 76 IU mL-1 intracellular enzyme. The mutant thus not only increased total production but also exported 83% of the total enzyme made compared to 55% in the parental strain.

Shin et £II. (1993) purified glucose oxidase upto 27.5 fold to apparent homogeneity with an overall yield of 23.8% from Pleurotus ostreatus, through a purification procedure of ammonium sulfate precipitation, gel permeation, anion exchange and hydrophobic-interaction chromatography. The molecular mass determined by gel filtration was found to be 290 kDa. SDS-PAGE revealed that the enzyme consists

12

of four subunits with a molecular mass of 70 kDa. The absorption spectra of the enzyme exhibited maxima at 280, 360 and 460 nm. The enzyme showed a fluorescence spectrum with an excitation maximum at 470 nm and an emission maximum at 530 nm. The enzyme was optimally active at 50°C and at pH 5.5·6. It exhibited broad affinity for various sugars and specificity for D-glucose with K; value of 1.34 mM. The enzyme was completely inhibited by mercuric chloride and partially by silver sulfate, sodium azide and 8·hydroxyquinoline.

Witteveen el al. (1993) reported the induction of glucose oxidase, catalase and lactonase activities studied both in wild type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various mutations. Glucose and a high oxygen level were necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effected regulation at the transcriptional level. The mutation resulted in constitutive expression of all three activities although modulated to some extent by the carbon source. The mutation only had an effect on lactonase and glucose oxidase expression and did not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain.

Fiedurek et al. (1994) used a new method for glucose oxidase production by Aspergillus niger. Conidia immobilized on seeds were also studied by the adsorption of Aspergillus niger. Spores on wheat seeds are very simple and inexpensive method of inunobilization and continued production of glucose oxidase was observed for 8 repeated batches.

Petruccioli el al. (1994) studied glucose oxidase production by Penicillium variabile P 16 immobilized on different carriers. Among different carriers, polyurethane proved to be the best for glucose oxidase production and the production continued for 7 repeated batches.

Hatzinikolaou and Maoris (1995) studied the certain factors affecting the production of extracellular and cell bound glucose oxidase by Aspergillus niger. The intension was to maximize total glucose oxidase activity of academic and potential commercial application by the selection of the appropriate strain and consecutive

13

optimization of growth media and conditions. It was possible to identify combination resulting in the utilization of molasses as the best carbon source and enhancing enzyme activity approximately 40 fold. Glucose oxidase activities as high as 5.7 U mL-1 were produced comparing favorably with those reported for other enzyme producing microorganisms. These activity levels were obtained with molasses indicating an economically attractive process for enzyme production in addition their work identified CaC03 as a particularly strong inducer of glucose oxidase activity.

Hellmuth et al. (1995) transformed wild type Aspergillus niger NRRL-3 with multiple copies of glucose oxidase structural gene. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to 4 times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 g Cl) to a mineral salt medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 30 and 50%, respectively.

Fiedurek and Gromada (1996) reported that a strain of Aspergillus niger mutant, most effective for simultaneous production of catalase and glucose oxidase was selected out of molds belonging to 15 different species by the method of test tube micro-culture. Conidia of the selected strain were subjected to mutagenesis with both UV and M1\TNG and the products were analyzed for catalase activity with their own diffusion plate methods. Among 1055 strains isolated after mutagenesis, eight showed higher catalase (from 44.4-102.1 %) and glucose oxidase (from 8.9-77%) activities than the starting Aspergillus niger mutant.

Gromada and Fiedurek (1996) reported the effect of some medium components and metabolic inhibitors on glucose oxidase production by mutant Aspergillus niger studied in shaken culture. Altering the composition of the basal medium, particularly substitution of (NH4)2HP04 for NaN03, and lack of Mg2+ ions caused an increase in glucose oxidase activity by 269.6%. The highest yield of extracellular glucose oxidase activity of A. niger mycelium was obtained in the presence of hematin (I mM), choline (40 mjvl) and Tween 80 (0.1 %), which improved the activity of this enzyme by about 31.4-53.9%. A significant increase (68.3%) in intracellular glucose oxidase activity was fOW1d in the presence of sodium orthovanadate (I mM). The time course of growth and enzyme production by A. niger in the optimized medium was also reported.

14

Hatzinikolaou et al. (1996) isolated and characterized a new glucose oxidase from Aspergillus niger that showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up and downstream of the open reading frame was much less, showing only 66% and 50% identity, respectively. Despite the low degree of similarity between the promoter region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, they showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6 phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

Gromada and Fiedurek (1997) obtained mutant Aspergillus niger strains resistant to certain agents after rnutangenizaticn and selection. Seven of the mutants showed increased extracellular glucose oxidase. The time course of growth and enzyme production by the most active mutant AM-II showed intra and extracellular glucose oxidase activities to have increased about 2.2 and 2.4 fold, respectively compared with the parental strain.

Kapat el al. (1997) expressed Aspergillus niger glucose oxidase II1 Saccharomyces cerevisiae under the regime of GAL-I 0 promoter and GAL-7 terminator of S. cerevisiae and a-amylase signal sequence of Aspergillus oryzae. The enhancement of the expression level was achieved in pH-stat feed back controlled fed-batch culture. The highest titre of extracellular glucose oxidase was 199 U mL-1 which marked 2 fold improvement over the batch (95 U mL-1) and 28% above that of non-feed back controlled fed-batch (154 U mL-1) operation.

Kohen et al. (1997) characterized three glycoforms of glucose oxidase, which varied in their degree of glycosylation and resulting molecular weight. Focusing on 2- deoxyglucose to probe the chemical step, they measured the temperature dependence of competitive HIT and OfT kinetic isotope effects and the enthalpy of activation using [1-

15

2HJ-2-deoxyglucose. The DIT isotope effect on the Arrhenius pre-exponential factor (AD/AT) is 1.47 (±0.09), 1.30 (±0.10) and 0.89 (±0.04) for the 136, 155, and 205 kDa glycoforms, respectively. The value obtained for the 136 kDa glycoforrn was well above the range expected for semiclassical-classical (no tunneling) reactions (upper limit of 1.22). The abnormal A(D)/A(T) was rationalized by extensive tunneling. The enthalpies of activation are 8.1 (±0.4), 11.0 (±0.3), and 13.7 (±0.3) kcal mol" for the 136, 155, and 205 kDa glycoforrns, respectively. Apparently, less glycosylation resulted in more tunneling and a lower enthalpy of activation. The crystal structure, kinetic analysis, and other studies suggested that the enzyme active site was not conformationally changed by the degree of glycosylation. Hence, the differences among the glycoforrns, which indicated that changes in the enzyme polysaccharide envelope led to a significant change in the nature of the hydrogen transfer step and suggested a dynamic transmission of protein surface affects to the active site.

Rando et al. (1997) investigated a number of nutritional factors influencing growth and glucose oxidase production by a newly isolated strain of Penicillium pinophilum. The most important factors for glucose oxidase production were the use of sucrose as the carbon source and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3, cation exchange and gel filtration chromatography. The relative molecular mass of native glucose oxidase was determined to be 1,54,700-4970, and 77,700 for the denatured subunit. Electron microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5-8 nm. Glucose oxidase was a glycoprotein that contained tightly bound FAD with an estimated stoichiometry of 1.76 mol mol" enzyme. The enzyme is specific for D-glucose, for which a Km value of 6.2 mM was determined. The pH optimum was determined in the range pH 4-6. Glucose oxidase showed high stability on storage in sodium citrate (pH 5) and in potassium phosphate (PH 6), each 100 mM. The half life of the activity was considerably more than 305 days at 4 and 30°C and 213 days at 40°C. The enzyme was unstable at temperatures above 40°C in the range pH 2-4 and at a pH above 7.

Fiedurek (1998) cultured a mycelium of Aspergillus niger under various osmotic stresses then total catalase activity was increased with increasing concentration of NaCI,

16

up 0.4 M, but glucose oxidase under this condition significantly decreased. Mycelial growth was repressed with increased media osmolarity. To release periplasmic glucose oxidase 72 hours old mycelium was suspended on a concentrated solution of NaCI (0.4 -

2.8 M). The highest yield of glucose oxidase activity was obtained at NaCI concentration of 1.2 M at pH 6, which improved the activity of this enzyme by about 2.1 fold in comparison with the control medium without this depressor.

Fiedurek et al. (1998) evaluated the production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants of A. niger. Optimization of stirrer speed, time cultivation and buffering action of some chemicals for glucose oxidase, catalase and gluconic acid production by the most active mutant At\!1-11, grown in a 3-L glass bioreactor was investigated. It appeared that 300 rpm was optimum to ensure good growth and best glucose oxidase production. but gluconic acid and catalase activity obtained maximal value at 500 and 900 rpm, respecti vely.

Liu et al. (1998) purified the H202 producing glucose oxidase from the phytopathogenic fungus Botrytis cinerea to homogeneity by anion exchange chromatography and chromatofocusing. The enzyme had its pH optimum at 7.5 and an isoelectric point of 4.2. The enzyme appeared to be a tetrarneric protein with a native molecular weight of about 160 kDa. Like the glucose oxidase of Phanerochaete chrysosporium, the glucose oxidase of B. cinerea was not glycosylated. Analysis of substrate specificity confirmed that P-D-glucosc was the specific substrate of the enzyme. The expression of the glucose oxidase of B. cinerea was induced by low glucose concentration in the culture medium. In this respect, glucose oxidase of B. cinerea was also similar to the glucose oxidase of P. chrysosporium and different from the glucose oxidases of Aspergillus and Penicillium, which requireed high glucose concentrations for expression.

Witt et al. (1998) reported that gene coding for Penicillium amagasakiense glucose oxidase was cloned by peR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasm ids were constructed from the heat-induced pCYTEXPI expression vector containing the mature

17

glucose oxidase coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per mL of E. coli culture containing more than 60% inactive glucose oxidase. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100 fold dilution to a final protein concentration of 0.05-0.1 mg mL-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide and glycerol. Reactivation followed first order kinetics and was optimal at 10°C. The reactivated recombinant glucose oxidase was purified to homogeneity by mild acidification and anion exchange chromatography. Up to 12 111g of active glucose oxidase could be purified from alL E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense glucose oxidases, The purified enzyme had a specific activity of 968 U mg' and exhibited kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native glucose oxidase from P. amagasakiense. In contrast to the native enzyme, recombinant glucose oxidase was nonglycosylated and contained a single isoform of pI 4.5. They further concluded that this was the first reported expression of a fully active, non-glycosylated form of a eukaryotic, glycosylated glucose oxidase in E. coli.

EI-Enshasy et af. (1999) investigated 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 glucose oxidase excretion by recombinant Aspergillus niger NRRL 3 (GOD 3-18). It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density resulted in higher yields of exocellular glucose oxidase. The pellets obtained in shake flask cultures, showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation were also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation OCCUlTed in two steps. Firstly, aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network. Secondly, aggregation of the primary aggregates to the final full size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with

18

respect to the production of exocellular glucose oxidase. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.

Liu et al. (1999) observed the synthesis of glucose oxidase by Aspergillus niger and noted that calcium carbonate induced the synthesis of enzyme while calcium chloride inhibited it. The optimal pH for the biosynthesis of glucose oxidase was 6. The biosynthesis of enzyme was promoted by MnCO:;, thioglucolic acid, pyroracemic acid and gluconic acid.

Petruccioli el al. (1999) studied the effects of the polysaccharides alginate and locust bean gum and oligosaccharides, oligornannuronate (OM) and oligoguluronate (OG) on glucose oxidase production by Penicillium variabile P16. Small increase observed when the cultures were supplemented with OG and OM blocks with an average degree of polymerization (DP) of approximately 10. With 200 mg i.' OM blocks addition at 0 hour, the increase reached to 32.1 % compared with the control. However, regardless of the time of addition, large increases (up to approximately 70%) in glucose oxidase production were obtained with 100 and particularly 200 mg i.' of alginatederived oligosaccharides (OG and/or OM blocks) with a DP of approximately 7. Where as no significant influence was observed on mycelial biomass.

Ray and Banik (1999) reported that of the factors tested, the source and concentration of carbon and nitrogen in the medium exerted maximum effect on growth and acid production. Glucose (15%) and urea (0.14%) induced glucose oxidase synthesis and optimum yield of calcium gluconate. KH2P04 (0.2%) and MgS04 (0.06%) stimulated glucose oxidase activity and calcium gluconate production. Borax at a concentration of 1.5 g L-1 induced maximum glucose oxidase and calcium gluconate production with increased glucose utilization.

Rothberg et al. (1999) worked on optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 L laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those

19

performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for A. niger strain used, was accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.

Fiedurek and Gromada (2000a) carried out the work on production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture. They showed that maximum oxygen concentration occurred in 50 mL of the medium containing 0.2% glucose at pH 5.

Fiedurek and Gromada (2000b) used the mutant strain of Aspergillus niger AM- 11 producing high glucose oxidase and catalase. A novel method for increasing dissolved oxygen concentration in culture media was developed. It involved adding H202 to the media, which was then decomposed to oxygen and water by catalase. Maximal oxygen concentration occurred in 50 mL of the medium containing 0.2 g wet mycelium and 0.2 % glucose at pH 5. A significant increase of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable.

Kapat et al. (2000) used recombinant strain of Saccharomyces cerevisiae harboring glucose oxidase gene originated from Aspergillus niger for the production of extracellular glucose oxidase. The effect of continuous galactose feeding on the induction of GAL-I 0 promoter was examined in a 5 L bioreactor. The highest enzyme production level (164 U ern -3) was achieved at 96 hours of cultivation. The production performance was compared with the results of fed-batch cultivations carried out in the same laboratory. Continuous feeding mode was found to be less productive due to excess ethanol formation and plasmid instability.

Park et al. (2000) cloned glucose oxidase gene of Aspergillus niger into the yeast shuttle vector YEp352 with combinations of various promoters and terminators, and then used to transform Saccharomyces cerevisiae. Expressed glucose oxidase was successfully secreted into culture medium due to the presence of the intrinsic signal peptide of glucose oxidase. Four different promoters fused to glucose oxidase were tested: bi-directional

20

galactose dehydrogenase I and 10 (GALl, GAllO) promoters, gIyceraldehyde-3- phosphate dehydrogenase (GPO) promoter and an yeast hybrid AOH 2-GPO promoter consisting of alcohol dehydrogenase II (AOH2) and GPO promoter. The intrinsic terminator of glucose oxidase as well as the GAL 7 terminator were also compared for better production of glucose oxidase. Deletion of most of the terminating region from glucose oxidase yielded onLy a slight amount of glucose oxidase while the presence of either terminator greatly increased glucose oxidase production. The GAll 0 promoter produced the least amount of glucose oxidase, GAll and GPO promoters were moderate and the ADI-I2-GPD hybrid promoter was the best among all tested. However, the hybrid promoter was tightly regulated by the presence of an excess amount of either glucose or ethanol and it appeared that 2% glucose and 1.5% ethanol supplement was the best concentration for glucose oxidase production. It was possible to produce 260 IU mL-1 of glucose oxidase an equivalent of 5 g L-1, under the presence of 2% glucose and 1.5% ethanol. UV mutagenesis of recombinant S. cerevisiae was also applied and it further increased the yield of glucose oxidase to 460 IU mL-1 in the presence of 2% glucose and 1.5% ethanol without any changes in cell growth. Corn steep liquor, which is commonly used in bio-industry was proved to be a good alternative substrate for high priced glucose for the hybrid promoter and suggested a cost effective means for commercial mass production of glucose oxidase using recombinant yeast.

Pickering (2000) reviewed the application of glucose oxidase in the food industry and discussed its potential commercial utility in winernaking. In particular, consideration was given to its application in the production of reduced alcohol wine and as an antioxidant to supplement or replace sulphur dioxide use in table wine.

El-Enshasy et al. (2001) produced gpdA-promoter-contro!led exocellular glucose oxidase by recombinant Aspergillus niger NRRL-3 (G003-18) and investigated the effects of glucose and non-glucose carbon sources during growth. Screening of various carbon substrates in shake-flask cultures revealed that exocellular glucose oxidase activities were not only obtained on glucose but also during growth on mannose, fructose and xylose. The performance of A. niger NRRL-3 using glucose, fructose or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled glucose oxidase synthesis was strictly coupled to

2]

cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid glucose oxidase catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and glucose oxidase production, resulted in low biomass yields and therefore, reduced the advantageous properties of glucose. The total (endo and exocellular) specific glucose oxidase activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific glucose oxidase activities nearly as high as reached during growth on glucose. Also, the portion of glucose oxidase excreted into the culture fluid reached similar high levels (90%) by using either glucose or xylose as substrate, whereas growth on fructose resu1ted in a more pelleted morphology with more than half the total glucose oxidase activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and consequently, the highest total volumetric glucose oxidase activity. These results showed that xylose was the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular glucose oxidase.

Eremin et at. (2001) studied the thermal stability of glucose oxidase at temperatures between 50 and 70°C by kinetic and spectroscopic (circular dichorism) methods. The stability of glucose oxidase was shown to depend on the medium pl-l, protein concentration, and the presence of protectors in the solution. At low protein concentrations «IS )lg mL-I) and pH>5.5, the rate constants (s-I), for thermal inactivation of glucose oxidase were high. Circular dichoric spectra suggested an essential role of r3-structures in stabilizing the protein globule. At a concentration of IS ug protein mL-1, the activation energy of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal mol". Other thermodynamic activation parameters estimated at 60°C had the values e.H of 78.4 kcal mol", e.G 25.5 kcal marl and e.S 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCI, polyethylene glycols and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol and citrate exerted destabilizing effects.

Kona et at. (2001) optimized the production of glucose oxidase using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL).

22

The culture produced 580 U mL-1 of the enzyme using 70 g L-1 sucrose as a carbon source. Using CSL as a sole nutrient source, enzyme synthesis was increased to 640 U mL-I. None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract and peptone) was beneficial to the enzyme synthesis.

Singh et al. (2001) produced Aspergillus niger ORS-4.410, a mutant of Aspergillus niger ORS-4, by repeated irradiation with UV rays. Treatment with chemical mutagens also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gluconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) obtained along with a conidial survival of 59% after second stage of UV irradiation, Comparison of gluconic acid production of the parent and mutant ORS-4.410 strains showed a signi ficant increase in gluconic acid production that was 87% higher than the wild type strain. Mutant ORS-4.4l 0 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory.

Ko et al. (2002) expressed the gene encoding glucose oxidase from Aspergillus niger, as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-lenninus of glucose oxidase to facilitate purification. The recombinant glucose oxidase-His6 secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger glucose oxidase, To investigate the effects of hyper-glycosylation on the secretion efficiency and enzyme properties, glucose oxidase-His6 \ .... as expressed and secreted in a S. cerevisiae mutant, in which the PMR 1 gene encoding Ca2+ ATPase was disrupted. The pmrl null mutant strain secreted an amount of glucose oxidase-His6 per unit cell mass higher than that in the wild type strain. In contrast to the hyperglycosylated glucose oxidase-His6 secreted in the wild type strain, the pmrl mutant strain secreted glucose oxidase-Hiso in a homogeneous form with a protein band pattern similar to that in natural A. niger glucose oxidase, based on SDS-PAGE. The hyper-glycosylated and pmr 1 0 mutant-derived glucose oxidase-His6 enzymes were purified to homogeneity by immobilized metal ion affinity chromatography and their specific activities and

23

stabilities were compared. The specific activity of the pmri 0 mutant derived glucose oxidase-His6 on a protein basis was very similar to that of the hyper-glycosylated glucose oxidase-His6, although its pH and thermal stabilities were lower than those of the hyperglycosylated glucose oxidase-Hiso.

Miron et al. (2002) studied the production of glucose oxidase by Aspergillus niger and found that both growth and production are diauxic processes, with logistic and linear phases. Gluconic acid that was 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 glucose oxidase, in spite of being a product of its activity. From a kinetic view point, the enzyme is a primary metabolite, but this can only be proved by including the Luedeking and Piret equation in a dynamic model that takes into account the effects of hydrogen peroxide on glucose oxidase as well as those of the catalase, which is also produced by the microorganism, on the hydrogen peroxide.

Smotrova and Kremenchutskii, (2002) worked on 13 strains of microorganisms producing enzyme glucose oxidase, which were isolated from various samples of soil. The ability of oxidase glucose in the presence of oxygen is most expressed in representatives of genera Aureobasidium, Aspergillus and Penicillium. Media containing the KJ-starch indicator were used at primary step for isolation of microorganisms from the envirorunent. The strain, identified as Aureobasidium pullulans (de Bary) Arnaud, isolated from soil had the greatest glucose oxidase activity, remained active after lyophilization grew on the medium, which contained low concentration of glucose and nitrate for candides, Use of its cells as a glucose oxidase sensor control allowed detecting glucose in the liquids with concentration from 0.027 ruM.

Zubair et al. (2002) reported that maximum activity of Aspergillus niger glucose oxidase was recorded after 36 hours of continuous shaking fermentation for optimum growth medium containing 2% rice polishing 0.3% urea, 0.05% CaC03 and 0.4% KH2P04 at pH 4 and 30°C. It was observed that addition of urea, CaCO) and KH2P04 enhanced the glucose oxidase production while addition of MgS04• 7H20 decreased it.

Godjevargova et al. (2003) studied the thermal stability of glucose oxidase in solutions containing water soluble hydrolyzed polyacrylonitrile (HPAN) and polyoxyethylene (POE) as a function of time and temperature between 28 and 60°C. The

24

results were compared with the thermal stability of glucose oxidase in solutions without polymers. The polymers studied were found to increase the enzyme thermal stability. The influence of the concentration of the water soluble polymers on enzyme thermal stability was also studied. The best protection effect on enzyme thermal stability had 3 wt% solution of HPAN and I w1% solution of polyoxyethylene. Solutions with higher concentrations led to a quick deactivation of the enzyme. It was proved that the effect of 3 \\1% HPAN solution was stronger than the effect of I wt% POE (59 versus 52%). The thermal transition of the enzyme was studied in both the presence and the absence of HPAN by DSC. The melting temperature of glucose oxidase in the presence of HPAN was shifted to an 11°C higher value. This sustained the supposition that HPAN always increased the therrnostability of glucose oxidase.

Gouda et al. (2003) found that thermal inactivation of glucose oxidase from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme. NaCI and K2S04 increased the half life of the enzyme by 3.5, 33.4 and 23.7 fold respectively, from its initial value at 60°C. The activation energy increased from 60.3 kcal morl to 72.9, 76.1, and 88.3 kcal mol ", whereas the entropy of activation increased from 104 to 141, 147 and 184 cal mol " deg " in the presence of 7.1 x 10-5 M lysozyme, 1 M NaCl and 0.2 M K2S04, respectively. The thermal unfolding of glucose oxidase in the temperature range of 25-90°C was studied using circular dichroism measurements at 222, 274 and 375nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from glucose oxidase. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59°C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62°e. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of glucose oxidase. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCI and lysozyme was primarily the result

25

of charge neutralization. K2S04 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.

Liu et at. (2003) applied response surface methodology (RSM) to optimize the speed of agitation and the rate of aeration for the maximum production of glucose oxidase by Aspergillus niger. A 22 central composite design using RSM was employed in this investigation. A quadratic model for glucose oxidase production was obtained. Aeration had more negative effect on glucose oxidase production than agitation. Significant negative interaction existed between agitation and aeration. The quadratic term of agitation presented significant positive effect. The maximum level of glucose oxidase was achieved when the speed of agitation and the rate of aeration were 756 rev min-I and 0.9 v v-I m', respectively. The fermentation kinetics of glucose oxidase by Aspergillus niger were also studied in a batch system. A simple model was proposed using the logistic equation for growth, the Luedeking-Piret equation for glucose oxidase production and Luedeking-Piret-like equation for glucose consumption. The kinetic model parameters Xo, Xm, Pm, a, k, Y XiS, m, and So were 0.24 mg mL -I, 1.65 mg mL-I. 0.23 h-I, 3.45 U rng", -0.81 U mL-I, 1.60 g g-I, 9.72 g g-I h·1 and 97.6 g L·I respectively. The model appeared to provide a reasonable description for each parameter during the growth phase and concluded that the production of glucose oxidase was growth linked.

Malherbe et at. (2003) elucidated that there was a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding glucose oxidase in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid and lactic acid bacteria during fermentation. The It niger structural glucose oxidase gene was cloned into an integration vector (YIpS) containing the yeast mating pheromone a-factor secretion signal (MFalS) and the phosphoglycerate kinase-I gene promoter (pGKIP) and terminator (PGKI T). The PGKI P-MFal S-gox-PGK I T cassette (designated GOXI) was introduced into a laboratory strain (S J 278) of S. cerevisiae. Yeast transformants were analyzed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was

26

active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid and acetic acid bacteria. This might be explained by the fact that a final product of the glucose oxidase enzymatic reaction was hydrogen peroxide. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2% less alcohol. 111is was probably due to the production of Dglucono-8-lactone and gluconic acid from glucose by glucose oxidase. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

Roth and Klinman (2003) studied the two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that converted FADI-r to FAD and formed H202 as a product. Limiting rate constants of kcmIK,nC02) = (5.7±1.8)xl02 M-1s-1 and kca,lKm(02) = (1.5±O.3)xI06 M-Is-I were observed at high and low pH, respectively. Reactions exhibited 018 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate limiting electron transfer from flavin to O2. Site-directed mutagenesis studies revealed that the pH dependence of the rates was caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicated that protonation of His-516 resulted in a reduction of the activation energy barrier by 6 kcalrnol" (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer was attributed to a 19 kcal morl (0.82 eV) decrease in the reorganization energy and a much smaller 2.2 kcal morl (0.095 eV) enhancement of the reaction driving force. An explanation was advanced that based on changes in outersphere reorganization as a function of pH. The active site was optimized at low pH, but not at high pH or in the H516A mutant where rates resembled the un-catalyzed reaction in solution.

Semashko et al. (2003) developed a method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1 using ultrafiltration membranes. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6. The effective rate constant of glucose oxidase inactivation at pH 2.6 and l6°C was 2.74 x 10-6 S-I. This constant decreased significantly as the pH of the medium increased (4-10). The temperature optimum for glucose oxidase catalyzed J3-D-glucose oxidation was in the range 30-65°C.

27

At temperatures below 30°C, the activation energy for j)-D-glucose oxidation was 6.42 kcal mol", while at higher temperatures this parameter was equal to 0.61 kcal mOrl. Kinetic parameters of glucose oxidase, catalyzed j)-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of2-deoxy-D-glucose, maltose and galactose.

Babu et al. (2004) concluded that an important requirement of immobilized enzyme based biosensors was the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase for biosensor applications. Immobilization of the enzyme was carried out using glass beads as SUpp0l1 and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of glucose oxidase has been investigated. Upon incubation at 70°C for 3 hours, the activity retention with I % silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70°C was carried out by using 4% silane and it was found that the enzyme was stable up to 75°C with an increased activity of 180% after 3 hours incubation. Although silanization had been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes was reported for the first time.

Beltrame et al. (2004) performed the selective oxidation of D-glucose to 0- gluconic acid at atmospheric pressure, controlled pH value and constant oxygen concentration, m the temperature range from 273.2-303.2 K, using Hyderase (a commercial glucose oxidase and catalase preparation). Measurements of initial rate as a function of initial glucose concentration were interpreted by a simplified version of the already proposed reaction mechanism, having the form of a simple equation of the Michaelis-Menten type, with two kinetic parameters, i.e., k, referring to the reaction between glucose and oxidized enzyme and k; referring to a combination of the other

28

steps. The activation energy for k, was found to be 49.6 kJ mol-I and an apparent activation energy of 26.7 kJ morl was obtained for k.,

Gulla et al. (2004) demonstrated that stability of glucose oxidase immobilized with lysozyme was considerably enhanced by modification of free thiols generated by reducing disulfide bonds using p-mercaptoethanol and N-ethylmaleimide in conjunction with additives like antibiotics and salts. Thermal stability of immobilized glucose oxidase was quantified by means of the transition temperature, Trrl and the operational stability by half-life (/112) at 70°C. Modification of the free thiols in the enzyme coupled with the presence of kanamycin, NaCI and K2S04, led to increase in Tm to 80, 82 and 84°C (compared to 75°C in control) and IIJ2 by 7.7, 11 and 22 fold respectively, indicated that this method could be effectively used for enhancing the stability of enzymes.

Leiter et al. (2004) purified and characterized a high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics (relative molecular mass 155000, subunit structure 76000, isoelectric point 5.4) were determined using SOS-PAGE and 2D-electrophoresis. N-tenninal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1 mg mL-I) prevented oxidative cell injuries triggered by 0.004 U glucose oxidase in Emericella nidulans cultures but bovine liver catalase was ineffective even at a glucose oxidase:catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen depleting glucose oxidasecatalase system was likely to replace the primary inhibition exerted by H202. So, Penicillium chrysogenum glucose oxidase possessed similar enzymological features to those described earlier for other Penicillium glucose oxidases. Its cytotoxicity was dependent on the inherent antioxidant potential of the test microorganisms. In short, Penicillium chrysogenum glucose oxidase could find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial andJor preservative agent.

Luque et af. (2004) synthesized the recombinant Aspergillus nidulans sVAL040, capable of secreting glucose oxidase derived from Aspergillus niger and used to study the

29

influence of pH and carbon source on enzyme production. Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnS gene. A maximum specific glucose oxidase activity of IOU mg' protein and a maximum volumetric productivity of 29.9 U L-1 h-l were obtained at pH 5.5 after 80 hour of growth by using xylose as inducer. Specific glucose oxidase activity obtained at pH 5.5 is 2-3 times more than those previously described for goxC multicopy transformants of A. nidulans.

Rodriguez-Nogales (2004) reported that glucose oxidase was encapsulated in Iiposomes (prepared from phosphat idyl choline and cholesterol) by the dehydrationrehydration method. The enzymatic activities of native and liposomal glucose oxidase were followed by the amount of H202 obtained in the enzymatic a-D-glucose oxidation. Some characteristics of the liposomal and free glucose oxidase were compared. The enzyme encapsulated in liposomes showed an apparent inhibition by glucose at concentrations higher than 0.28 mol dm' with a substrate inhibition constant of 0.95± 0.12 mol dm·3. The enzyme entrapped showed an apparent Kill value higher than that of the free enzyme. The apparent Villar of liposomal enzyme decreased by a factor of 0.35 with respect to that of the native enzyme. The optimum temperature of the free and entrapped enzymes remained similar but the liposomal enzyme showed maximal activity at a more acidic pH (5.2). The thermal and proteolytic stabilities were enhanced by encapsulation in liposomes. The stabilization factors (relationship between half lives of entrapped form and free enzyme) at 45, 50 and 55°C for liposomal glucose oxidase were 2.6, 1.6 and 1.6 respectively.

Semashko et al. (2004) studied the main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM 95.132 and 46.1. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes and extracellular glucose oxidase were found to be maximum between 14 and 18 hours, between 48 and 52 hours and by the 961h hour of cultivation, respectively. Among the mutants studied, P. funiculosumi 46.1 showed the maximal rates of growth and glucose oxidase synthesis.

30

Sukhacheva et af. (2004) proposed a method for isolation of extracellular glucose oxidase from Penicillium funiculosum 433 and its purification. The enzymatic preparation was produced with a yield of 56% and a specific activity of 3730 AU mg" protein. The enzyme studied displayed a high therrnostability, resistance to metal ions and performance in a wide pH range and was equal in its properties to foreign analogues.

Zoldak et al. (2004) found that glucose oxidase from Aspergillus niger was a dimeric flavoprotein with a molecular mass of 80 k Da/monomer. Thermal denaturation of glucose oxidase was studied by absorbance, circular dichroism spectroscopy, viscosimetry and differential scanning calorimetry. Thermal transition of this hornodimeric enzyme was irreversible and surprisingly independent of glucose oxidase concentration (0.2-5.1 mg ml."). It had an apparent transition temperature of 55.8±I.2°C and an activation energy of ~280 kJ mol", calculated from the Lumry-Eyring model. The thermally denatured state of glucose oxidase after recooling had the following characteristics: (i) It retained ~ 70% of the native secondary structure ellipticity (ii) it had a relatively low intrinsic viscosity (7.5 mL gO]) (iii) it could bind ANS (iv) it had a low Stern-Volmer constant of tryptophan quenching and (v) it formed defined oligomeric (dimers, trimers and tetramers) structures. It was significantly different from chemically denatured (6.67 M GdmHCI) glucose oxidase. Both the thermal and the chemical denaturation of glucose oxidase caused dissociation of the flavin cofactor, however only the chemical denaturation is accompanied by dissociation of the hornodimeric glucose oxidase into monomers. The transition temperature was found to be independent of the protein concentration and the properties of the thermally denatured protein indicated that thermally denatured glucose oxidase had a compact structure, a form of molten globule like apoenzyme. Glucose oxidase was thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.

Ferreira et al. (2005) reported the operating conditions for the extraction and recovery of glucose oxidase by reversed micelles from mixtures of commercial enzyme and Aspergillus niger homogenates. For this purpose, the influence of the main operating parameters (pH, surfactant concentration and presence of cell debris or not) on enzyme extraction was investigated at 25°C_ Without cell debris, the highest yield of glucose

31

oxidase activity recovery (90.8%) was obtained performing (a) the forward extraction in isooctane as solvent and hexanol and butanol as cosolvents at 76/6118 ratio, pH 7, 0.2 M cetyl trimethylammonium bromide as cationic surfactant and electric conductivity of 5 mS ern" and (b) the backward extraction at pH 5.5. Forward and backward extractions furnished comparable results when using raw homogenate, which demonstrated a negligible impact of the presence of cell debris on the process. The highest extraction yield (94%) was obtained under the same forward and backward conditions adopted without cell debris. The promising results of this work suggested that the proposed methodology could be profitably exploited at an industrial level.

Khattab and Bazaraa (2005) screened various strains of Aspergillus niger for extracellular glucose oxidase activity. The most effective producer, strain FS-3 (15.9 U mL~l), was mutagenized using U'V-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy D-glucose and 17 of these exhibited higher glucose oxidase activities (from 114.5 to 332.1 %) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5-394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher glucose oxidase activity than their corresponding higher producing parental strain.

Vasileva and Godjevargova (2005) studied the relative activity and thermal stability of native and covalently immobilized glucose oxidase onto polyamide-6 (PA-6) membrane at temperatures of 28, 45 and 60°C for 10 hours and in the presence of organic solvents (methylol, ethylene glycol and glycerol) with concentrations 10, 30 and 60%. It was proven that immobilized glucose oxidase had better stability than the native one in all the three organic solvents. At 28°C, the strongest activating and stabilizing effect on the free glucose oxidase was observed with 10% rnethylol and on the immobilized glucose oxidase with 10 and 30% methylol. The addition of certain concentrations of ethylene glycol and glycerol to the enzyme at higher temperatures was found to stabilize the enzyme molecule. At temperatures of 45 and 60°C, the strongest stabilizing effect on both forms of the enzyme was exerted by 10% glycerol. It was concluded that the most

32

stable form of the enzyme was glucose oxidase covalently immobilized onto PA-6 membrane in the presence of 10% solution of glycerol.

Betancor el al. (2006) immobilized glucose oxidase on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermostable preparation of glucose oxidase. The enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, highest stability of all the immobilization systems was achieved, showing a half life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionic ally adsorbed enzyme or the soluble one. Therefore, the adsorption of glucose oxidase on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.

Wong et al. (2006) developed a statistical method named MAP (Mutagenesis Assistant Program) to equip protein engineers with a tool to develop promising directed evolution strategies by comparing 19 mutagenesis methods. Instead of conventional transition/transversion bias indicators as benchmarks for comparison, they proposed to use three indicators based on the subset of amino acid substitutions generated on the protein level as (1) protein structure indicator (2) amino acid diversity indicator with a codon diversity coefficient and (3) chemical diversity indicator. A MAP analysis for a single nucleotide substitution was performed for four genes (I) heme domain of cytochrome P450 BM-3 from Bacillus megaterium (2) glucose oxidase from Aspergillus niger (3) arylesterase from Pseudomonas fluorescens and (4) alcohol dehydrogenase from Saccharomyces cerevisiae. Based on the MAP analysis of these four genes, 19 mutagenesis methods have been evaluated and criteria for an ideal mutagenesis method have been proposed. The statistical analysis showed that existing gene mutagenesis methods are limited and highly biased. An average amino acid substitution per residue of only 3.15-7.4 can be achieved with current random mutagenesis methods. For the four investigated gene sequences, an average fraction of amino acid substitutions of 0.5-7%

33

resulted in stop codons and 4.5-23.9% in glycine or proline residues. The diversity remained low even when applying a non-biased method: an average of seven amino acid substitutions per residue, 2.9-4.7% stop codons, 11.1-16% glycine/proline residues, 21- 25.8% preserved amino acids and 55.5% were amino acids with chemically different side chains. Statistical information for each mutagenesis method can further be used to investigate the mutational spectra in protein regions regarded as important for the property of interest.

2.3 Glucose diagnostic kit

Enzymatic methods for the determination of sugars are of advantage over chemical methods in that (I) they are far more specific, (2) they can be used to follow reactions in which sugar is liberated, and (3) they allow the determination of sugar in the presence of protein. There are also some other enzymatic methods for glucose estimation (I) Glucose dehydrogenase from liver that requires pyridine nucleotides, has a much lower affinity for glucose and can not be used for a quantitative assay (2) another enzymatic procedure for the detection of glucose is to use yeast hexokinase and then measure the glucose-6-phosphate with glucose-6-phosphate dehydrogenase.

Okuda et al. (1977) produced a modification, utilizing mutarotase of an enzymic colorimetric systems determining D-glucose with D-glucose oxidase and peroxidase. ABTS was satisfactory for the assay of D-glucose in aqueous solutions. The time required for the assay is about 10 minutes and the lower limit of D-glucose is 0.4 ~lg.

Wong et al. (1981) reported that horseradish peroxidase, in the presence of H202, ammo antipyrine and chromotropic acid catalyzed the formation of a deep blue compound having an absorption with maximum at 590 nm wavelength. By coupling glucose oxidase, peroxidase and the chromogen, it was possible to measure glucose enzymatically at levels of 10-100 ug,

Marks (1996) described a simple, accurate and rapid method of determining glucose specifically in blood, C.S.F. and urine, using glucose oxidase and peroxidase. A comparison between glucose and non-glucose reducing fractions before, during and after insulin administration was made, in which it was shown that non-glucose reducing substances in blood were diminished by insulin over a prolonged period. From the data

34

presented it was suggested that the older non-specific methods of glucose estimation should be replaced by one of the newer enzyme methods.

Scognamiglio et al. (2004) showed recent progress in the field of glucose sensing based on the utilization of enzymes and proteins as probes for stable and non-consuming fluorescence biosensors. They developed a new methodology for glucose sensing using inactive forms of enzymes such as the glucose oxidase from Aspergillus niger, the glucose dehydrogenase from the thermophilic Thermoplasma acidophilum and the glucokinase from the thermophilic Bacillus stearothermophilus. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting ape-glucose oxidase still could bind glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-l-naphthalene sulfonic acid was found to bind spontaneously to ape-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose and the mean life time of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10-20 mM glucose, which was comparable to the kD of holo-glucose oxidase. The ANSlabeled ape-glucose dehydrogenase from Thermoplasma acidophilum also displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease could also be used to measure the glucose concentration. The thermophilic ape-glucose dehydrogenase was also stable in the presence of organic solvents, allowing determination of glucose in the presence of acetone. TI1e third enzyme used for glucose sensing was the glucokinase from Bacillus stearothermophilus. A fluorescence competitive assay for the determination of glucose was developed based on the utilization of this thermostable enzyme. Taken together the results showed that enzymes, which used glucose as their substrate can be used as reversible and non-consuming glucose biosensors in the absence of required co factors. Moreover, the possibility of using inactive apo-enzyrnes for a reversible sensor greatly expanded the range of proteins, which could be used as sensors, not only for glucose but also for a wide variety of biochemically relevant analytes.

Wu e/ al. (2004) fabricated a glucose biosensor using an enzyme immobilized eggshell membrane and oxygen electrode for glucose determination. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross

35

linking agent. The glucose biosensor was fabricated by positioning the enzyme immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor was studied in detail. Common matrix interferents such as ethanol, D-fructose, citric acid, sodium benzoate, sucrose and L-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (l00 s), high sensitivity (8.3409 mg L-1 oxygen depletion/mmol L-J glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response was 1 x 1 0.5 to L 3x 1 0-3 mol L-J glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.

Tsuchiya el al. (2005) developed a compact human blood sampling device used for the Self Monitoring of Blood Glucose (SMBG). The 5MBG comprised (I) an indentation system using a shape memory alloy (SMA) actuator to force a microneedle through the skin (2) a micro electrical pumping system to extract blood using a birnorph type piezoelectric microactuator (3) a biosensor using an enzyme such as glucose oxidase to detect and evaluate the amount of glucose in extracted blood. A titanium microneedle the same size as a female mosquito'S labium (60 urn outer diameter, 25 urn inner diameter) was produced by the sputter deposition method. The mechanical design of the device was based upon the mosquito'S blood sampling mechanism. They measured the performance of the principal components: the indentation load for a microneedle of external diameter 100 urn was found to be 0.1 N. The pumping system has an extraction speed of about 2 ~L min-I for whole blood. This is similar to that achieved by the mosquito.

Zhu el al. (2006) reported the progress in miniature chip-design raised demands for implantable power sources in health care applications such as continuous glucose monitoring of diabetic patients. Pioneered by Adam Heller, miniaturized enzymatic biofuel cells (mBCs) converted blood sugars into electrical energy by employing for example glucose oxidase the anode and bilirubin oxidase on the cathode. The power output had been limited by the performance of glucose oxidase on the anode. They

36

developed a glucose oxidase detection assay (GODA) as medium-throughput screening system for improving glucose oxidase properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation, which was recorded at 340nrn. For validating the screening system, a mutagenic library of glucose oxidase from Aspergillus niger was generated and screened for improved activity using Saccharomyces cerevisiae as host. Directed evolution resulted in a glucose oxidase mutant I 115V with 1.4-1.5 fold improved activity for P-Dglucose (Vma r from 7.94 to 10.81 umol min-I mg'"; Km 19-21 mM) and oxygen consumption kinetics correlated well [Vmax (02) from 5.94 to 8.34 umol min-l mg "; Kin (02) from 700 to 474 ).!M].

Summarizing this chapter its important to know that, during the past years numerous studies have been presented on Aspergillus niger, presumably the most important fungus for production and secretion of glucose oxidase. The utilization of A. niger as a host organism for production and enhance production demonstrates many advantages. Traditionally, strain development requires painstaking, lengthy and tedious procedures to identify superior isolates among a mutagen-treated population. Several attempts have been made to improve glucose oxidase production by strain selection using classical screening and mutagenesis techniques. Special environmental conditions, toxic to the majority of the cell type (wild type) but less toxic or non-toxic to a desired minority of cells (mutant), have often been applied to enrich a cell population to obtain desired mutants (Direct selection). The greatest advantage of this screening method is its simplicity that does not require any profound understanding of the molecular biology and physiology of the microorganisms being manipulated. In this work, the efforts have been made to improve the methods of mutant selection from a large population of microorganisms. Much improved purity and thermodynamic parameters have been studied which will explore a new window for its utilization at industrial scale. This research was carried out to produce the mutant of Aspergillus niger for the hyperproduction of glucose oxidase, optimization of conditions for the production of glucose oxidase and thermal characterization of glucose oxidase. Finally the enzyme was used to optimize the conditions to measure the blood/serum glucose level.

37

Chapter 3

MATERIALS AND METHODS

3.1 Chemicals and Enzymes

All the chemicals, reagents and enzymes were of analytical grade and mainly purchased from Sigma.

3.2 Microorganism

The test organism used in this research work, Aspergillus niger, was procured from the National Fungal Culture Collection of Pakistan (NFCCP), Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan. The stock culture was maintained on potato-dextrose-agar at 4°C in a refrigerator.

Table 3.1. Composition of potato-dextrose-agar medium (PDA)
S. No. Ingredients Quantity (g 100 ml,' )
Potato starch 2
2 Glucose 2
3 Agar 2
4 Urea 0.3
5 ZnS04.7H20 0.001
6 KH2P04 0.008
7 KCl 0.015
8 MgS04.7H2O 0.05 pH 4 and temperature 30°C

38

3.2.1 Counting of spores

For counting of spores, 0.1 mL of spore suspension was poured onto haemocytometer under the cover slip. Using low power of microscope, spores were counted (Kolmer et al., 1959).

3.3 Strain improvement techniques

The spores of Aspergillus niger were prepared in Vogel's media (pH 5.5), using 250 mL Erleruneyer flasks with working volume of 50 mL in rotary shaker (Gallenkamp) operating at 220 rpm and temperature was adjusted at 30°C (Hag et al., 200 I).

Table 3.2. Composition of Vogel's media
S. No. Components Quantity (g 100 mL- )
KH2P04 0.5
2 NH4N03 0.2
3 (NH4)2S04 0.4
4 MgS04.7H2O 0.02
5 Peptone 0.1
6 Trisodium citrate 0.5
7 Yeast extract 0.2
8 Glucose 50% (w/v) pH 5.5 and temperature 30°C

3.3.1 Radiation mutagenesis

3.3.1.1 Mutagenesis using gamma radiation

Aspergillus niger spore suspension (l x 107 spores mL-1) in a 36 hours Vogel's broth was transferred in 10 mL McCartney vials. Seven mL of spore suspension was transferred in each vial, sealed with plastic cover and para film. These vials were exposed to gamma radiation using Co60 irradiator at Nuclear Institute of Agriculture and Biology (NIAB), Faisalabad, Pakistan. The radiation source was calibrated with Fricke dosimeter.

Seven different doses of gamma radiation were selected, which were 20, 40, 60, 80,100, 120 and 140 k Rad. As dose depends on time i.e. 1.25 k Rad hour-I, so 40 k Rad

39

sample was collected on first day where as 120 k Rad sample was collected on 41h day (Gromada and Fiedurek, 1997).

Eighty k Rad dose was selected as optimized dose for mutation, as it gave 3 log kill. It means that dose at which 1000 cells mL-1 of the spore suspension was killed due to the exposure of gamma radiation. As the log of 1000 = 3, so it is called as 3 log kill. The kill/survival curve was prepared and time of exposure giving (1 x 104 CFU mCI) 3 log kill was selected for mutation of the Aspergillus niger.

3.3.1.2 Mutagenesis using UV lamp

UV germicidal lamp of 20W (Phillips) was used for the mutation of Aspergillus niger spores (lxl07 spores mL-1) for enhanced production of glucose oxidase. Ten mL spores were transferred to sterile petri plates, which were exposed to UV light for 30, 60, 90, 120, 150, 180, 210, 240 and 270 minutes. The exposure was carried out at distance of 20 ern of its radiation from the center of germicidal lamp. A dose (240 min.) producing 87% killing was selected as optimum dose, after preparing kill curve. The kill/survival curve was prepared and time of exposure giving (lAx 1 04 CFU ml,") 3 log kill was selected for mutation of the Aspergillus niger for hyperproduction of glucose oxidase enzyme (Gromada and Fiedurek, 1997; Khattab and Bazaraa, 2005).

3.3.2 Chern ical m u tagenesis

N-methyl-Nt-nitro-N-nitrosoguanidine (M1\TNG) (Musilkova et al., 1978; Gromada and Fiedurek, 1997) and ethidium bromide (Grornada and Fiedurek, 1997; Khattab and Bazaraa, 2005) were used to induce mutagenesis in Aspergillus niger for hyperproduction of glucose oxidase.

3.3.2.1 Mutagenesis using MNNG

To prepare the stock solution, 0.15 mg of N-methyl-N'-nitro-N-nitrosoguanidine (NfNNG) was dissolved per mL of buffer saline. Different time intervals were selected for chemical mutagenesis. One mL of i\11\TNG stock solution and 9 mL of Vogel's media containing spores of Aspergillus niger (IXl07 spores mL-I) were added in flask and kept in water bath (Mernmert), at 37°e. After intervals of 30, 60, 90, 120, 150 and 180 minutes, 1 mL sample withdrawn and centrifuged (Sanyo) thrice for 15 minutes at 10,000 rpm to remove the mutagen from spore suspension. A dose (after 120 min.) producing 82% kill, by kill curve was proved to be the best.

40

3.3.2.2 Mutagenesis using ethidium bromide

A stock solution of 0.5 mg mL-1 ethidium bromide was prepared and 1 mL of ethidium bromide solution was added to 9 mL of Vogel's media containing spores of Aspergillus niger (lxl07 spores mL-1). After specific time intervals of 30,60,90, 120, 150 and 180 minutes of incubation, it was centrifuged (Sanyo) three times at 10,000 rpm for 15 min. A dose (after 120 min.) producing 76% kill, by making kill curve, was proved to be the best.

3.4 Selection of mutant

Following procedures/steps were adopted in the procedure to select the specific mutant having the ability to hyperproduce glucose oxidase.

3.4.1 Selection of colony restrictor

In order to restrict the formation of fungal colonies, ox gall (0.1-1 % w/v) and triton X-I00 (1-2% v/v) were used as colony restrictor (Khattab and Bazaraa, 2005). Finally, on the basis of results, ox gall (1 %) was used in PDA medium obtaining the best results.

3.4.2 Selection of 3 log kill mutant dose by kill curve

After treating spores with 4 different mutagens, 100 fold serial dilutions of mutated spores (each mutagen treated) were prepared to give approximately 30 colonies or less per plate. In a dark room, the spore dilutions (0.1 mL) were spread onto PDA media containing 1 % ox gall as colony restrictor. Non-irradiated spores were also plated as control. All processes were carried out in strict aseptic conditions in a laminar air flow. The plates were covered with aluminum foil, placed in an incubator (Mernmert), set at 30°C for 3-7 days or till colony formation (different in each case). More than 1000 colonies were screened for selection of each mutant (each mutagen treated) and few mutants were isolated on PDA plates to study their enzyme activities. The best mutant was selected from a number of variants (Petruccioli et al. 1999; Khattab and Bazaraa,

2005).

3.4.2.1 Calculation of colony forming units (C.F.U. mL·1) The colony forming units were calculated as follows:

Number of colonies on agar plate

C.F.U mL-1

x

Amount plated (O.lmL)

Dilution factor

41

3.4.3 Screening procedures 3.4.3.1 Plate screening method

The basal medium used for selection of mutant was potato-dextrose-agar supplemented with 2% glucose as carbon source with 1 % ox gall as colony restrictor. After 2-3 days of incubation at 30°C in dark, the size of clearing zones was determined. The colonies showing bigger zone were further sub-cultured. A few colonies were obtained showing large clearance zones than wild type (Petruccioli et al. 1999; Khattab and Bazaraa, 2005).

3.4.3.2 Isolation of mutants by selective marker

For selection of resistance to catabolite repression, 2-Deoxy-D-glucose was used at 1 mg mL-1 (Gromada and Fiedurek, 1997; Khattab and Bazaraa, 2005). The mutant spores were allowed to grow in PDA at 30°C for 4-8 days. The colonies that appeared as background growth were picked and subjected to the preliminary glucose oxidase identi fication.

3.4.4 Identification of mutant

For the identification of specific mutant of Aspergillus niger following two tests were performed:

3.4.4.1 Enzyme diffusion zone test

Glucose oxidase positive strain was identified on agar plate containing 0.1 g L-1 0- dianisidine and 310 U rng" of purified horseradish peroxidase. If glucose oxidase is formed, then enzymatic reaction will occur giving rise a brown color (El-Enshasy, 1998; Petruccioli et al., 1995; Khattab and Bazaraa, 2005)_ The strains showing the greatest diffusion areas (mm) were further studied.

3.4.4.2 Analytical test

The larger zone producing strains were scratched, dissolved and homogenized in buffer, filtered and then the reaction for glucose oxidase activity by enzyme assay was determined spectrophotometerically (Hitachi).

3.5 Production of glucose oxidase

3.5.1 Preparation of inoculum

The strain selected on the basis of above described criteria, was further examined more accurately by cultivation in basal medium (PDA) consisted of glucose (2%) as a

42

carbon source and the pH of the medium was maintained at 5.5 before sterilization. The medium was autocla v ed (Sanyo) at 121°C for 15 minutes at IS lbs. pressure and then inoculated with 2% (2x I 07 spores mL-1) spore suspension (gamma rays induced mutant, obtained at 80 k Rad dose). The inoculated flasks were incubated in orbital shaker (Gallenkamp) (ISO rpm) at 30°C for 72 hours (Gromada and Fiedurek, 1997).

3.5.2 Enzyme production by liquid-state fermentation

The selected mutant BCG-4 (gamma rays at 80 k Rad) was used for growth in liquid-state fermentation in order to analyze the glucose oxidase activity. Moreover, parental fungus was also used to produce the enzyme so as the comparison between both was recorded (Fiedurek et al., 1998).

Abundantly available agriculture residue, corn steep liquor 2 % (w/v) was used as an economical substrate along with glucose 2, urea 0.3, CaCOJ 0.05 and KH2 P04 0.04 % to achieve higher glucose oxidase yield using liquid-state fermentation. These were autoclaved at 121°C for 15 minutes. Then, 5% inoculum (2x107 spores mL-I) was added aseptically in each flask (triplicate were used) for incubation in shaker at 30°C and 120 rpm for 36 hours.

3.5.3 Optimization of conditions for glucose oxidase production

Various parameters were optimized with parental as well as mutant strains, as to obtain the maximum yield of glucose oxidase. The parameters were as, substrate concentration (I, 2, 3, 4 and 5%)~ fermentation period (24, 36, 48, 60 and 72 hours); pH (4,4.5,5,5.5,6,6.5 and 7); temperature (20, 30, 37 and 45°C); urea (0.1,0.2,0.3,0.4 and 0.5%); KH2P04 (0.1, 0.2, 0.3, 0.4,0.5,0.6,0.7 and 0.8%); CaC03 (0.01,0.02,0.03, 0.04,0.05 and 0.06%); MgS04.7H20 (0.01, 0.02, 0.03, 0.04 and 0.05%) and glucose (1, 2,3,4 and 5%).

After each step of optimization of media for maximum production of glucose oxidase (parental and mutant derived), it was compared with non-optimized conditions and hence showed a highly significant results at optimized conditions.

3.5.4 Sample harvesting

After growth upto 36 hours, the culture was subjected to homogenization by a glass cell homogenizer for 10 minutes and the resulting suspension was subjected to

43

centrifugation at speed 10,000 rpm for 15 minutes at 4°C, in order to disrupt/remove the cell membranes (Gromada and Fiedurek, 1997). Then the suspension was filtered through a Whatman filter paper and the separated mycelia were washed twice with distilled water and suspended in 0.1 M potassium phosphate buffer (pH 6). Extracellular and intracellular glucose oxidase activity was determined by enzyme assay.

3.6 Enzyme assay

One unit (lU) of the enzyme activity was defined as the amount that produced I urnol of H202 per minute at 30°C. The assay for glucose oxidase was followed by Worthington (1988). It includes the following reagents and steps.

3.6.1 Preparation of 0.1 M phosphate buffer

Potassium dihydrogen phosphate (13.18 g) and 0.67 g of dipotassium hydrogen phosphate were dissolved in distilled water and volume was made upto 1000 mL. pH of the buffer was adjusted at 6 with the help of 0.1 N HCllNaOH.

3.6.2 1 M ncr

Eighty three mL of 37% pure HCl was added to distilled water and volume was made upto 1000 mL.

3.6.3 1 M NaOH

Forty g of NaOH was dissolved in distilled water and volume was prepared until 1000 mL.

3.6.4 1 % o-Dianisidine

Point three (0.3) g of o-dianisidine was added to 0.1 N HCl upto the volume of 30

mL.

3.6.5 18 % D-glucose solution

D-glucose of 18 g was added to distilled water and volume was prepared upto 100 mL. It was kept overnight at room temperature, for the spontaneous mutarotation.

3.6.6 Peroxidase

Purified peroxidase (310 U mg'), from horseradish, was used in enzyme assay. 3.6.7 Dianisidlne-buffer mixture

It was prepared by dissolving 0.1 mL of 1 % o-dianisidine in 12 mL of 0.1 M phosphate buffer of pH 6 and saturated the solution with oxygen for 10 minutes within 30 minutes of use.

44

3.6.8 Preparation of buffered-substrate solution Dianisidine-buffer mixture (PH 6) Peroxidase

18% glucose

3.6.9 Preparation of blank solution

Dianisidine-buffer mixture (PH 6) Peroxidase

Distilled water

3.6.10 Procedure

To get temperature equilibration, the sample was incubated in spectrophotometer

2.5 mL 0.1 mL 0.3 mL

=

2.5 mL

=

0.1 mL

0.3 mL

for 3-5 minutes. Then, 0.2 mL glucose oxidase was added in buffered-substrate solution and recorded the increase in absorbance for 5 minutes, at spectrophotometer (Hitachi) set at 460 nm wavelength, after inserting the blank solution in cuvette (Worthington, 1988).

3.7 Biomass estimation

Culture samples were taken after every 6 hours of optimized fermentation media and cell dry weight determination were recorded. Mycelial biomass resuspended after centrifugation and filtration was dried in an oven (Memmert) at 60°C to a constant weight (petruccioli et al., 1999; Kona et al., 2001).

3.8 Carbohydrate analysis

During the course study of optimized fermentation period (36 hours), the amount of glucose utilized or that remained was also determined. In case of soluble glucose, the remaining unutilized glucose was determined by estimating the sugars by dinitro-salicylic acid (DNS) method using glucose as the standard (Miller, 1959).

3.8.1 Preparation of DNS reagent

35-Dinitro-salicylic acid

Potassium sodium tartrate tetrahydrate Phenol

Sodium sulfate

=

109 182 g 2g 0.5 g

=

NuOH 10 g

Above mentioned contents were mixed in 600 mL distilled water, stirred and the volume was made upto 1000 mL.

45

3.8.2 Procedure

Standard curve was prepared by using glucose, with concentrations of 0.5-5 g L -I.

I mL of each standard dilution was mixed with the same volume of DNS reagent. It was heated in boiling water bath (Memmert) in an incubator for 15 minutes. After it, optical density (00) was recorded against blank on spectrophotometer at 540 run wavelength and a standard curve was plotted between the concentration and absorbance. Same procedure was repeated for the test solutions and then sugar contents were calculated (Fig. 3.1).

3.9 Estimation of protein contents

Biuret method (GornaII et al .. 1949) was applied for the determination of protein. 3.9.1 Preparation of Biuret reagent

Both 3 g copper sulfate pentahydrate and 12 g of sodium potassium tartrate tetra hydrate were dissolved in distilled water up to the volume of 500 mL. To this 300 mL of 10% NaOH solution was poured and then distilled water was added to a final concentration of 1000 mL (This solution is light sensitive so it was stored in dark brown bottle at 4°C).

3.9.2 Procedure

Standard curve was prepared by using bovine serum albumin, with concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 mg mL -I, 1 mL of each standard dilution was mixed with the same volume of Biuret reagent. It was incubated in an incubator for 15 minutes. After it, optical density (00) was recorded on spectrophotometer (Hitachi) at 540nm wavelength and a standard curve was plotted between the concentration (mg ml.") and absorbance. Same procedure was repeated for the test solutions and then protein contents were calculated (Fig. 3.2).

3.10 Determination of growth kinetic parameters

For determining kinetic parameters for batch fermentation process, the procedures described previously (Lawford and Rouseau, 1993; Miron et al., 2002; Semashko et al., 2004) were adopted. Dry cell mass (g L-1) of A. niger cultures after growth, was determined on triplicate samples as described by (Petruccioli et al., 1997; Petruccioli et al., 1999; Kona et al., 2001) and residual carbohydrates (g Cl) were determined on dry mass (Miller, 1959). The growth yield coefficient (Y XiS) was calculated as the dry cell

46

5 _'

o ,-'

Fig. 3.1.

0].1

012 '

01 I

0) -

Fig. 3.2.

•

•

•

•

•

•

•

----~ r- -----,~---,-

-_-- ~,-.---

OS

2S

15

Standard curve of glucose for carbohydrates estimation by DNS method

•

•

•

•

[O-~

Standard curve of bovine serum albumin for protein estimation by Biuret method

47

mass per mass of saccharide utilized from the test substrate following fermentation. Rate of substrate consumption (Qs) and volumetric rate of product formation (Qp) was determined from the maximum slope in plot of substrate and enzyme produced vs, time of fermentation. Various parameters as product yield (YP/S), specific product yield (Y PIX). specific rate of substrate consumption (qs) and specific rate of enzyme production (qp) etc. were determined from the calculations.

3.11 Purification of glucose oxidase

Purification of intracellular glucose oxidase enzyme (having many fold increased activity as compared to extracellular enzyme) was carried out by ammonium sulfate precipitation, ion exchange and gel filtration chromatography.

3.11.1 Ammonium sulfate precipitation technique

Crude enzyme was subjected to ammonium sulfate precipitation by the method of Shin et al. (1993).

3.11.1.1 Salting out of other proteins

Solid ammonium sulfate was added to the crude extract until it was 60% saturated by adding 42 g 100 mL-I. It was kept for 4 hours at 4°C, then centrifuged at 10,000 rpm and 4°C for 15 minutes. The supernatant was separated from the sediment.

3.11.1.2 Salting out of glucose oxidase

The supernatant of above step was adjusted to 85% ammonium sulfate saturation by adding 17.5 g more salt. It was centrifuged at ! 0,000 rpm for 15 minutes after 4 hours. The sediment was separated from the supernatant. which were re-dissolved in minimum distilled water.

3.11.1.3

Desalting of glucose oxidase

The re-dissolved sample was dialyzed In the dialysis bag against continuous stirring distilled water for some hours. All the fractions i.e. supernatant, sediment and desalted samples were subjected to enzyme assay (Section 3.6) and protein estimation (Section 3.9).

3.11.2 Purification by ion exchange chromatography

3.11.2.1 Preparation of 0.5 N net

41.5 mL of HC! (37%) was added to distilled water and volume was made upto 1000 mL.

48

3.11.2.2

Preparation of 0.5 M N aOH

Twenty g of NaOH was dissolved in distilled water and volume was made upto 1000 mL.

3.11.2.3 Preparation of column

A column of DEAE-(Diethyl amino ethyl) cellulose was prepared by the method of Kelley and Reddy (1986) and Sukhacheva et al., (2004). The resin was gradually added to the 0.1 M phosphate buffer (PH 6) until slurry was prepared. It was heated in a water bath, set at 95°C for 5 hours, without drying the slurry. Buffer was passed through the column, to fill the outlet tube and slurry was poured into the column of 2x25 em specifications. It was kept on a leveled place for 24 hours. Then, the buffer was removed from the colwnn after opening the outlet tube and closed when just small amount present on top of the column.

3.11.2.4 Washing the column with base

The column washed with 50 mL of 0.5 M NaOH, which was allowed to flow throughout the column. After the complete removal of base, distilled water was added to column and it allowed to pass until the pH of eluent was 7.

3.11.2.5 Washing the column with acid

An amount of 50 mL of 0.5 N HCl was poured on the column. It was allowed to flow throughout the column and then distilled water was passed until the eluent pH was 7.

3.11.2.6 Equilibration of column

The column was equilibrated with phosphate buffer of pH 6. It was achieved by washing it continuously with buffer, overnight as/or the pH of eluent should be the same as that of buffer.

3.11.2.7

Application of sample

The outlet tube was opened and the buffer already present at the surface of column was allowed to flow, until a small amount on the top. Using a fine pipette, the desalted enzyme sample of 1.5 mL was poured on the surface of column. The outlet tube was opened and sample was penetrated into the column bed. The elution of sample was carried out with 0.1 M phosphate buffer (pH 6). The drop rate of eluted sample was kept

49

constant and 100 fractions of 2 mL each were collected. All these fractions were subjected to enzyme assay and protein estimation.

3.11.3 Gel filtration chromatography

A column of sephadex 0-150 (Pharrnacia) was prepared by the method of Jakoby (1971) and Sukhacheva et ai. (2004).

3.11.3.1 Swelling of the resin

The dry sephadex 0-150, 1 g was suspended into IS mL of phosphate buffer. It was heated in a water bath for 3 hours at 95°C (without drying the slurry).

3.11.3.2 Filling the column

The column was placed vertically on stable stand. Distilled water was added to column as to fill the empty outlet tube. The slurry was poured in order to completely fill the column of I ern diameter and 50 em length specifications. It was left undisturbed for some hours, as distinct layers of gel and water were appeared.

3.11.3.3 Application of sample

The outlet tube was opened and the distilled water present in column was removed until a small layer on the top of column. The sample having the maximum specific activity (obtained after ion exchange chromatography) was applied on it and outlet was opened. The sample was allowed to penetrate in packed column. Elution was carried out by 0.1 M phosphate buffer (pH 6) at a constant drop rate. A total 100 fractions of 2 mL each were collected which were then subjected to enzyme assay and protein estimation.

3.12 Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAOE; 10%) of different glucose oxidase preparations was done as described by Laemmli (1970) to analyze the purity and homogeneity of the enzyme.

3.12.1 Stock solutions

I. 30% (w/v) acrylamide + 0.8% (w/v) bis-acrylarnide

2. 1.5 M TrislHCI pH 8.8 + 0.3% (w/v) SDS

3. 0.5 M Tris/HCl pH 6.8 + 0.4% (w/v) SDS

50

3.12.2 Preparation of resolving gel

The following reagents were mixed together in a 250 mL Buckner flask and degassed for 5 minutes by a vacuum pump.

1. 2. 3.

Stock solution-I Stock solution-2 Distilled water

13.3 mL 10.0 mL 16.7 mL

=

After degassing, the following reagents were added to initiate polymerization.

4.

2% (w/v) aqueous ammonium persulfate freshly prepared TEMED

=

133 f.lL

5.

The resolving gels were prepared by pouring the above mentioned mixture into gel apparatus which was assembled by sandwiching 2 spacers between two glass plates (l0 ern x 8 em x 1.5 em), After polymerization, l-butanol was layered on the top of gel to get even surface. Then l-butanol was removed and top of the gel surface was washed many times with distilled water.

3.12.3 Preparation of stacking gel

The following reagents were mixed together in a 250 mL Buckner flask and

degassed for 5 minutes.

1. 2. 3. 4.

Stock solution-l "" 1.5 mL
Stock solution-3 2.5 mL
Distilled water 6.0 mL
2% (w/v) aqueous ammonium 100 ilL
persulfate (APS) freshly prepared
TEMED = 10 ilL 5. The stacking gel mixture was then poured on the top of polymerized resolving gel. The comb (well maker) was immediately inserted and the stacking gel was allowed to polymerize.

3.12.4 Sample buffer

The following reagents were mixed to prepare sample buffer.

1. 2.

0.75 M TrislHCl buffer pH 6.5 Distilled H20

200 ilL 6.3 mL

51

3. 4. 5. 6.

Glycerol

10% wlv aqueous SDS Bromophenol blue p-mercaptoethanol

2.5 mL I mL

2.5 mg 5% (v/v)

=

Non-covalently attached sub-units of proteins dissociated into monomers on heating in the presence of SDS. p-mercaptoethanol was mixed to break the S-S bridges between the subunits, if any.

3.12.5 Stock electrode buffer

The following reagents were mixed to prepare stock electrode buffer and this

stock solution was diluted 10 fold in distilled water just before use.

1. Tris base 30 g

2. Glycine 144 g

3. SDS 109

4. Distilled water 1 L

3.12.6 Preparation of glucose oxidase for SDS-PAGE

Glucose oxidase (10 mg mL-1) was mixed in SDS sample buffer (4X) and boiled for 3-5 minutes.

3.12.7 Preparation of protein markers ladder for SDS-PAGE

Protein markers ladder was used as standard for SDS-PAGE. The ladder consisted of 4 bands ranging from 45-200 kD. The protein ladder was added in gel loading buffer (50% glycerol, 2% SDS, 30 mM NaCI, 1 mM NaN3, 62.5 mM Tris/HCl pH 7,0.01% bromophenol blue and 50 mM DTT) and applied directly to an SDS-polyacrylamide gel after slight warming.

3.12.8 Running of PAGE

SDS gel was run at a constant voltage of 100 volts. The PAGE was stopped when tracking dye reached at the bottom of gel.

3.12.9 Protein staining of SDS-polyacrylamide gels

The gel was treated with 20% (v/v) isopropyl alcohol in 50 mM sodium acetate buffer, pH 5 to remove SDS and washed thrice for 15 minutes. Then the gel was immersed in 50 mM sodium acetate buffer, pH 5 to remove isopropyl alcohol and three changes of 40 minutes each were given.

52

The polyacrylamide gels were finally stained with coomassie brilliant blue R- 250 (Kelley and Reddy, 1986). Gel was dipped in stain so protein bands were clearly visible after 10 minutes. Then, gel was washed with dist. water after staining to improve the visualization of bands.

3.13 Molecular mass determination

3.13.1 Native molecular mass

The glucose oxidase and protein standard markers were poured on sephadex G- 150 gel filtration column to determine native molecular mass (Sukhacheva et al., 2004). Standard proteins of 1 mg mL-1 each, was loaded and eluted with 0.1 M phosphate buffer, pH 6. The flow rate was adjusted to 0.5 mL min-l and 1 mL of size fractions were collected.

Different marker proteins such as egg albumin (45 kDa), bovine serum albumin (67 kDa), alcohol dehydrogenase (150 kDa) and p-amylase (200 kDa) were subjected to gel filtration chromatography. The retention times of blue dextran (2000 kDa) and Tyrosine (0.2 kDa) were used to evaluate the void and total internal volumes of the column, respectively. The Kd (distribution coefficient) for all protein markers and glucose oxidase was calculated using the following equation:

Kd=Vc-Vo/Vi-Vo

Where,

Vc = Elution volume/retention time (glucose oxidase or protein marker)

Vo = Void volume/retention time of blue dextran

Vi = Total internal volume/retention time of tyrosine

3.13.2 Subunit molecular mass

The subunit molecular mass of glucose oxidase was determined by subjecting glucose oxidase to 10% SDS-PAGE. Proteins were heated in SDS sample buffer (4X) to attach SDS with proteins. SDS bound proteins have same charge to mass ratio, therefore, they move on the basis of size. Subunit molecular mass of glucose oxidase was determined from standard curve drawn by plotting R. values vs. log molecular mass of marker bands of ladder.

53

3.14 Kinetic a nd Thermodynamic Studies

3.14.1 Optimum pH

Parent and mutant derived glucose oxidase were assayed at different pH ranging from 3-9 to seek the optimum pH (Sukhacheva et al., 2004).

3.14.2 Optimum Temperature

Parent and mutant derived glucose oxidase were assayed at different temperature (Sukhacheva et al., 2004) ranging from 20-80°C at pH 5 (pH 5.5 for parent enzyme). The assay methodology was the same as described earlier.

3.14.3 Activation energy (£a)

Activation energy was determined by assaying glucose oxidase at vanous temperatures ranging from 20-S0°C (Sukhacheva et al., 2004; Siddiqui et al., 1997). The data was plotted according to Arrhenius as described by Siddiqui et al. (1997) and Rashid and Siddiqui (1998).

3.14.4 De te r m in a tion of Michaelis-Menten constants

The Michaelis-Menten kinetic constants (Vmax and Kill), catalytic constant (kcal) and specificity constant (kcaIIKIll) were determined by using the different concentration of glucose ranging from 4-20% wlv (Witt et aI., 1998; Siddiqui et al., 1997). Glucose oxidase activity was determined in each glucose concentration keeping enzyme concentration constant.

3.14.5 Irrevcrsible thermal dcnaturation

Irreversible thermal denaturation of glucose oxidase was determined by incubating enzyme in phosphate buffer (PH 5) at different temperatures (45-65°C). Time course aliquots were withdrawn, cooled in ice for 30 minutes and then assayed for enzyme activity at 40°C (Witt et al., 1998). This procedure was repeated at five different temperatures (45. 50, 55, 60 and 65°C). The data was fitted to first-order plots and analyzed as described by Munch and Tritsch (1990); Montes et al. (1995); Witt et aI., (1998).

3.14.6

Activation cnergy of thcrmal denaturation

The first-order rate constants for irreversible thermal denaturation (Kd) of glucose oxidase were determined and Arrhenius plot was applied to determine the activation energy for denaturation (£a).

54

3.14.7 Thermodynamics of irreversible therma I inactiva tion

The thermodynamic parameters for thermostability were calculated by rearranging the Eyring's absolute rate equation derived from the transition state theory (Eyring and Stearn, 1939; Steam, 1949; Tan ford , 1968) as described by Siddiqui et at. (1999).

Kd = (kB/h) e (-o.H*/RT).e ('),S'·/R) •.•.•..•.. (l) Where,

h = Planck's constant = 6.63 x 10-34 Js

kB = Boltzman's constant (R/N) = 1.38 X 10-23 JK-1

R = Gas constant = 8.314 JK-! morl

N = Avogadro's No. = 6.02 X 1023 morl

T = Absolute temperature

6H* = E; - R T (2)

Where,

/'o.H* = Enthalpy of activation of denaturation

E; = Activation energy for denaturation

/'o.G* = -RT In(Kd, h/kB.T) (3)

Where,

/'o.G* = Free energy of activation of denaturation

/'o.S* = (/'o.H* - 6G*)/T (4)

Where,

/'o.S* = Entropy of activation of denaturation 3.15 Determination of stability

The stability of glucose oxidase from gamma rays mutated Aspergillus niger against different agents like zinc sulfate, sodium fluoride, copper chloride and silver sulfate was determined (Sukhacheva et al., 2004).

3.16 Glucose estimation kit

The conditions of the indigenously hyperproducedJmutant derived glucose oxidase enzyme, along with mutarotase and peroxidase (procured from other members of

55

research group) was standardized to measure the serum glucose level. The standard solution of a-D-glucose of 100 mg dL-1 was prepared in distilled water and following parameters were applied in this concern (Zia, 2002).

3.16.1 Enzymes concentrations

The four sets of the enzymes, having different concentrations were applied, designated as A to D.

GrouQs A D C D
Mutarotase (ul.) 10 10 10 10
Glucose oxidase (Mutant) (ul.) 5 10 20
Glucose oxidase (Parent) (ul.) 20
Peroxidase (ul.) 20 20 20 20
3.16.2 Enzymes schedule Three fashions were selected in this parameter to utilize the reagents, for groups A

to C as; Group A:

Group B:

Mutarotase: 10 ul. + Buffered chromogen 1 mL then other enzymes added separately).

Mutarotase: 10 ul, + Mutant Glucose oxidase: 5 ul, + Buffered chromogen 1 mL then Peroxidase added separately).

Mutarotase: 1 0 ~~L + Mutant Glucose oxidase: 5 uL + Peroxidase: 20 ul. + Buffered chromogen I mL, added collectively in a single vial.

Group C:

3.16.3 Guaiacol concentration

Guaiacol as chromogen was used and its concentration to utilize in enzymatic reaction for glucose determination was optimized at 18, 25 and 40 ul.,

3.16.4 Amount of sample

To optimize the amount of serum sample, various concentrations of standard glucose as 5, 10,25, 50 and 100 ul, were used.

3.16.5 Comparison with standard kit

56

The standardized fractions (i.e. obtained after all above mentioned trails) were then compared with standard glucose estimation kit of "Human, Germany' upon the serum of diabetic patients. The comparison (in both cases) was carried out by their respective schemes.

3.16.6 Determination of sensitivity of kit

The sensitivity of indigenously prepared kit for standard glucose was determined by using different concentrations as 200, 175, 150, 125, 100, 75, 50, 25, 10, 5 and I mg .n.'. The results obtained are arranged in the form of a curve and the sensitivity 'vas measured.

3.17 Statistical analysis

The data obtained was subjected to statistical design using softwares MINIT AB- 14 and SAS while graph were preduced through slidewrite plus-6 (SWWL-6) and MS Excel.

57

chapter 4

RESUL TS AND DISCUSSION

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. Chemical and physical mutagens induce mutations, non-randomly distributed within a sequence and occur more frequently in some sites than expected, originating mutagen-specific patterns of mutations. In recent new procedures such as rational screening and genetic engineering have begun to make a significant contribution to this activity but mutagenesis and selection so called "random screening" is still a cost effective procedure and for reliable short term strain development is frequently the method of choice (Rowlands, 1984).

Although several organisms have been reported to produce glucose oxidase, however, Aspergillus niger is the main organism used for industrial production. Screening, improvement and evaluation of new glucose oxidase overproducing strains is very important in improving the efficiency and economics of the industrial process. Several attempts have been made to improve glucose oxidase production in A. niger by strain selection using random/classical screening and mutagenesis techniques, optimizing of cultivation conditions and genetic engineering (Khattab and Bazaraa, 2005).

In these studies the purpose of mutagenesis was to select the colonies of Aspergillus niger which could hyperproduce glucose oxidase enzyme. Mutagenic procedures can be optimized in terms of the type of mutagen and dose. Mutagen specificity can be taken into account and mutagenesis itself can be enhanced or directed in order to obtain the maximum frequency of desirable mutant type among the isolates to be screened.

The physiological growth of wild type strain Aspergillus niger is depicted Fig 4.1.

58

(A)

Fig. 4.1. Aspergillus niger

(A) Mycelial grow th

(8) Typical characteristic spore

4.1 Kill Cu rve Determination

(8)

Gamma rays, high energy beams, very penetrating and need substantial thickness of heavy metals as lead, steel or concrete to shield them. Initially a kill curve was prepared using gamma radiation as a mutagen. For this purpose, various doses of "(-rays were used to induce mutation in Aspergillus niger. It was found that a dosage of 80 k Rad. produced 88.53% killing (1 x 104 CFU mL-I) as 3 log kill of the fungal spores (Fig. 4.2). Mutants of Aspergillus awamori were isolated with enhanced production of extracellular xylanase and p-xylosidase by Smith and Wood, (1991) using gamma irradiation (C060 irradiator) that was carried out in a semi-darkened room. The dose of 80 k Rad. produced a 90% killing of spore suspension.

UV irradiation, carried out in a semi-darkened room, was used to increase the glucose oxidase activity of Aspergillus niger. In order to optimize the treatment i.e, different doses of UV were compared evaluating spore survival and the frequency of positive and negative mutations. UV radiations produced 87% killing (1.8 x 103 CFU ml.' I) at 240 minutes of exposure where it produced 3 log kill as optimum dose and the detailed findings are arranged in Fig. 4.3.

59

Higher doses of irradiation decreased the frequency of positive mutations and the number of viable colonies as described by Petruccioli et al. (1995). Khattab and Bazaraa (2005) reported that after exposure of A. niger to UV treatment, 11 out of 200 mutants colonies were isolated, resistant to 2-deoxy-D-glucose. The data also revealed the number of resistant colonies increased by increasing the exposure time, and then a sharp decline was noted. They further showed that the highest level of glucose oxidase was produced by mutant at 46.3 U mL ~I (13 mm enzyme' di ffusion zone, 291.2% production compared with the original untreated strain), only 2 did not produce glucose oxidase enzyme and 12 mutants produced glucose oxidase in amounts less than their original parental strain. Our results are in this agreement that positive and negative effects were observed. Park et al. (2000) also carried out UV mutagenesis of the recombinant S. cerevisiae strain containing A. niger gene, and obtained 460 U mL-1 activity with 80% increase as compared to parental strain.

It has been well documented that N-methyl-N'-nitro-N-nitrosoguanidine (M1\TNG) IS one of the strong and multipotential carcinogen that has been frequently reported inducing mutations (Zhu et al., 2000). Chemical mutagenesis of Cellulomonas was carried out with ethyl methane sulfonate (EMS) and MNNG as mutagens to obtain a hyper-xylanoytic mutant with 2.5 times higher enzyme production than parent strain when grown on sugarcane bagasse as a carbon source (Lino and Teresa, 1998).

Chemical mutagenesis of A. niger to hyperproduce glucose oxidase was carried out by ethyl methane sulfonate (EMS) by Khattab and Bazaraa (2005). After screening 200 EMS treated colonies, only 21 were characterized as 2-deoxy-D-glucose resistant. It also showed an increase in the number of resistant colonies with increasing exposure to EMS mutagen and then there was a sharp decline. Highest glucose oxidase level was observed at 52.8 U mL ~I (with 14 mm of enzyme zone size, 332.1 % production compared to original), whereas only 1 did not produce and 12 mutants produced glucose oxidase in less amounts than their parental strain.

To obtain an instant mutant, high dose of mutagen is required. For this purpose, 0.15 mg mL-1 MNNG for 120 minutes dose rate, produced 82% killing (lxl04 CFU ml.' \ This showed, exposure of spore suspension to MNNG gave 3 log kill which was determined as the best mutant having the ability to hyperproduce glucose oxidase (Fig.

60

4.4). Musilkova et al. (1978) reported that MNNG strongly influenced the variability of Aspergillus niger as the number of spores capable of growth, decreased with the duration of treatment while morphological/biochemical mutants considerably increased.

Ethidium bromide is reported to be a strong mutagen that was used to obtain a

-I mutant having the ability to hyperproduce glucose oxidase. For this purpose, 0.5 mg mL

ethidium bromide for 120 minutes exposure, producing 76.13% killing!23.87% survival (1.53x 1 04 CFU mL-1) as shown in Fig. 4.5. These results are in agreement with Witteveen et at. (1990) who isolated glucose oxidase overproducing mutants of A. niger when conidial survival ranged between 33 and 78%. Production of glycosidases by P. canescens mutants obtained the best frequency of mutation when percentage of viability was between 65 and 30% as reported by Lomkatsi et al. (1990),

4.2 Selection and evaluation of mutant

After mutagenesis, serial dilutions were made of the suspension in such that 0.1 mL was plated on PDA media. The number of colonies were restricted to 30 or less than this.

4.2.1 Colony Restriction

With the help of colony restrictors, colonies can be observed very clearly for their selection. In order to restrict the fungal colonies to small size on selection medium, ox gall and triton Xvl Of), were used. The use of ox gall (1%) was found to be optimal for colony restriction and clearance as the colony size was small and showed good ones around colonies. Due to the presence of bile salts, ox gall is being used as colony restrictor (Khattab and Bazaraa, 2005). All further studies were based on this concentration for the selection of colonies.

Brown et at. (1987) used ox gall (0.2%) as a colony restrictor for the isolation of Penicillium pinophilum mutant Kumar et al. (1995) used 0.1 % sodium taruroglycocholate as colony growth restrictor, for hyper-xylanolytic mutant of Fusarium oxysporum. Gadgil et al. (1995) used triton X-laO (0.0 I %) to limit the colony size, to facilitate the screening and isolation of mutants of Trichoderma reesei for enhanced cellulase production. Khattab and Bazaraa (2005) spread the mutagen treated A. niger spore suspension on to the media containing 0.1 % (v/v) triton X-IOO to restrict radial colony growth, for glucose oxidase production.

61

20

16

-;" 12 J

~ I

;: I

w

-= gJ

•

•

•

•

O<----~ o

20

40

60

so

100

120

14D

Dose rate (k Rad.)

Fig. 4.2. Effect of ,,(-radiation to formulate tbe s u rv iv a l curve

30
25
•
20:
t, •
E
;;;; 15 •
;...
w •
-= •
10
•
:L~ -r+ --.-
0 30 60 90 120 150 ISO 210 240 270
Dose rate (min.) Fig. 4.3. Effect of UV-radiation to formulate tbe s u rv iv a l curvc

62

25 .

5

-~ [5 E

~

....

u

.s [0

o

o

30

60

90

[20

[50

180

Dose nile (min.)

Fig. 4.4

Effect of MNNG to formulate the survival curve

20 ~

[6

-s [2 •
E •
~
...
u
.= 8 a

20

~o

60

so

[00

[20

I~O

160

180

Dose rate (min.)

Fig. 4.5

Effect of ethidium bromide to formulate the su rv iv a l curve

63

4.2.2 Selection of glucose oxidase hyperproducing Aspergillus niger mutants using 2-deox)'-D-glucose

A number of specific selection schemes have been adapted to improve the biosynthetic capacity of production strains. Literature reports that resistance to toxic glucose analogue i.e. 2-deoxy-D-glucose has been used as a criterion to select the mutants showing increased rates of glucose oxidase (Gromada and Fiedurek, 1997), glucoamylase (Fiedurek et al.. 1987) and cellulase (Labudova and Farkas, 1983). The mutants resistant to 2-deoxy-D-glucose were obtained by mutagenic activation and passage in the medium with gradually increasing concentrations of this nonmetabolizable agent (Fiedurek et al., 1987). Selective isolation medium was utilized to isolate 2-deoxy-D-glucose-resistant mutants and was prepared by the addition of 2- deoxy-D-glucose at the level of 1.5 g L-1 by Khattab and Bazaraa (2005).

In order to produce depressed mutants for enzyme production, 2-deoxy D-glucose was used at 1 rug mL-1 concentration. Each mutagen treated spores were spread, separately on to PDA plates with 2-deoxy-D-glucose. A few colonies were selected based on large clearance zones than wild type microorganism. Some of the mutant colonies showed variations and rough/velvety appearance on PDA plates. Moreover, some colonies showed considerably smaller size of colonies as compared with the other darker colonies with much larger size (Fig. 4.6-4.9). These colonies were subjected to enzyme diffusion zone analysis to select the best one.

4.2.3 Selection of specifie mutants by enzyme diffusion zone

Enzyme diffusion zone analysis is the specific procedure to screen and to identify the specific mutant based on enzymatic reaction on plate media. Glucose oxidase positive strain was identified on agar plate containing 0.1 g L-1 o-dianisidine and 310 U mg" of purified horseradish peroxidase. The size and intensity of zone color is an index of the formation of glucose oxidase. The following enzymatic reaction occurs giving rise a brown color:

P-D-glucose + O2 o-dianisidine red + H202

Glucose oxidasi Peroxidase

8-D-gluconolactone + H202 o-dianisidine ox + 2H20

These results indicated that mutant BCG-4 obtained at 80 k Rad dose of gamma rays produced 15 nun enzyme diffusion zone as compared to wild type (2 mm) with

64

processes to get the enhanced production of iit.icose oxidase. -Howe~er, ~nother test was also employed on the colonies obtained by zone analysis.

Petruccioli et al. (1995) found the distribution of the irradiated colonies according to the size of halos of glucose oxidase diffusion into the agar plates and 54 colonies showed diffusion halo larger than 7 mm in diameter as compared to parent of 3-5 mm. Malherbe el af. (2003) screened for the secretion of biologically active A. niger glucose oxidase by selecting the colonies surrounded by a brown halo in the glucose oxidase agar plate assay and were identified as positive.

According to Khattab and Bazaraa, (2005) the glucose oxidase activities of the mutants selected were also determined by the violet-blue zone method. The results demonstrated a very high correlation between the diameter of the zones formed on screening medium and glucose oxidase activities measured using the spectrophotometric assay, especially in the range of 0-10 nun (glucose oxidase zone). Park et al. (2000) also adapted the enzymatic diffusion zone method to select the active mutant for glucose oxidase activity.

4.2.4 Analytical test

The larger and darker zone producing strains were scratched, dissolved into 0.1 M phosphate buffer pH 6, filtered, homogenized and then the reaction tor glucose oxidase activity was determined spectrophotometerically and the results are arranged in Table 4.1. Depending upon these trials, it was suggested that Aspergillus niger BCG-4 would be a potential mutant for the maximum production of glucose oxidase.

65

Fig. 4.6.

Screening of glucose oxidase hyperproduced by gamma rays using 2- deoxy-D-glucose resistant mutant derivative

Gamma rays (Control)

Gamma rays (80 k Rad)

Gamma rays (80 k Rad)

66

Fig. 4.7.

Screening of glucose oxidase hyperproduced by UV -rays induced mutant resistant to 2-deoxy-D-glucose

UV rays (Control)

UV rays (240 min)

UV rays (240 min)

67

Fig. 4.8.

Screening of glucose oxidase hyperproduced by MNNG induced mutant resistant to 2-deoxy-D-glucose

MNNG (Control)

MNNG (120 min)

68

Fig. 4.9.

Screening of glucose oxidase hyperproduced by ethidium bromide induced mutant resistant to 2-deoxy-D-glucose

Ethidium bromide (Control)

Ethidium bromide (120 min)

Ethidium bromide (120 min)

69

Fig. 4.10.

Selection of glucose oxidase hyperproducing gamma rays induced mutants by enzyme diffusion zone

Enzyme Diffusion Zone (Control)

Enzyme Diffusion Zone (80 k Rad) (BCG-4)

Enzyme Diffusion Zone (80 k Rad) (BCG-5)

70

Fig. 4.11.

Selection of glucose oxidase hyperproducing mutants by enzyme diffusion zone

Enzyme Diffusion Zone (UV rays 240 min.) (DCU-5)

Enzyme Diffusion Zone (MNNG 0.15 mg mL·1 : 120 min)

(DCM-8)

Enzyme Diffusion Zone (Ethidium bromide 0.5 mg mL-1 : 120 min) (8CE-6)

71

Table 4.1. Activity of glucose oxidase shown by various mutants by analytical
test
A. niger mutant strains Zone size Activit), %age increased
{mm} {U mL-1} aetivi!):'
Wild type/Control 2 3.89 roo
BCG-J (Gamma: 80 k Rad) 7 7.89 205
BCG-2 (Gamma: 80 k Rad) 8 11.25 289
BCG-3 (Gamma: 80 k Rad) 5 4.60 I 18
BCG-4 (Gamma: 80 k Rad) 15 25.67 660
BCG-5 (Gamma: 80 k Rad) 14 22.61 583
BCG-6 (Gamma: 80 k Rad) 1 I 17.54 451
BCG-7 (Gamma: 80 k Rad) 7 9.36 241
BCG-8 (Gamma: 80 k Rad) 8 11.28 290
BCG-9 (Gamma: 80 k Rad) 12 19.24 495
BCG-I0 (Gamma: 80 k Rad) 8 10.08 259
BCU-l (UV: 240 min.) 5 4.71 121
BCU-2 (UV: 240 rnin.) 7 4.90 126
BCU-3 (UV: 240 min.) 5 4.09 105
BCU-4 (UV: 240 min.) 8 6.54 168
BCU-5 (UV: 240 min.) 11 15.48 398
BCU-6 (UV: 240 min.) 6 4.55 117
BCU-7 (UV: 240 min.) 2 3.11 80
BCU-8 (UV: 240 min.) 9 13.59 349
BCM-J (MNNG: 120 min.) 3 2.53 65
BCM-2 (MNNG: 120 min.) 5 5.66 146
BCM-3 (M1\JNG: 120 min.) 4 4.02 103
BCM-4 (MNNG: 120 min.) 6 5.78 149
BCM-5 (MNNG: 120 min.) 8 10.00 257
BCM-6 (MNNG: 120 min.) 6 8.97 231
BCM-7 (MNNG: 120 min.) 3 4.75 122
BCM-8 (MNNG: 120 min.) 9 10.96 282 72

74

BCE-I (EB: 120 min.) 5 4.33 1 II
BCE-2 (EB: 120 min.) 5 5.14 132
BCE-3 (EB: 120 min.) 3 1.96 51
BCE-4 (EB: 120 min.) 4 3.52 91
BCE-5 (EB: 120 min.) 2 2.32 86
BCE-6 (EB: 120 min.) 6 7.87 202
BCE-7 (EB: 120 min.) 4 5.01 129
BCE-8 (EB: 120 min.) 5 4.45 115
Key:
BCG: Gamma irradiated BCU: UV rays treated
BCM: MNNG treated BCE: Ethidium bromide treated 4.3 Production of enzyme in shake flask

As compared to its parental strain, a mutant can have a new genotype and therefore, reoptimization of the culture conditions is usually necessary to show its real potential. Thus, a series of preliminary experiments in shaken culture was performed to determine the effect of medium composition on glucose oxidase activity of parent as well as mutant strain (Petruccioli et al., 1997). Shake flask experiments (in triplicate) were carried out in 250 mL Erlenmeyer flasks. Cultivation was carried out on a rotary shaker at 120 rpm and 30°C with 100 mL working volume as described previously (EIEnshasy, 1998). It should be noted that, the mode of combination of factors during optimization was not used, but the optimization was carried out in a sequential order.

The selected mutant BCG-4 in comparison with parental Aspergillus niger, were used for the production of enzyme, glucose oxidase, in shake flask culture. Shake flasks carrying 2% corn steep liquor as substrate in 100 mL of fermentation medium were inoculated with 5 mL of inoculum from the mutant strain as well as wild type as described earlier (Fiedurek et al., 1998).

Various parameters were optimized in order to get the enhanced production of glucose oxidase.

4.3.1 Effect of substrate

The selection of a suitable substrate for fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial

73

to 7.88 U mL-1 of parental strain, after 36 hours incubation, the biosynthesis of enzyme decreased with increase in time (Fig. 4.14 and 4.15). Moreover, ANOV A table also supported the better increase in enzyme production after 36 hours fermentation (Tables 4.4 and 4.5).

Petruccioli et 01. (1995) reported that maximum production of glucose oxidase from UV -mutant of Penicillium varia bile M-80.10, was achieved after 96 hours. Pazur (1966) and Kona et 01. (2001) optimized the fermentation media for the production of glucose oxidase by Aspergillus niger and obtained highest glucose oxidase yield after 48 hours of fermentation. Maximum intracellular enzyme reached its highest value after 60 hours as reported by Fiedurek and Gromada (2000a). These values reported earlier are different to our findings due to di fferent conditions as organism, substrate etc.

4.3.3 Effect of pH

After selecting the suitable substrate level and fermentation period, pH of medium was optimized to get maximal enzyme yield. The maximum glucose oxidase production from parental strain, was obtained at pH 5.5. The results are shown in Fig. 4.16. While, mutant strain showed optimum production at pH 5 (149.39 U mL-J) (Fig. 4.17). These findings were also supported by statistical analysis through ANOVA (Tables 4.6 and 4.7)

Optimal pH is very important for growth of microorganism and its metabolic activities. As the metabolic activities of the microorganism are sensitive to changes in pH, so glucose oxidase production by A. niger, found to be affected at higher or lower pH as compared to optimum one. This is attributed for production of a PaCC protein by different hydrogen ion in the medium and do not allow the expression of the same to take place.

The results showed a good coincidence with the reports from Kusai et al. (1960) and Petruccioli and Federici (1993) that optimal pH for the synthesis of glucose oxidase by the most fungi was 5-5.5. Liu et al. (1999) obtained the highest biosynthesis of glucose oxidase A. niger at pH 6, showing as optimum. Semashko et al. (2004) also subjected the medium to pH 5 for glucose oxidase production by mutant strain of Penicillium.

75

7.00
~
..J
E 5.60 -
;J
'-'
Co
~ 4.20
<..I
~
~
'"
~ 2.80
:s!
)o'!
0
~
'"
0
<..I 1.40
:::I
e
0.00
0 Fig. 4.12.

::;-..J

E 100

::l

.....

,C •

. ;; 75- '';::

<..I

~

~

'"

cos

:s! 50

y. o <OJ

'" o

~ 25-

c

Fig. 4.13.

5

2

Substrate conc. ('}'o)

Production of glucose oxidase by parental culture with varying substrate levels

o

Substrate COliC .(%)

Production of glucose oxidase by mutant culture with varying substrate levels

76

Table 4.2.

Analysis of variance for glucose oxidase production by parental culture at different substrate concentrations

SoY df SS MSS F-val'ue Probability
Treatment 5 52.771 10.554 145.73 0.000
L 1.705 1.705 23.68
Q 24.835 24.835 344.93
Lack of Fit 3 26.231 8.744 121.44
Error 12 0.869 0.072
Total 17 53.640 Standard deviation: 0.269

CV: 10.72%

Treatment Mean

o 0.96

2.96

2 6.06

3 2.22

4 1.59

5 1.29

SE of mean: 0.155

SE of difference: 0.219

y = 1.394 + 2.174X -0.471 X2
SE = 0.703 0.662 0.127
T = 1.98 3.29 -3.71
p = 0.066 0.005 0.002
(where, Y= Response; X= Substrate concentration) 77

Table 4.3.

Analysis of variance for glucose oxidase production by mutant culture at different substrate concentrations

SoY df SS MSS F-value Probability
Treatment 5 29919.86 5983.97 981.34 0.000
L 1982 1982 325.45
Q 25322 25322 4157.96
Lack of Fit 3 2615.9 871.97 143.18
Error 12 73.17 6.09
Total 17 29993.03 Standard deviation: 2.47

R-Square: 0.9976

CV: 4.10%

Treatment Mean

o 1.02

2

3

4 79.23

5 25.72

41.68

1 17.42 96.27

SE of mean: 1.426
SE of difference: 2.016
y = -5.263 + 81.326X -15.036X2
SE = 7.005 6.59 1.265
T = -0.75 12.34 -11.89
P = 0.464 0.000 0.000 (where, Y= Response; X= Substrate concentration)

78

10
--
.J
E 8
~
'-'
,Co
:~ 6
u
cos
101
or.
~ 4
~
';:1
0
101
III
0
u 2
..::
r.,)
0 Fig. 4.14.

ISO
~
~ J20
E
~
'-'
c·
:~ 90
u
cos
101
'"
~ 60
:=
><e
0
...
III
0
u 30
..::
r.,)
0 Fig. 4.15.

-r-
I .~
I
-=r=_
I .--1-
-=--
I .- I -

1 I
I 24

36

72

48

60

Fermentation period (h)

Effect of fermentation period on enzyme production by parental culture

---r-
! __r::::::
,.
-=c-
_:..L,..
-,-
-=::c=_ I I ~I,
I ~
I I
,.
I - ~
-
,
;,
, I
"'I, ,. I 36

48

60

72

Fermentation period (h)

Effect of fermentation period on enzyme production by mutant culture

79

Table 4.4.

Analysis of variance for glueose oxidase production by parental derivative at different fermentation periods

SoY df SS MSS Fvvalue Probability
Treatment 4 74.552 18.638 48.21 0.000
L 17.618 17.618 45.52
Q 28.785 28.785 74.379
Lack of Fit 2 28.149 14.07 36.36
Error 10 3.866 0.387
Total 14 78.418 Standard deviation: 0.622

CV: 14.89%

Treatment 24

Mean 3.05

36 7.88

48 5.56

60 2.5

72 1.9

SE of mean: 0.359

SE of difference: 0.507

y = -4.348 + 0.488X -0.0057X2
SE = 3.748 0.169 0.00175
T = -1.16 2.87 -3.28
p = 0.269 0.014 0.007
(where, Y= Response: X= Fermentation period) 80

Table 4.5.

Analysis of variance for glucose oxidase production by mutant derivative at different fermentation periods

SoY df SS MSS Fvvalue Probability
Treatment 4 11197.5 2799.4 1435.13 0.000
L 2666.6 2666.6 1333.3
Q 7264.8 7264.8 3632.4
Lack of Fit 2 1266.1 633.05 316.53
Error 10 19.5 2
Total 14 11217 Standard deviation: 1.397

CV: 1.85%

Treatment 24

Mean 61.38

36 48

111.94 100.49

60 67.2

72 36.61

SE of mean: 0.806

SE of difference: 1.14

y = -70.89 + 7.982X -0.0913X2
SE = 23.75 1.076 0.01 [1
T = -2.98 7.42 -8.23
P = 0.11 0.000 0.000
(where, Y= Response; X= Fermentation period) 81

15~---------------------------------------------'

Co

:~ 9

u ~ 4; ." ~

"'0 6 ';:(

e

~

~

g J

o

;--
..J
E 12
:J
'-' ".5

5,5

6

6,5

7

pH

Fig. 4.16.

Optimization of pH for maximal yield of glucose oxidase by parental culture

Fig. 4.17.

Optimization of pH for maximal yield of glucose oxidase by mutant culture

82

Table 4.6.

Analysis of variance for glucose oxidase production by parent derivative at different pH

SoY df S8 MSS F-value Probabilit)'
Treatment 6 236.29 39.82 81.42 0.000
L 5.27 5.27 10.89
Q 196.31 196.31 405.59
Lack of Fit 3 34.71 8.68 17.93
Error 14 6.77 0.48
Total 20 243.06 Standard deviation: 0.695

CV: 10.29%

Treatment 4

Mean 3.81

4.5 5.05

5 11.01

5.5 11.75

6 7.94

6.5 5.61

7 2.13

SE of mean: 0.402

SE of difference: 0.567

y = -93.75 + 38.33X -3.53X2
SE = 11.34 4.22 0.38
T = -8.27 9.08 -9.23
P = 0.000 0.000 0.000
(where, Y= Response; X= pH) 83

Table 4.7.

Analysis of variance for glucose oxidase production by mutant derivative at different pH

SoY df SS MSS Fvvalue Probability
Treatment 6 11344.9 1890.8 1072.76 0.000
L 971.7 971.7 539.83
Q 9988.7 9988.7 5549.28
Lack of Fit 3 384.5 96.125 53.40
Error 14 24.7 1.8
Total 20 11369.6 Standard deviation: 1.328

CV: 1.11%

Treatment 4

Mean 97.85

4.5 123.6

5 5.5 6 6.5

149.39 141.35 137.15 106.81

7 81.39

SE of mean: 0.767

SE of difference: 1.083

y = -579.55 + 270.21X -25.183X2
SE = 35.62 13.26 1.202
T = -16.27 20.38 -20.96
P = 0.000 0.000 0.000
(where, Y= Response; X= pH) 84