Studies on the production of alpha amylase by Aspergillus oryzae using submerged fermentation

ROHEENA ABDULLAH

10 Bot

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY GC UNIVERSITY LAHORE

A THESIS TITLED

Studies on the production of alpha amylase by Aspergillus oryzae using submerged fermentation

Submitted to GC University Lahore in fulfillment of the requirements for the award of degree of

Doctor of Philosophy
IN BOTANY By ROHEENA ABDULLAH

10 Bot

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY GC UNIVERSITY LAHORE

ii

DECLARATION

I Miss Roheena Abdullah Roll No.10-Bot-PhD-2005 student of PhD in the subject of botany, hereby declared that the matter printed in this thesis titled Studies on the production of alpha amylase by Aspergillus oryzae using submerged fermentation is my own work and has not been printed, published and submitted as research work, thesis or publication in any form in any university, research institution etc. in Pakistan or abroad.

Date:_______________

______________________

Signature of Deponent

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RESEARCH COMPLETION CERTIFICATE

Certified that the research work contained in this thesis titled Studies on the production of alpha amylase by Aspergillus oryzae using submerged fermentation has been carried out and completed by Miss Roheena Abdullah Roll No 10-Bot phD-05 under my supervision during her Ph.D studies in the subject of Botany.

_________________

_______________________

Date

Prof. Dr Ikram-ul-Haq (S.I) Supervisor

Submitted through

___________________ Prof. Dr Ikram-ul-Haq (S.I) Director, Institute of Industrial Biotechnology GC University, Lahore

_____________________ Controller of Examinations GC University, Lahore

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ACKNOWLEDGEMENTS
All praise for the, ALMIGHTY ALLAH who is the only supreme Authority and whose presence has been figured on the two words i.e. KUN FAYAKUN. Every tiny or massive entity moves with His permission. Countless thanks to Him for accrediting me to accomplish this important task with in this specified time. All my respect and regards to the Holy Prophet Hazrat Muhammad (peace be upon him) who is forever a torch of guidance and knowledge for humanity. In view of his saying: He who does not thank to people is not thankful to Allah I am highly obliged in paying deepest gratitude to my respected teacher and research supervisor Prof. Dr. Ikram-ul-Haq, SI (Director, Institute of Industrial Biotechnology, GCU, Lahore for his valuable guidance, encouragement, cooperation and discussion. His enthusiastic inspiration and fatherly affection enabled me to attain the objectives without any difficulty. I most great fully acknowledge my indebtedness to Dr. M. A. Qadeer and Dr. Muhammad Yaqub, Dr. Sikander Ali, Dr. Hamid Mukhtar, Dr. Mohsin Javed, and Dr. Numan Aftab for their scholarly, scientific discussions and generous advices when needed, during the entire period of my research work. I am thankful to highly esteemed Dr. Zaheer-ud-Din Khan, (Chairperson Department of Botany, GCU, Lahore) and Dr. Amin-ul-Haq Khan, Dean, Faculty of Science and Technology, GCU, Lahore for providing all the necessary facilities through out my research duration. I am grateful to Dr. Khalid Aftab, Vice

v Chancellor, GC University, Lahore for providing me this opportunity to work in this great Institute The words are inadequate to express my heartfelt thanks to my friends and fellows Aafia Aslam, Zahid Butt, Shazia Malik and Tehreema Iftikhar, for their moral support in the research work. I feel pleasure to acknowledge Dr. Shakeel (Assistant Professor Department of Pathology Punjab University) for helping in the identification of strain. I am also thankful to laboratory staff especially Mr. Fasial, Mr. Usman , Mr Ramez and all others for their full cooperation during the whole period of my research. Although feelings are deep but unfortunately words are too shallow, that cannot follow the depths of my deep gratitude to my loving mother and father Mr. and Mrs. Abdullah. My fortune is due, to their prayers.

ROHEENA ABDULLAH

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CONTENTS
Minor contents Title Declaration Research Completion Certificate Acknowledgements Contents List of Tables List of Figures Abstract Major contents Chapter# 1 : INTRODUCTION Objective Chapter# 2: REVIEW OF LITRATURE Uses of alpha amylase Chapter# 3 : MATERIALS AND METHODS 3.1. Materials 3.2. Methods 3.2.1. Isolation of organism 3.3. Fermentation 3.3.1. Inoculum preparation 3.3.1.1. Conidial inoculum 3.3.1.2 Conidial count 3.3.1.3. Vegetative inoculum 3.3.2. Fermentation media 3.4. Shake flasks studies 3.5. Fermenter studies 3.6. Nutritional and cultural requirement of Aspergillus oryzae 3.6.1. Fermentation media 3.6.2. Incubation period 3.6.3. Effect of initial pH 3.6.4. Effect of temperature 3.6.5. Effect of volume 3.6.6. Effect of inoculum size 3.6.7. Effect of agitation 3.6.8. Evaluation of carbon sources 3.6.9. Evaluation of nitrogen sources 3.7. Induction of mutation 3.7.1. Minimal inhibitory concentration of 2-deoxy-D-glucose 3.7.2. Ultraviolet (UV) irradiation 1 8 9 44 49 49 49 49 51 51 51 51 51 52 52 53 53 53 53 54 54 54 54 55 55 55 55 55 56 Page No i ii iii iv vi ix x xiii

vii 3.7.3. Nitrosoguanidine treatment 3.7.4. Nitrous acid treatment 3.7.5. EMS treatment 3.7.6. Selection of mutants 3.8. Analytical techniques 3.8.1. Estimation of alpha amylase 3.8.2. Estimation of total protein contents 3.8.3. Determination of mycelial morphology 3.8.4. Estimation of dry cell mass (DCM) 3.9. Statistical analysis 3.10. Kinetic study 3.11. Purification of alpha amylase 3.11.1. Separation of fungus from fermented broth 3.11.2. Ammonium sulfate precipitation 3.11.2.1. Anion exchange chromatography 3.11.2.2. Gel filtration 3.11.3. Dialysis 3.11.4. Electrophoresis 3.11.5. Protein Marker 3.12. Gel Preparation 3.12.1. Separating gel 3.12.2. Stacking gel 3.13. Characterization of enzyme 3.14. Standard curves 3.14.1. Maltose 3.14.2. Bovine serum albumin 3.15. Preparation of reagents/ buffers 3.15.1. DNS reagent 3.15.2. Brad ford reagent 3.15.3. Starch Solution 3.15.4. Acetate Buffer (pH 5.0) 3.15.5. Phosphate Citrate Buffer (pH 7.5) 3.15.6. Tris HCl buffer (pH 7.5) 3.15.7. Acrylamide bis acrylamide (30%) 3.15.8. Separating buffer (1.5 M Tris HCl, pH 8.8) 3.15.9. Stacking buffer (1M Tris HCl, pH 6.8) 3.15.10.Tank Buffer 3.15.11.Gel loading buffer 3.15.12. SDS Solution (10%) 3.15.13. Ammonium per sulfate 3.15.14. Staining and Destaining solution Chapter # 4: RESULTS AND DISCUSSION 4.1. Identification, isolation and screening of organism 4.2.Strain improvement 4.2.1. Physical mutagenesis 56 57 57 57 58 58 58 59 59 59 59 60 60 61 61 61 62 62 62 62 63 63 64 64 64 64 65 65 65 65 66 66 66 66 67 67 67 67 67 68 68 71 71 75 75

viii 4.2.1.1. Screening of UV treated isolates 4.3. Chemical Mutagenesis 4.3.1. Screening of NG treated isolates 4.3.2. Screening of nitrous acid treated isolates 4.3.3.Screenning of EMS treated isolates 4.4. Optimization of cultural conditions in shake flasks 4.4.1. Screening of Culture media 4.4.2. Rate of alpha amylase production 4.4.3. Effect of incubation temperature 4.4.4. Effect of different initial pH 4.4.5. Effect of different volume of medium 4.4.6. Effect of inoculum size 4.5. Optimization of nutritional requirements of A. oryzae in shake flasks 4.5.1. Effect of starch from different sources 4.5.2. Effect of different concentrations of corn starch 4.5.3. Evaluation of additional carbon sources 4.5.4. Evaluation of inorganic nitrogen sources 4.5.5. Evaluation of organic nitrogen sources 4.5.6. Effect of surfactants 4.6. Optimization of cultural conditions in stirred fermenter 4.6.1. Rate of alpha amylase production 4.6.2. Effect of pH 4.6.3. Effect of aeration levels 4.6.4. Effect of dissolved oxygen 4.6.5. Effect of inoculum size 4.6.6. Effect of agitation intensity 4.7. Purification of alpha amylase 4.7.1. Ammonium sulfate precipitation 4.7.2. Step wise purification 4.7.2.1. Ammonium sulfate precipitation 4.7.2.2. Anion exchange chromatography 4.7.2.3. Gel filtration 4.8. Characterization 4.8.1. Temperature optima of purified alpha amylase 4.8.2.Effect of time of incubation on the activity of purified alpha amylase 4.8.3. Effect of distilled water and buffer on the activity of purified alpha amylase 4.8.4. Effect of pH on the activity of purified alpha amylase 4.8.5. Effect of metal ion on the activity of purified alpha amylase Discussion Conclusion Chapter # 5 : References 75 78 78 78 79 86 86 86 87 87 87 88 95 95 95 96 96 97 97 109 109 110 110 111 111 112 125 125 125 125 125 126 132 132 132 132 133 133 139 148 149

ix LIST OF TABLES

Table 4.1 4.1.1 4.2 4.2.1 4.2.2 4.3 4.3.1 4.3.2 4.4 4.4.1 4.4.2 4.5 4.5.1 4.5.2 4.6

4.7

4.8

4.9

4.10

4.11

4.12 4.13

Title of Table Page Isolation and screening of A. oryzae for the alpha amylase 72 production Sub grouping of alpha amylase producing isolates of A. oryzae 74 Screening of UV isolates of A. oryzae IIB-30 for alpha amylase 76 production UV treated survivors at different exposure time 77 Range of alpha amylase activity of UV isolates 77 Screening of NG treated A. oryzae UV-23 isolates for alpha 80 amylase production NG treated survivors of A. oryzae 81 Range of alpha amylase activity of NG isolates 81 Screening of nitrous acid treated strains of A. oryzae NG-18 for 82 the alpha amylase production Nitrous acid treated survivors of A. oryzae 83 Range of alpha amylase activity of nitrous acid treated isolates 83 Screening of EMS treated A. oryzae NA17 for the alpha 84 amylase production EMS treated survivors of A. oryzae 85 Range of alpha amylase activity of EMS treated isolates 85 Kinetic evaluation of rate of fermentation for the alpha amylase 114 production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different pH values of media for the alpha 116 amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different aeration for the alpha amylase 118 production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different levels of dissolved oxygen for the 120 alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different inoculum sizes for the alpha 122 amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different agitation speeds for the alpha 124 amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter Purification summary of alpha amylase produced by mutant 127 strain of A. oryzae EMS-18 by using ammonium sulfate Step wise purification profile of alpha amylase produced by 128 mutant strain of A. oryzae EMS-18.

LIST OF FIGURES

Figure 3.1 3.2 4.1

Title of Figure Standard curve of maltose Standard curve of bovine serum albumin (BSA) Screening of fermentation media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of incubation temperature on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different initial pH of fermentation medium on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different volume of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different inoculum sizes on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of raw starch from different sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. Effect of different concentrations of starch on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. Effect of additional carbon sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different concentrations of lactose on the alpha amylase production by A. oryzae IIB-30 and its

Page 69 70 89

4.2

90

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91

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92

4.5

93

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94

4.7

99

4.8

100

4.9

101

4.10

102

xi mutant derivative A. oryzae EMS-18 4.11 Effect of inorganic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different concentrations of ammonium sulfate on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of organic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different concentrations of peptone on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different surfactants on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. Effect of different concentrations of Tween 80 on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. Comparison of rate on the alpha amylase production by wild (IIB-30) and mutant strain of A. oryzae (EMS-18) in stirred fermenter Effect of initial pH of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different aeration levels on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different level of dissolved oxygen on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different inoculum size on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different agitation intensity on the alpha 103

4.12

104

4.13

105

4.14

106

4.15

107

4.16

108

4.17

113

4.18

115

4.19

117

4.20

119

4.21

121

4.22

123

xii amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 4.23 4.24 4.25 Elution pattern on Sephadex DEAE The elution profile on Sephadex G-100 SDS-PAGE analysis of pooled fractions of ion exchange chromatography and ammonium sulfate fractionation. Effect of temperature on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 Effect of different pH on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 Effect of metal ions on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18. 129 130 131

4.26

134

4.27 4.28

135 136

4.29 4.30

137 138

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Abstract
The present study, deals with the isolation, screening and selection of Aspergillus oryzae for the alpha amylase production. Seventy eight isolates of A. oryzae were isolated from different soil samples. The strains were initially selected qualitatively on starch agar medium and screened quantitatively for enzyme production in shake flasks and a strain producing 130 0.1U/ml of enzyme was selected which was assigned the code IIB-30. The selected strain was subjected to physical and chemical mutagenic treatments in order to improve its amylolytic potential. During the treatments, isolates were qualitatively and quantitatively screened. Among these, EMS-18 exhibited the highest enzyme activity (3471.2 U/ml). This mutant showed 2.6 fold increased activity over the parental strain in terms of enzyme production. The cultural conditions and nutritional requirements of the selected strains (both wild and mutant) were optimized in 250 ml Erlenmeyer flasks prior to scale up studies in a fermenter. Six different fermentation media were evaluated for the alpha amylase production by both wild and mutant strains of A. oryzae in shake flasks fermentation. Of all the media, M4 containing (g/l); starch 20, yeast extract 8.5, NH4Cl 1.3, MgSO4.7H2O 0.12, CaCl2 0.06 gave maximal enzyme production i.e., 1682 (wild) and 3852 (mutant) which was highly significant (p0.05). The effect of incubation temperature, initial pH, volume of media and inoculum size was investigated on the enzyme production. The optimal enzyme production was obtained at 30C, pH 5, volume, 10 % and inoculum size 4 %, by both wild and mutant strains. The rate of fermentation was also studied and the highest yield of enzyme was obtained 72 h after inoculation.

xiv Corn starch (2 %) and lactose (1.5 %) as carbon sources while, ammonium sulfate (0.3 %) and peptone (0.2 %) as nitrogen sources were also optimized. Different surfactants were added to the fermentation media and Tween 80 at the level of 0.1% was found to be the best for enzyme production. The scale up studies for alpha amylase production was carried out in a 7.5 L stirred fermenter. The rate of fermentation for enzyme production by both wild and mutant strains was investigated.) It was found that the enzyme production increased gradually and reached maximum (335 U/ml and 608 U/ml) after 64 h (wild) and 48 h (mutant). The kinetic depiction of results showed optimal fermentation period for enzyme production to be 64 h and 48 h, respectively. The other cultural conditions such as initial pH (5), aeration level (1.5 vvm), dissolved oxygen (15 %), inoculum size (10 %) and agitation intensity (200 rpm) were optimized for enzyme production. The fermented broth was subjected to ammonium sulfate precipitation at different saturation levels (20-90 %). The optimum level of ammonium sulfate saturation was found to be 70 % that gave 1.3 fold purification. By using SephadexDEAE column, the active fractions were eluted using 0.05 M Tris-HCl buffer containing 0.30 M NaCl at pH 7.5. The molecular weight of alpha amylase was found to be 48 kDa on SDS-PAGE after gel filtration. A total of 9.5 fold enzyme purification was accomplished. The effect of time, temperature, pH and metal ions on purified enzyme was also investigated and maximum activity was achieved after 30 min at 40C and pH 5 in the presence of Ca+2 ion.

INTRODUCTION
The starch degrading enzyme alpha amylase (-1,4 glucan-glucanohydrolase EC 3. 2. 1. 1) is widely distributed in nature. This extracellular enzyme hydrolyses -1,4 glucosidic linkages randomly throughout the starch molecule in an endo-fashion producing oligosaccharides and monosaccharides including maltose, glucose and alpha limit dextrin (Omemu et al., 2005; Bhanja et al., 2007; Leman et al., 2009). Alpha amylases are one of most important and widely used enzymes whose spectrum of application has widen in many sectors such as clinical, medicinal and analytical chemistry. Beside their use in starch saccharification they also find applications in food, baking, brewing, detergent, textile and paper industries. These are important enzymes used in starch processing industries for hydrolysis of polysaccharides such as starch into simple sugar constituents. Increasing utility and consumption of alpha amylase in different industries has placed a greater stress on increasing indigenous enzyme production and search of more rapid processes (Carlsen et al., 1996; Ramachandran et al., 2004; Kathiresan and Manivanan, 2006; Gupta et al., 2008). Alpha amylase can be derived from several sources such as plants, animals and microorganisms, but production from first two groups is limited for several reasons. The concentration of enzymes in the plant material is generally low so the processing of large amount of plant material is necessary; on the other hand enzyme of animal origin is by- product of meat industry. In contrast, microbial source of alpha amylase can be produced in amount meeting the demands of market. Different fungal and bacterial strains have been extensively used for the enzyme production (Pandey et al.,

2 2000). Filamentous fungi have been well known for the starch and cellulose degrading enzymes they naturally secrete. The ability of filamentous fungi to secrete large amounts of extracellular protein has made them well suited for the industrial enzyme production. The commonly used fungi included Trichoderma sp. Themomyces lanuginosus, Penicillium griseoreseum, Fusarium moniliformis, Actinomycetes sp. and Alternaria sp. (Arnesen et al., 1998; Ray 2001; Poornima et al., 2008). Many species of Aspergillus such as A. niger, A. tamarii, A. awamori and A. oryzae have received most attention to obtain many kinds of hydrolytic enzymes like alpha amylase, lipase and protease. However, A. oryzae is the organism of choice because of its ubiquitous nature, non fastidious nutritional requirements and high productivity of alpha amylase (Abe et al., 1988; Archer and Wood, 1995; Agger et al., 2001; Zangirolami et al., 2002). Strain selection is a critical step in the development of a biotechnological process, and it is based on a number of factors such as physiological stability, yield consistency, incubation time required for maximum production as well as the tolerance to temperature, aeration and shear stress etc. (Laluce et al., 1991). Production of enzyme is greatly effected by the cultivation method. Alpha amylase can be produced both by solid state and submerged fermentation technique (Prescott and Dunn, 1987; Nielsen et al., 1995; Yovita et al., 2005). The liquid culture used in submerged fermentation was usually preferable to solid state culture not only due to it allowing better aeration and proper agitation but also the separation of enzyme from the solid substrate is more difficult than submerged fermentation (Alazard and Raimbault, 1981). Morphological variety is a typical feature of filamentous fungi. Its

3 morphology has distinct effects both on the enzyme production and rheological nature of a fermentation broth. In submerged fermentation, two extreme types of morphology are generally known, pellets and free filaments. Between these two extremes lies an intermediate aggregated morphology called clumps (Wang et al., 2005). The morphology of filamentous organism during enzyme production varies from round pellets to free filaments depending upon the cultural conditions and strain genotype. Traditional methods for strain improvement, such as ultra violet (UV) radiation, use of alkylating agents like N-methyl N-nitro N-nitroso guanidine (NG), ethyl methane sulphonate (EMS) and nitrous acid to obtain superior mutants have been proved successful by subjecting the microorganisms to these mutagens, followed by suitable selection and screening of the survivors (Szafraniec et al., 2003). However, strain improvement is trial and error process involving laborious procedure. Rational selection procedures are more efficient and usually have a biochemical basis (Elander 1982). In primary screening prior to laboratory fermentations, rational selection is achieved by the use of techniques allowing visual identification of superior mutations. The selection of alpha amylase producers using the size of the zone of hydrolysis of starch is an example. However zonation can not in any way be correlated quantitatively with the amount of alpha amylase produced because the hydrolytic activity of other amylolytic enzymes such as glucoamylase. Therefore isolation of improved producers of alpha amylase using starch plate can only be partially selective (Kuek and Kidby, 1984). Mutant strains of Aspergillus oryzae were found to be best for enzyme production compared to wild strain. It was studied a mutant strain of A. oryzae showed more dextrinizing and saccharogenic activity than the parental strain.

4 In case of mutagenic application to the wild strain, better initial improvement can be expected. A strain of A. oryzae treated with NG gave better enzyme production compared to the parental strain. A best mutant for alpha amylase production can be obtained by irradiating the fungal strain to the UV irradiation and then successive treatment with mutagenic chemicals like NG, EMS etc. (Spohr et al., 1998; Qirang and Zho, 1994; Azin and Noroozi, 2001) Selection of suitable fermentation medium and initial pH is very important for the enhanced alpha amylase production. All microorganisms require energy and certain minerals for growth and metabolism. The energy for growth generally comes from the oxidation of medium components. The presence of carbon, nitrogen sources and mineral nutrients such as phosphorous, potassium, magnesium, and calcium are essential for the growth of fungi as well as enzyme production (Hughes and Poole 1991). The enzyme production has been greatly affected by the addition of different carbon sources. The carbon sources affect not only the mode of amylase formation but also the rate with which carbohydrates are metabolized (Dubey et al., 2000; Abdullah et al., 2003). The influence of different carbon sources such as glucose, maltose, fructose, galactose and sucrose on the alpha amylase production by A. oryzae was studied and it was found that starch and maltose strongly increased enzyme productivity by A. oryzae where as glucose led to very low productivity (Lachmund et al., 1993; Carlsen and Nielsen., 2001). So it is important to select suitable carbon source for the enhanced enzyme production. Fungal strains have been grown on starch, maltodextrin, dextrin, maltose, amylopectin, glucose and dextran. All these

5 substrates exhibited good alpha amylase production. The optimum pH of fermentation medium was found and fixed to 4.9 by using 100 mM citrate buffer for the enzyme production. Various concentrations of soluble starch and soybean meal were used in cultivating the organism. The highest enzyme activity was recorded with starch (Omidiji et al., 1997; Moreira et al., 1999). A strain of Aspergillus sp. was able to produce enzyme in mineral media supplemented with 1.0 % (w/v) starch or maltose as carbon source. The alpha amylase production was found to be tolerant to a wide range of initial pH values (4.0-10) and temperature (25-42C). Aspergillus sp. isolated from soil produced extracellular glucoamylase and alpha amylase using wheat starch as a carbon source. The enzyme productivity was doubled by the addition of -methyl-Dglucoside to the medium (Junichi et al., 1988). Different inorganic and organic nitrogen sources and their concentrations have major influential impact on their ability to synthesize the enzyme as well as on the growth of organism (Bailey and Ollis, 1977; Bajpai and Sharma, 1989; Hashim et al., 1993). Both inorganic and organic nitrogen sources were tested for alpha amylase production. Among the inorganic nitrogen sources, nitrate has been shown to be inferior to ammonia. A mixture of ammonia and complex nitrogen sources such as yeast extract or casein hydrolysate was found to be better than ammonia as nitrogen source. Low concentration of casein hydrolysate resulted increase in alpha amylase productivity (Pedersen and Nielsen, 2000). The organic nitrogen sources such as peptone, yeast extract, tryptophan and corn steep liquor are widely used for enzyme production. By the use of these nitrogen sources organism grew better and produced higher levels of enzyme activity. However, urea and casein hydrolysate showed

6 marked effect on enzyme production by A. oryzae (Kammoun et al., 2008). Influence of inoculum age and size on alpha amylase production should be optimized in depth investigation before scaling up a high-yielding fermentation process (Bokosa et al., 1992). The amount of inoculum introduced into the culture medium determines the extent and quality of enzyme produced. So, there exists a correlation between amount of inoculum and substrate concentration in context to alpha amylase production by A. oryzae. Surfactants play an important role in increasing the enzyme production. Alpha amylase activity was increased in the presence of surfactants because surfactants increase the cell membrane permeability as a result enzyme secretion increased. Different surfactants such as Tween 80, Triton X-100 and poly ethelyen glycols were used to increase the permeability of cell membrane (Arnesen et al., 1998; Yoon et al., 2005) Fermenters of different working volumes may be used for the large scale alpha amylase production as an industrially important enzyme under controlled conditions. By optimizing the cultural conditions such as inoculum size, nutritional requirements, temperature, pH, agitation, aeration, and dissolved oxygen etc. the enzyme production can be enhanced by many fold (Gigras et al., 2002). Enzyme production commences at a low rate during the logarithmic growth phase but reaches its maximum value during the stationary phase towards onset of sporulation. Time course study and agitation determines the efficacy of the batch process and subsequent product formation. The pattern of accumulated reducing sugar after specific incubation time is characteristic to the species (Matrai et al., 2000). Alpha amylase production at different agitation rates (100-300) at 30C were tested and maximum amount of

7 enzyme was obtained at 150-200 rpm after 72 h. According to Francis et al. (2002), the maximum alpha amylase production was obtained after 120 h in a fermenter operating at 300 rpm and airflow of 11/L/min in a limited dissolved oxygen concentration. It was determined that the increase in agitation rate was not favorable for enzyme production; despite of this an increase was verified in dissolved oxygen. Enzyme production was superior with the A. oryzae NRRL 6270 at 30C after 96 h when spore suspension used 1 x107 spores/ml. Industrial enzymes produced in bulk generally require little downstream processing and hence are relative crude preparations. The applications of enzyme in pharmaceutical and clinical sectors etc. require high purity amylase. The enzyme in purified form is also a prerequisite in studies of structure function relationships and biochemical properties. The purification of enzyme is to remove as completely as possible all the proteins except which possess the specific enzyme activity desired. A frequently used method in enzyme purification is salt fractionation. Ammonium sulfate is often used for this purpose because of its high solubility (700 g/l) which permits the salting out of any protein. The properties of alpha amylase in culture broth were examined by partially purified enzyme with 60 % ammonium sulfate. Alpha amylase from Aspergillus sp. subjected to purification and characterization under optimum conditions. The enzyme was purified by ammonium sulfate precipitation and Sephadex G200 filtration. The purification of alpha amylase resulted 9.97 fold purification. The optimum substrate (starch) concentration was 0.2 % (W/V) while the optimum incubation temperature was 35C. The purified enzyme had maximum activity at pH 6.2, after 30 h of incubation (El-Safey and Ammar, 2002; Pimpa 2004).

8 Alpha amylase purified from the cultural broth of A. oryzae indicated 12.6 fold purification and yield being 25.3 %. The molecular weight of alpha amylase from A. oryzae was estimated to be 50 kDa. The purified enzyme was most active at pH 4.5 and temperature 55C (Kariya et al., 2003). The production and stability of the enzyme is very sensitive to pH and temperature. Fungal alpha amylase was unstable above 45C but at 25C attack raw starch granules more efficiently than enzyme from Bacillus amyloliquefaciens. The optimum growth conditions for enzyme production by A. oryzae was pH 5.0 and 35oC (Fairbairn et al., 1986; Jin et al., 1998). The enzyme retained 94 % activity in 1 h at 60C. The alpha amylase is an unusual enzyme which converts starch to maltose in > 75 % yield. The purified enzyme, obtained in 11 % yield had optimal temperature and pH 50-55C and 5.0- 6.0, respectively. It may be of industrial value in the production of low viscosity corn syrups (Hidaka et al., 1980). Objectives Specific objectives of present work are as follows 1-Isolation, identification and screening of A. oryzae strains.

2-Random mutagenesis by UV and chemicals to improve the fungal strain as well as

enzyme production. 3-Optimization of cultural conditions for the selected strain of A oryzae in shake flasks. 4-Scale up studies of enzyme production in a laboratory scale stirred fermenter. 5- Purification and characterization of alpha amylase.

REVIEW OF LITERATURE
Starch degrading amylolytic enzymes is of great importance in biotechnological application ranging from food, fermentation, and textile to paper industries etc. Alpha amylase is a key enzyme in metabolism of spacious diversity of living organisms which utilize starch as carbon and energy sources. It can hydrolyze starch, glycogen and related polysaccharides by randomly cleaving internal -1,4-glucosidic linkages to produce different sizes of oligosaccharides. Amylases are enzymes which hydrolyze the starch molecules in to polymers consists of glucose units. Alpha amylase is ubiquitous in distribution, with plants, bacteria and fungi being the major sources. Most of the microbial alpha amylases belong to the family 13 glycosyl hydrolases, and they contributed numerous common properties. But different reaction specificities have been observed across the family members. Structurally alpha amylase possesses barrel structures and is responsible for hydrolysis or formation of glycosidic bonds in the conformation. Stability of alpha amylase has extensively been studied; pH and temperature have very vital roles to play. Alpha amylase acts on starch and breaking them up into sugars (hence the term saccharification). Starch is a carbohydrate source consisting of two molecules amylose and amylopectine. Amylose is formed from chains of glucose linked 1,4 and amylopectine is formed from 1,4 linked chains of glucose with 1,6 linked branch points. The amylases are enzymes that work by hydrolyzing the straight chain bonds between the individual glucose molecules that make up the starch chain. A single straight chain starch is called an amylose. A branched starch chain (which can be

10 considered as being built from amylose chains) is called an amylopectin. These starches are polar molecules and have different ends.

Alpha amylase can be derived from several sources such as plants, animals and microbes. The microbial enzyme meets the industrial demands a large number of them are available commercially and have almost replaced chemical hydrolysis of starch processing industry (Pandey et al., 2000). The major advantage of using microorganisms for the amylase production is economical bulk production capacity and microbes are also easy to manipulate to obtain enzymes of desired characteristics (Lonsane and Ramesh, 1990). Alpha amylase has been derived from several fungi, yeasts, bacteria and actinomycetes, however, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. Fungal sources are mostly terrestrial isolates such as Aspergillus species. Mode of action, properties and product of hydrolysis differ, some what and depend on the source of enzyme. Two types of enzymes have been recognized called as liquefying and saccharifying. The main difference between them is that the saccharifying enzyme produces a higher yield of reducing sugar than liquefying enzyme. Many scientists carried out extensive work on

11 alpha amylase production. The enzyme production is dependent on the type of strain, composition of media and methods of cultivation. Generally fungi secrete alpha amylase (dextrinizing enzymes) although a few fungi have been known to secreted alpha amylase and beta amylase (saccharifying enzymes). A. oryzae EI 212 secrete alpha and beta amylase or both depending upon the composition of media and fermentation conditions. The nature and amount of extracellular amylase produced by Aspergillus species determine the efficiency of conversion of starch to oligosaccharides. Tokhadze et al. (1975) isolated 86 strains of the Aspergillus producing maximum acid stable alpha-amylase. Repeated cultivation of the selected strains in the Minoda agar medium along with sodium nitrate during submerged cultivation showed a 3-fold increase in the alpha amylase production. Yabuki et al. (1977) studied rapid induction in the alpha amylase production by A. oryzae using inducer such as maltose. The mycelia were taken from 20 h old cultures and cultivated on the medium containing peptone and glycerol. Afterwards these cultures were starved for 5 h; in this case maltose was added as inducer. During first hour of induction, both extra and intracellular alpha amylases were produced with the same rate (70-80/g of cells/h). After 1.5 h remarkable increase in alpha amylase production takes place and enzyme production reached at optimum rate. No significant increase was occurred in the weight of mycelia during 2 h of induction. When the purified samples of these intra and extracellular enzymes were tested by using diethylaminoethylcellulose column and techniques of gel filtration, both enzymes were showed similar properties in all respects. Vallier et al. (1977) observed alpha amylase production after the lysis of

12 mycelia. For this purpose, mineral medium was used which consist of starch and glucose. The lysis of mycelium seems to be due to the action of hydrolyzing enzyme dextranase and levulanase on the cell wall. The pH of the media has great impact on the lysis of cell wall and alpha amylase secretion. With increase in the pH of mineral medium up to 8.8 the secretion of enzyme and lysis of mycelial wall were greatly increased. This method makes it easy to get 3 times more enzyme production. Sinha and Chakrabrty (1978) reported Aspergillus wentii hydrolysed the soluble starch in to maltose. The optimum amylase production by using A. wentii was obtained when fermentation medium consisted of Tryptophan as nitrogen source along with 1 % starch which was incubated for 72 h at 20C and pH of medium was adjusted at 6. The enzyme activity was greatly inhibited with the addition of 1mM sodium iodoacetate. However, enzyme production was increased 3.51 to 6 mg/ml with the addition of 10 mM sodium citrate. Varnavskaia et al. (1978) studied the impact of pH on the protein conformation and alpha amylase activity produced by using Aspergillus terricola. Dispersion of optical rotation technique showed that macromolecule of alpha amylase consists of alpha helix and beta structures. The change in the values of pH resulted in two conformational forms. When decrease in pH occurred from 4-2 alpha helix structure uncoiled and degradation of beta forms occurred with the increase in the pH from 8-12. Mahmoud et al. (1978) reported the use of different agricultural by-products and wastes such as wheat bran, rice bran, cane molasses, corn bran, glucose syrup, corn starch as a substitute of original carbon source in the fermentation medium for the synthesis of alpha amylase by Aspergillus niger NRRL-337. The medium containing

13 rice bran showed maximum alpha amylase activity. The nitrogen source also substituted by such type of material that makes the medium economic such as corn steep liquor, corn steep precipitate, dried yeast and gluten-30 and 50. Corn steep precipitates give highest alpha amylase production compared to other nitrogen sources. From these results, it was concluded the medium containing rice bran 7.2 %, corn steep precipitate 2.5 %, magnesium sulfate 0.1 %, potassium di hydrogen phosphate 0.1 % and calcium carbonate 0.1 % showed maximum activity. The fungal amylase was isolated and purified from this medium. The purified enzyme showed optimal activity at 40C and pH 4.3. Allen and Thoma (1978) studied alpha amylase produced from A. oryzae acts on reducing ends, and maltotriose which was uniformly labeled. The enzyme breaksdown the glycosidic bonds during enzyme substrate formation. Augustin et al. (1981) examined the activity and production of alpha amylase and alpha glucosidase in the some members of ascomycetes, imperfect and mucoral fungi. The factor of polysaccharide system which was responsible for the consumption of alpha(1 to 4) glucans was described along with screening of the growth of organism or fungi on soluble starch. Forty nine strains were tested for the production of amylolytic activity and only twenty nine strains showed this activity. Kasim (1983) investigated the biosynthesis of alpha-amylase and amyloglucosidase (EC.3.2.1.3) by A. oryzae in submerged fermentation. For this purpose different sources of carbon and nitrogen were tested. The medium which shows maximum production of alpha amylase and glucoamylase was not very costly and consists of following components in (%) corn steep liquor 3, magnesium sulfate 0.1, potassium dihydrogen phosphate

14 0.1, defatted rice bran 8 and calcium chloride 0.1. The pH of medium was adjusted at 5. The optimum conditions for enzymes production were incubation at 28C for 96 h and the inoculum consists of 0.5 % mycelial suspension. Erratt et al. (1984) reported that starch was used as inducer for alpha amylase production from the A. oryzae. When glucose was used as carbon source the production of both intra and extracellular amylase was very low. While starch was used as carbon source increase in the activity of alpha amylase was noticed. In glucose grown cultures intracellular activity of alpha amylase increased 6.5 fold; however, 20 fold increase was observed in extracellular activity. Regardless of type of carbon source used, the active protein react only those antibodies which showed specificity only for alpha amylase and active protein have molecular weight 52 500 +/- 1800. Ustiuzhanina et al. (1985) studied the regularities in the biosynthesis of protease and alpha amylase by using washed cells of selected strain of A. oryzae. The results enabled us to compare the constitutive characters of protease and alpha amylase by selected strain of A. oryzae. Carbon, nitrogen and sulfur play very important role in the regulation of protease synthesis. However, in alpha amylase synthesis, merely carbon source played an important role. Phosphorous was vital for the synthesis of both alpha amylase and protease. Removal of phosphorous from the medium adversely affects the production of both enzymes. The alpha amylase and protease production was stimulated by the addition of celatin. Hayashida and Teramoto (1986) reported that a protease negative mutant M33 of A. ficum was obtained by treating A. ficum with MNNG. This strain showed highest alpha amylase activity compared to parent strain in submerged fermentation at optimal

15 condition i.e. 30C for 24 h. The molecular weight of purified enzyme was 54, 0000. MacGregor (1988) studied two computerized methods which explain the sequence of amino acid in the secondary structure of protein in alpha amylase which was produced by A. oryzae. Alpha-amylase produced by A. oryzae, showed three dimensional structures. The computerized methodology explained the position of amino acid and gave the predictions about the structure of alpha amylase from different sources. It was noticed all alpha amylase having known amino acid sequence possess same basic structure, these alpha amylase possess barrel shape structure which was surrounded by eight helices. The strong resemblance were found in those part of protein which take part in binding the Ca+2 ions and active site of enzyme which play important role in catalyzing the substrates hydrolysis. The active site was composed of amino acids which were specifically found in the loop joining the adjacent helix. The changes in the length and sequence of amino acid created the differences in binding the substrate and produced modifications in the action pattern of alpha amylase from different origins. Ali and Abdel-Moneim (1989) reported that the best temperature for the preservation of A. flavus var. columnaris alpha-amylase was -5C followed by 5C. CaCl2 at 0.005 M had no effect on the activity in both temperatures. Repeated freezing (-5C) and thawing followed by freezing (-5 C) had no effect on stability of alphaamylase. On the other hand, 25C was the lowest preservation temperature without any effect on the stability of alpha-amylase. 0.005 M CaCl2 decreased the activity of alpha amylase and reached a 100 % inhibition at 35th day. The fungal alpha amylase had an optimum temperature of 55C at pH 4.6, but had 60C in buffer containing

16 0.005 M CaCl2 and 50C in buffer containing 0.005 M Na2-EDTA. The addition of 0.01 M CaCl2 greatly increased the thermostability of alpha amylase at 40, 45, 50, 55 and 60C for 30 min. Optimum pH for alpha amylase was 5, but in the presence of 0.01 M CaCl2 or Na2-EDTA 5.6. The enzyme was only stable for 4 h at 25C. Whereas, addition of 0.01 M CaCl2 showed a loss of 4 % compared to a 22 % loss in the presence of 0.01 M Na2-EDTA after 4 h at 25C and 65 % loss in the presence of 0.01 M CaCl2 together with 0.01 M Na2-EDTA in the beginning and a 100 % loss after 4 h at 25C. The optimum temperature for the activity of alpha-amylase at pH 5 was 50C for the enzyme only but 55C in the presence of 0.01 M CaCl2. However, at pH 6 and 7 optimum temperature was 55C for the activity of the enzyme only or with 0.01 M CaCl2. The presence of 0.01 M CaCl2 at pH 5, 6 and 7 resulted in increase of enzyme activity at the temperatures above 50, 40 and 25C, respectively. However, 0.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures below 55 and 45C, respectively. Rousset and Schlich (1989) screened different species of A. niger for the synthesis of amylolytic enzymes i.e., alpha amylase and glucoamylase by using the submerged fermentation. Statistical analysis was used to explain the behaviour of culture instead of explaining optimization of fermentation conditions. Principal component analysis (PCA) was used to explain the affect of three agitation rates on amylase production and the formation of many other factors which affect the growth in indirect way. The result of Principal component analysis (PCA) describes the transfer of oxygen at different agitation rate influences enzyme production and carbon dioxide. The production of carbon dioxide was indirect growth measurement.

17 Maximum alpha amylase production was obtained at lower agitation speed while in case of glucoamylase intermediate agitation speed gave maximum alpha amylase production. Shah et al. (1991) optimized the conditions for the synthesis and recovery of alpha amylase from A. oryzae. A. oryzae alpha amylase retained 100 %, 61 % and 58 % respectively when preserved for 12 months at 4C, ambient temperature and 37C. Harway (1991) has isolated thermophilic bacteria from the soil which was preliminary enriched with 0.6 % starch broth at 55C. These bacteria had ability to hydrolyze the starch. Of the entire isolated cultures one was Bacillus coagulans, which was best producer of alpha amylase. The maximum production was obtained in optimal condition which consists of incubation temperature 55C, 200 rpm agitation speed, 48 h incubation period and broth extract starch agar medium. Tsekova et al. (1993) studied the ability of Aspergillus genus for alpha amylase production. When 3 % soluble starch was used in Czapek-Dox agar and in liquid Czapek-Dox media maximum alpha amylase production was obtained. Sudo et al. (1993) studied the fermentation medium containing all the components which were necessary for the production of acid stable alpha amylase (asAA) by A. kawachii using submerged fermentation. One hundred and thirty milligram of acid stable alpha amylase per liter of medium was produced after 5 days of inoculation at 30C in submerged fermentation. Glycogen was present as stored polysaccharide. When the amount of stored glycogen (CSG) decreased and inducer such as dextrin was present synthesis of acid stable alpha amylase started. Maximum production of as AA was obtained when amount of CGS reaches at zero. When the amount of CGS increased production of acid stable alpha amylase tend to be decreased. The amount of glucose

18 in the medium and growth of mycelia was strongly influence by the concentration of CGS. The quantity of acid stable alpha amylase was directly proportional to the production of mycelia. Soccol et al. (1994) tested two species of Rhizopus for protein enrichment of both cooked and raw cassava and also for the synthesis of amyloglucosidase and alpha amylase in solid and submerged fermentation. The protein enrichment and maximum enzyme synthesis were obtained in solid state fermentation. Cooked cassava showed optimum production in solid state fermentation; however, maximum synthesis of amyloglucosidase by R. oryzae was obtained when raw cassava was used. Khoo et al. (1994) achieved fifty units per milliliter amylolytic activity by using A. flavus in liquid medium containing topica starch. The culture filtrate was subjected to electrophoretic analysis. This analysis showed filtrate contains only one type of amylolytic enzyme named alpha amylase. The following factor support the identification of alpha amylase (i) iodine stained starch quickly become colourless (ii) starch digestion resulted in the formation of a mixture of glucose, maltose, maltotriose and maltotetrose. Purification of enzyme was involve the use of ammonium sulfate precipitation, ion exchange chromatography and gel

electrophoresis. The purified enzyme showed 52.5 2.5 kDa molar mass with an isoelectric point at pH 3.5. Characterization of enzyme showed the maximum activity of purified enzyme was noticed at pH 6 and 55C. Omori et al. (1994) isolated acid labile alpha amylase (A-3) from A. kawachii in barley koji. The enzyme was purified by using the different techniques such as ion exchange chromatography and gel filtration. The changes in new alpha amylase production was compared with two known alpha amylase represented as A-1 and A-2.

19 Sodium dodecyl sulfate poly acrylamide gel eletrophoresis showed A-3 has molecular weight 56,000. The enzyme showed constant production at 40C approximately for 54 h. However, in traditional method formation of A-3 was not detected after 36 h. In the presence of 2 % citric acid in barley A3 was formed upto 36 h. The results indicated production of A3 was influenced both by temperature and initial concentration of citric acid. Chang et al. (1995) reported that alpha amylase produced by A. oryzae was purified by passing through the different steps in a specific sequence such as amylopectin affinity adsorption, DEAE-Sepharose ion exchange chromatography and sephacryl S-200 HR gel filtration. After passing through these steps the enzyme showed 16 fold increase in the purity and 45 % of enzyme was recovered. The optimum conditions for purified enzyme was pH range 4-5, temperature 50C and km value for starch hydrolysis was 0.22 %. Incubation for 30 min at 50C result 80 % lose in enzyme activity. The heat denaturation constant and molecular weight by gel filtration was 0.024/m and 52 kDa, respectively. The enzyme activity was inhibited by using Mercuric ion (0.3m M), DNFB# (6mM), NBSI (6mM) and NAI (6mM). The hydrolysis of maltoheptaose by the enzyme resulted in the formation of maltotriose and maltotetraose. Donmez and Melike (1996) isolated bacteria showing amylolytic activity from different samples and grouped them on the basis of showing amylolytic activity in the solid and liquid fermentation media. Of all the isolated strains Bacillus subtilis produce 24 U/ml alpha amylase. Different carbon sources were added to the fermentation media to check effect of these carbon sources on alpha amylase production. The maximum activity of alpha amylase 360 U/ml was obtained in the

20 presence of dextrin and optimum temperature was 50C for enzyme production. However, if enzyme was incubated for 2 h at 100C 23 % of this activity was lost. Carlsen et al. (1996) tested the stability of alpha amylase produced by A. oryzae at different pH. The enzyme showed highest stability at neutral pH (5-8); however, beyond this pH range a great loss in the activity of enzyme was noticed. On line FIA system was used and a rate constant was obtained by the empirical expression k = 1.19 107 [H+]1.99 (h1) explained the inactivation of enzyme was greatly influenced by pH values. The inactivated enzyme again obtained some of its activity at pH 6 and this reactivation steps also obey the first order kinetics rules. The contamination of protease in the protein sample was not result to the irreversible loss of activity. Abou Zeid (1997) isolated filamentous fungi from cereals and screened to test the alpha amylase producing potential. The strain which showed highest ability for alpha amylase production was identified as A. flavus. The enzyme was purified by using starch adsorption methodology. The polyacrylamide gel electrophoresis (PAGE) indicated the molecular weight of A. flavus was 75, 000 3,000. The optimum temperature for purified enzyme was 7 and 30C, respectively. The use of potassium ions increased the activity of alpha amylase. However, magnesium ions did not extremely influence the enzyme activity. The activity of alpha amylase was greatly inhibited in the presence of manganese, zinc, copper and ferric ions. The hydrolysis of native starch by A. flavus resulted in the formation of glucose and some other oligosaccharides Kajiwara et al. (1997) studied the production of acid stable alpha amylase from A. kawachii during production of shochu-koji. From barley shochu-koji two types of

21 the acid stable alpha amylase (as AA) represented as as A-1 and as A-2 were puified. The asA-1 and asA-2 showed different adsorption characteristics on raw starch. The activity of as A-1 slowly increased during the process of shochu-koji production but dropped after incubation of 24 h. Contrary to as A -1 the activity of as A-2 was increased with increase in the incubation time. Temperature affect ratio of as A1 to total as AA activity. When acid protease and as A-1 were incubated along with each other and this sample was analyzed by SDS-PAGE. A known band was appeared in the place of as A-1 band after 12 h of incubation. The unknown protein showed all the characteristics which were present in as A-2. The result showed acid stable alpha amylase was found in different form just like the glucoamylase produced by A. awamori during the production of shochu-koji. Spohr et al. (1997) examined alpha amylase producer strain of A. oryzae for the production of recombinant protein and affect of growth on the production of protein. The comparison of these strains for morphology and impact of morphology on the protein indicated the mutant strain having denser mycelium, produce more alpha amylase compared to other strains. Arnesen et al. (1998) cultivated thermophilic fungus in the presence of dextran (having low molecular weight) along with Tween 80 or Triton X-100. The fermentation was carried out in shake flasks for more than 120 h. The 2.7 fold increase in the activity of alpha amylase was observed in medium containing Tween 80 compared to the medium with out Tween 80. The medium containing Tween 80 showed increase in the alpha amylase production after 48 h; while general protein secretion was stimulated after 24 h of inoculation. The Tweeen 80 also influences the production of biomass. The production of biomass increased gradually with the

22 increase in the concentration of Tween 80. Contrary to this Triton X-100 produce reverse effect. It was noticed increase in the amount of Tween 80 resulted greater than 3 fold increase in the total extracelluar protein. The Tween 80 had no effect both on the hyphal length and diameter. Glycosylation degree was also not effected by the Tween 80. Anidyawati et al. (1998) purified three forms of alpha amylase to homogeneous state by using the methodology of column chromatography. These forms of alpha amylase were produced from A. awamori. These forms were designated as Amyl1, Amyl 11, and Amyl 111. The SDS PAGE indicated these three forms possess 49,000, 63,000 and 97,000 molecular weight, respectively. The optimum pH for Amyl 11 and Amyl 111 was 5.5 while in the case of Amyl 1 the pH was 4. Maltose and maltosetriose were formed by the hydrolyzing action of Amyl 1 on malto-tetraose-pentose,-hexaose,-heptose and and -cyclodextrin. However Amyl 1 produces no hydrolyzing effects on raw corn starch, maltose, maltotriose, isomaltotriose, isomaltosse, and -cyclodextrin. Unlike Amyl 1 both Amyl 11, and Amyl 111 have ability to hydrolyze maltotriose, raw corn starch and alpha, beta, gamma cyclodextrin resulting in the formation of maltose along with minor products of glucose and maltose. The range of soluble starch hydrolysis through Amyl 1, Amyl II and Amyl III was 33, 35 and 38 %, respectively. Jin et al. (1998) used A. oryzae for alpha amylase production and microbial biomass protein (MBP) from starch processing waste water (SPW) in air lift bioreactors. The production of MBP and fungal alpha amylase was carried out under the optimized conditions i.e., pH 5 and 35C. Bioproduct yield obtained from 12h batch culture was 6.1 g/l. This yield consists of 55 EU/ml of alpha amylase and 38 %

23 protein. The enzyme showed stability at pH 5-9 and 25-35C. Spohr et al. (1998) tested three different strain of A. oryzae having the ability to form recombinant protein with respect to growth and alpha amylase production. One was wild strain and the second strain was a transfomant strain which consists of additional copies of alpha amylase gene while third strain was morphological mutant. It was observed the production and growth of organism were correlated. Comparison of production and morphology of these strains indicated the variations in the morphology had direct impact on enzyme production in submerged fermentation. Moreira et al. (1999) isolated a fungal strain from the soil having the ability to produce amylolytic enzymes. This strain was identified as A. tamari. A. tamari formed both alpha amylase and glucoamylase in the mineral medium concomitant with carbon source i.e., 1 % starch or maltose. The formation of alpha amylase and glucoamylase indicated tolerance to wide range of initial fermentation medium pH (4-10) and temperature (25 - 42C). Ion exchange chromatography was used for the separation of alpha amylase and glucoamylase. Partially purified alpha amylase and glucoamylase showed maximum activities at pH 4.5 and 6 and stability at pH 4-7. The temperatures at which enzymes showed highest activities was between 50 - 60C. Pedersen and Nielsen (2000) reported the effect of organic and inorganic nitrogen sources on alpha amylase production by A. oryzae in continuous cultivations. Both nitrogen sources were tested along with glucose. In case of inorganic nitrogen source ammonia was better than nitrate. The comparison between organic and inorganic nitrogen sources indicated organic nitrogen for example yeast extract or casine hydrolysate was superior to ammonia. In the presence of 0.05 g/l casine

24 hydrolysate 35 % increase in alpha amylase production was found. The transcription of the alpha amylase genes were not involved in the increase production of alpha amylase the basic reason was the grater secretion of alpha amylase from the biomass. Nguyen et al. (2000) optimized the composition of fermentation media for increasing amylases production through Thermomyces lanuginosus by using the different ways. The influence of different carbon and nitrogen sources was tested. The carbon and nitrogen sources, which proved to be good substrates for the growth of T. lanuginosus and exbhited maximal alpha amylase (92-125 U/ml) and glucoamylase (6-13 U/ml) activites included starch, maltodextrin, dextrin, maltose, amylopectin, glucose dextran and L-asparagine. L-asparagine at the level of 6.5 % was good for alpha amylase production and 2 % L-asparagine was optimum for glucoamylase production. The pH of medium was adjusted by using hundred millimolar citrate buffer for amylases production. Response surface method (RSM) was used to find out the suitable concentration of medium component for the synthesis of amylolytic enzymes. A second order polynomial model was used at significance level 95 % (p<0.05) for alpha amylase and glucoamylase. The selected composition of media was tested with respect to synthesis of amylolytic enzymes. Mariani et al. (2000) studied impact of Amaranth seed meal and the aeration on the productiviy of alpha amylase by A. niger NRRL 3112. The assays for the selection of fermentation media was carried out by using the rotary shaker at 250 rpm and 2.5 cm stroke. The selection of aeration conditions were carried out in New Brunswick mechanically stirrer fermenter A fermentation medium containing 5.0g/l Amaranthus cruentus seed meal produce 2750 U.Dun/ml alpha amylase with dry weight of 8.0 g/l

25 after 120 h, of inoculation. The optimum condition for alpha amylase production in fermenter were fermentation period of 120 h, agitation rate 300 rpm and an air flow of 11/l/ min in limited concentration of dissolved oxygen. Although increase in agitation speed increases the dissolve oxygen but it was not suitable for the formation of alpha amylase. Morphology of A. niger such as long and branched hyphae was very important to obtain the maximum alpha amylase production. Petrova et al. (2000) reported the purification of wild and mutant strains of Thermomyces lanuginosus ATCC 34626 a thermophilic fungus. The purification was carried out to homogeneity by using the different techniques in a sequence such as precipitation with ice cold propanol, anion exchange and molecular sieve chromatographic methods. The SDSPAGE results indicated purified alpha amylases (both with PI values of 3.0) have molecular mass 58 kDa. The optimum pH for the activity of wild and mutant strains was 5 and 4.5, respectively. 1 Cyclohexyl - 3 - (2-morpholinyl 4 - ethyl) carbodiimide (40 100 mM) and N- bromo succinimide (0.1 1mM) produce inhibitory effect on the enzymes activity due to the presence of carboxylic groups and tryptophan residues in the catalytic process. Madihah et al. (2000) isolated and partially purified alpha amylase from the fermentation of sago starch to solvent by C. acetobuylicum P262. The characterization of partially purified enzyme showed the following optimal conditions. The highest activity of alpha amylase was observed at pH 5.3 while enzyme showed stability from pH 3-9. The highest activity of alpha amylase was found at 40C; however, if alpha amylase was placed at 60C for 60 min merely 50 % of its original activity was retained. The Km and Vmax values of alpha amylase for soluble starch were 0.31 g/l and

26 10.03 U/ml, respectively. Viswanathan and Surlikar (2001) designed the medium by the use of fractional factorial method and Plackett-Burman design to study the influence of component of Amaranthus paniculatas (Rajgeera) medium on alpha amylase production by A. flavus. Fifteen components were used in developing the medium. Out of these components only four i.e., CSL, NaCl, CaCl2 and NH4HPO4 were choosed on the basis of contrast coefficient values and selected as independent variables for the Box-Behnken design. By using SPSS/PC +(version 7.5) statistical analysis a polynomial multiple regression model was prepared. CSL, NaCl, CaCl2 and NH4HPO4 increased the yield up to 81.3 % however, NaCl, CaCl2 influence the product to the tune of 68.3 %. The comparison of control and optimized medium exhibited 8 fold increase in production of enzyme in the optimized medium. Carlsen and Nielsen (2001) tested the effect of different carbon sources such as fructose, galactose, mannitol, glucose, glycerol, sucrose, and acetate on alpha amylase production by A. oryzae in carbon limited chemostat cultures. A. oryzae was not able to grow on such a medium which contain galactose as only carbon sources; however, a combination of glucose and galactose allow the fungal strain to grow and produce alpha amylase. Medium containing maltose and maltodextin indicated more alpha amylase production during growth of A. oryzae compared to medium containing glucose concentration less than10 mg/l. Sucrose, glycerol and mannitol showed low alpha amylase production. Acetate alone did not show any production of enzyme but acetate along with little quantity of glucose exhibited alpha amylase production. It was observed alpha methyl-D-glucoside was acted as an inducer for alpha amylase production but it was not as good as glucose.

27 Agger et al. (2001) evaluated the influential impact of formation of biomass on the synthesis of alpha amylase by using the wild strain of A. oryzae and recombinant strain of A. nidulans in submerged fermentation. It was noticed specific rate of alpha amylase production was inversely proportional to the concentration of biomass formation. When the concentration of biomass was increases 2-12 g dry weight/kg the specific rate of enzyme production was decreased. However in case of recombinant strain of A. nidulans in which gene creA was removed (which cause carbon catabolite repression) no marked decrease in the specific rate of enzyme formation was observed. The results indicated less alpha amylase production at high biomass formation was due to slow mixing rate of vital components in viscous culture medium. Ray (2001) isolated Penicillium sp possessing the ability to form alpha amylase and xylanase in the presence of starch and xylan respectively, in fermentation. It was noticed the optimum amylolytic activity and xylanolytic activity was obtained on 4th and 6th day of fermentation respectively. The quality of alkalophilic strain of Penicillium sp to hydrolyze starchy and hemicellulosic wastes made them a potent strain for the large scale economic production of both enzymes using the cheap substrates. Bogar et al. (2002) tested different strains of A. oryzae on spent brewing grain (SBG) and corn fiber for alpha amylase production. A Plackett-Burman experimental design was practiced to develop optimized media for alpha amylase production using best producer strain. A. oryzae NRRL 1808 strain produced 4519 U of alpha amylase/g of dry matter substrate in stationary 500 ml Erlenmeyer flask culture after 72 h. The crude enzyme, in situ enzyme produced in solid substrate fermentation material was economic biocatalytic product for animal feed and for the

28 production of bio alcohol from starchy substrate. Francis et al. (2002) investigated the effect of spent brewing grains on alpha amylase production by A. oryzae NRRL 6270 when spent brewing grains utilized as sole carbon source. Maximum alpha amylase production was obtained at 25C. At 30C almost similar results were obtained. Optimum alpha amylase production [6870U/g dry substrate (gds)] was obtained in solid state fermentation at 30C after 96 h by the use of suspension containing 1107 spores/ ml. Addition of any external carbon source in the spent brewing grain resulted decreased in alpha amylase production. Arnesen et al. (2002) used thermophilic fungus T. lanuginosus for alpha amylase production in shake flasks. The fermentation medium contained carbon source in the form of low molecular dextran. The fermentation was carried out up to 120 h. The results showed maximum alpha amylase activity after 96 h of inoculation during stationary phase while the production of maximal biomass takes place after 48 h of fermentation. A same pattern was observed in the case of total extra cellular protein. It was found many unidentified proteins and alpha amylase were de novo synthesis by using pluse labeling techniques of proteins. The sequencing of alpha amylase from T. lanuginosus using specific primmer and RT-PCR technique indicated that transcription of alpha amylase was not start before the late growth phase and reached at its highest value more than 24 h after maximum biomass was produced. Gigras et al. (2002) used the central composite design along with 3 variables i.e., starch, yeast extract, and di potassium hydrogen phosphate for alpha amylase production by A. oryzae in shake flasks and bioreactor. The alpha amylase production was 133U/ml in shake flasks while in case of bioreactor production was 161 U/ml.

29 However, there was great difference in the fermentation period of shake flasks and bioreactor. The fermentation period for the maximum alpha amylase production by A. oryzae in shake flasks was 120 h but in case of bioreactor this time period was reduced to only 48 h. A high concentration of phosphate in the fermentation medium and use of low inoculum size was essential to prevent the unnecessary foaming in bioreactor; but managing the pO2 level and agitation rate was not compulsory for alpha amylase production. The enzyme production increases with the increase in the pH of medium and reached at its peak at pH above than 7.5. Thus in present study pH act as sign of commencement or ending of the enzyme production. Huang et al. (2003) developed a segregated model to explore the intrinsic associations between growth, substrate consumption, cell differentiation and enzyme formation by Bacillus subtilis in bioreactor. The segregated model represented three different states of cell and the change from vegetative stage to sporangium and lastly to mature spore. An age-based population balance model was used to explain the maturity of sporangium in the direction of the formation of spores. Parameters in the model were found out by placing the experimental data in the model. The model has ability to describe the temporary behavior of B. subtilis in both batch and fed-batch cultures. Francis et al. (2003) optimized incubation temperature, initial moisture contents and inoculum size by application of BoxBehnken design under the response surface methodology for the highest production of alpha amylase by A. oryzae NRRL 6270. The experimental data was added into a polynomial model to find out alpha amylase production. A PlackettBurman design was used to test the influence of nineteen

30 nutrient components on alpha amylase production by the A. oryzae. Soybean meal, CaCl2 and Mg SO4 were chosen on the basis of their positive affect on alpha amylase production. A BoxBehnken design was used to select the best condition for alpha amylase production. Incubation temperature 30C, initial moisture contents 70 % and an inoculum size of 1107 spores/g dry substrate were the optimum conditions for the alpha amylase formation by A. oryzae NRRL 6270 on SBG. Under selected conditions of solid state fermentation SSF, about 20 % enhancement in enzyme production was found. Kariya et al. (2003) purified alpha amylase from culture broth of A. oryzae MIB 316. The enzyme had ability to efficiently hydrolyzed amylopectin, amylose and starch and break down maltopentose to produce a maltotriose and maltose. However, maltose did not produce glucose. The N-terminal sequence of first 10 residues and many other molecular characteristics were similar to Taka-amylase. Kusuda et al., (2003) isolated alpha amylase from an immobile culture filtrate of Tricholoma matsutak. The enzyme was purified to homogeneity by sequential steps of Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The alpha amylase showed 3580 fold purity and 10.5 % recovery. SDS-PAGE analysis resulted in a single protein band. The characterization of purified alpha amylase showed that it was most active at pH 56 and having stability between wide range pH i.e., 410. The experimental results also indicated the alpha amylase was somewhat thermostable and showed thermostability at 50C while the optimal temperature was 60C. The sizeexclusion chromatography and SDS-PAGE showed that purified alpha amylase had molecular mass 34 kDa and 46 kDa, respectively. The mercuric ion did not inhibit the activity of enzyme. Measurement of viscosity, TLC and HPLC analysis indicated

31 amylases from T. matsutake was of endo type. The specificity of alpha amylase was tested by using amylose along with various polysaccharides. This alpha amylase rapidly hydrolyzed the -1,4 glucoside linkage in soluble starch and amylose A (MW,2900), but was not able to hydrolyze the -1,6 linkage and cyclic polysaccharides e.g - and -cyclodextrin. Kanwal et al. (2004) extracted alpha amylase from Malus pumila (apple) by homogenizing the apple in buffer for alpha amylase. After extraction, the enzyme was purified by passing sequential steps of purification. The crude extract exhibited 3.09 U/ml alpha amylase activity was subjected to ammonium sulfate precipitation. This partially purified enzyme produces 4.76 U/ml and showed 5.01 U/mg specific activity. The enzyme was further purified by gel filtration chromatography (Sephadex G-150). After gel filtration chromatograph it produces of 5.025 U/ml and specific activity 38.95 U/ml along with 20-fold purification. SDS-PAGE of enzyme removed the undesirable proteins and single band of enzyme was appeared. Molecular weight of alpha amylase was 51,180 D which was finding out by Sephadex G-150 column. Amylase exhibited optimal pH 6.8, incubation temperature 37C, Km value 2.0x10-3 g/ml, max 540nm and incubation time for enzyme assay was ten min. Apar and Ozbek (2004) studied the effects of temperature on the enzymatic hydrolysis of starch from different sources such as corn, rice and wheat. Three commercial alpha amylases produced from Bacillus sp. A oryzae and B. licheniformis were employed for hydrolysis of starch. In every starch hydrolysis process, the concentration of residual starch and the residual activity of alpha amylase in percentage were determined at 50 and 60C temperature based upon the processing

32 time in a stirred batch reactor. Mathematical models were developed for using experimental data of residual starch concentration from each source. Some inactivation models were used to understand the relation between temperature and stability of enzyme during hydrolysis of starch from enzymes having different origins. El-Safey and Ammar (2004) reported that the amylolytic family has great importance due to its wide spectrum of application. Alpha amylase produced from Aspegillus flavus var.columinaris was isolated and characterized. The enzyme was purified by using ammonium sulfate precipitation and Sephadex G200 filtration method. The purified enzyme showed 9.97 fold purification and 6471.6 (units/mg port/ml) specific activity. The alpha amylase activity amplified with the enhancement of enzyme concentration. The optimum condition for the production of alpha amylase was 0.2% (w/v) starch, while the optimal temperature was 35C. The purified alpha amylase showed maximum activity at pH 6.2 after 30 h of incubation. Pimpa (2004) reported that the highest alpha amylase production by Aspergillus sp. was obtained after 24 h. Addition of suitable nitrogen sources and inorganic salts to the medium appreciably increased the enzyme production. The maximum enzyme yield 36.5 U/ml was obtained in the media containing wastewater, defatted soyabean 10 g/l, potassium di hydrogen phosphate 10g/l, magnesium sulfate 5 g/l, zinc chloride 0.1 g/l. The alpha amylase produced by Aspergillus sp. showed catabolic repression. The enzyme was partially purified by subjecting into 60 % ammonium sulfate. The optimal pH and temperature of partially purified enzyme was 5 and 50C, respectively. Chavez et al. (2004) screened different carbon sources namely sorghum, soluble potato, corn and cassava starches as well as maltose for the concurrent cultivation and

33 production of both alpha amylase and glucoamylase by a novel Trichoderma sp. even though maltose gave better results compared to other carbon sources with respect to activity of alpha amylase (about 28,000 U/l) and alpha amylase production (about 390 U/l/h), cassava and corn starches showed maximum glucoamylase activities (17,00018,000 U/l) and production of enzyme was almost similar to those obtained with maltose (about 100 U/l/h). Because of its capability to produce both alpha amylase and glucoamylase, the Trichoderma sp used in this study proved to be beneficial in a direct process of raw starch saccharification with no preliminary gelatinization. Konsula and Kyriakides (2004) isolated a somewhat thermophilic Bacillus subtilis strain, from fresh milk of sheep possess the ability to produce extracellular thermostable alpha amylase. The medium containing low starch concentration showed maximum alpha amylase production at 40C. The enzyme exhibited highest activity at 135C and pH 6.5. The thermostability of alpha amylase increased in the presence of calcium or starch. This thermostable alpha amylase was employed for the hydrolysis of different starches. Ammonium sulfate crude enzyme preparations as well as the cell-free supernatant actively break down the starches. The use of the clear supernatant as enzyme source was highly advantageous mainly because it decreases the cost of the hydrolysis. When the reaction temperature increased up to 70C, all of the substrate showed higher rates of hydrolysis. Potato starch upon hydrolysis produced higher concentration of reducing sugars compared to other starches at all tested temperatures. Soluble and rice starch came at second and third position respectively, with respect to reducing sugar liberating ability. However, in case of corn and oat starch alpha amylase showed somewhat less affinity.

34 Ramachandran et al. (2004) investigated alpha amylase production by A. oryzae in solid state fermentation (SSF).The substrate used was coconut oil cake (COC). Raw COC was a good substrate and 1372 U/gds alpha amylase was produced in 24 h. As a result of optimization of media component alpha amylase production was increased (1827 U/gds) when solid state fermentation was carried out at 30C for 72 h along with media contained 68 % moisture contents. Addition of glucose and 0.5 % starch further increased the alpha amylase production (1911 U/gds). However, maltose repressed the alpha amylase production. Alpha amylase production increased upon adding the organic and inorganic nitrogen sources. When peptone at the level of 1 % was added in the fermentation media 1.7-fold increase in enzyme activity (3388 U/gds) was observed. The enzyme production and growth were correlated. The

activity became maximal when the fungal biomass was at its peak at 72 h. Kunamneni et al. (2005) employed the response surface methodology to study the collective impact of the nutritional parameters and to increase extracellular alphaamylase production in solid-state fermentation by T. lanuginosus. These nutritional parameters consist of carbon source (soluble starch), nitrogen source (peptone) and a concentrated mineral medium. A twenty three factorial central composite design using response surface methodology was used to optimize the above three variables. The best calculated values of these variables for optimal amylase production were soluble starch 71.10 g/Kg, peptone 91.97 g/Kg and mineral salts solution 175.05 ml/Kg with

an estimated alpha amylase activity of 5.085 105 U/Kg of wheat bran. These parameters were checked in the laboratory and ultimate alpha amylase activity obtained, 4.946 105 U/Kg of wheat bran, was very near to the calculated value.

35 Kiran et al. (2005) isolated the thermophilic Bacillus sp. K-12 from soil samples having the ability to produced amylolytic enzyme. Effects of different carbon sources and chemicals on production of alpha amylase were checked in the laboratory. Medium consist of 1 % starch showed maximum alpha amylase activity after 60 h of fermentation. However manganese sulfate, zinc sulfate and EDTA showed inhibitory effect on alpha amylase production by Bacillus sp. Haq et al., (2005a) reported the choice of the appropriate surfactant for alpha amylase production by Bacillus subtilis GCBM-25 during shake flasks. Various surfactants (laundry soap, detergent powder, sulphonic acid, acyle benzene sulphonic acid, liquid soap, Tween 80, sodium silicate, bath soap, sodium tripolyphosphate, sodium lauryl ether sulphate or sodium lauryl sulphate) at rate 2.0 % (w/v) were screened for synthesis of enzyme. Among all the surfactants, tested laundry soap proved to be superior with respect to alpha amylase production (605 U/ml/min) after 44 h of fermentation using 4.0 % inoculum. The enzyme production was enhanced (857 U/ml/min) with the addition of Millon soap at rate 3.2 % (w/v) in the medium. However, addition of surfactants in the medium reduced the thermostability from 70 to 50C. Haq et al. (2005) reported the use of agricultural starchy substrate for alpha amylase production by Bacillus licheniformis. The use of agriculture by products made the medium economic. Soluble starch, hordium, pearl millet, rice, corn, gram and wheat starch were screened for the alpha amylase production by parental and its mutant derivative. The mutant strain B. licheniformis GCUCM-30 exhibited 10 fold more enzyme production compared to parental strain when1.5 % pear millet and 0.25 % of nutrient broth was added to fermentation medium.

36 Anto et al. (2006) reported the alpha amylase production by B. cereus MTCC 1305 in solid state fermentation using wheat bran and rice flake manufacturing waste as substrates. Maximum alpha amylase activity (942) U/g was obtained when wheat bran was used as a substrate. Optimal conditions for alpha amylase production were inoculum size 10 % substrate moisture ratio 1:1 and glucose, (0.04 g/g). Addition of different nitrogen sources (0.02 g/g) showed decrease in enzyme production. Optimal alpha amylase activity was observed at 55C and pH 5. Swain et al. (2006) reported the alpha amylase production by B. subtilis isolated earlier from cow dung microflora. The optimum temperature, pH and incubation period for amylase production were 5070C, 5-9 and 36 h, respectively. Enzyme secretion was very similar in the presence of any of the carbon sources tested (soluble starch, potato starch, cassava starch, wheat flour, glucose, fructose, etc.). Yeast extract and ammonium acetate (1 %) as nitrogen sources gave higher yield compared to other nitrogen sources (peptone, malt extract, casein, asparagine, glycine, beef extract) whereas ammonium chloride, ammonium sulfate and urea inhibited the enzyme activity. Addition of Ca+2 (10-40 mM) to the culture medium did not result in further improvement of enzyme production, whereas the addition of surfactants (Tween 20, Tween 40, Tween 80, and sodium lauryl sulphate) at 0.02 % resulted in 2-15 % increase in enzyme production. There were no significant variations in enzyme yield between shake flask and laboratory fermenter cultures. The purified enzyme was in two forms with molecular mass of 18.0 1 and 43.0 1 kDa in native SDS-PAGE. Kathireasan and Manivannan (2006) isolated Penicillium fellutanum from coastal mangrove soil and screened out the sound effects of different variables such

37 as pH, temperature, incubation time, salinity, carbon and nitrogen sources in shake flasks fermentation for alpha amylase production. The fermentation medium with no addition of seawater and supplemented with maltose and peptone as carbon and nitrogen source was incubated for 96 h, at pH 6.5 and temperature 30C, gave maximum alpha amylase production by P. fellutanum. Djekrif-Dakhmouche et al. (2006) studied the alpha amylase production and optimization from A. niger ATCC 16404 .The statistical analysis has revealed that variation in agitation from 150 rpm 200 rpm has no effect on the alpha amylase production but it increased biomass. As far as variation in pH from 5 to 6 has positive effect on alpha amylase production while its effect on the biomass was negative. The addition of starch at 10 g/l to the fermentation medium (an inductive substrate and carbon source) stimulated the alpha amylase production, while it has no effect on biomass production. Calcium chloride at 1 g/l (a structural and stabilizing element for the alpha amylase) solely affect the enzyme production. The use of other salts (manganese, iron sulfates as well as magnesium chloride) seemed to be increased alpha amylase production but did not effect either the production of protein or biomass. Prakasham et al. (2007) reported fractional factorial design of Taguchi methodology for the optimization of medium along with eight variables soluble starch, corn steep liquor, casein, potassium dihydrogen phosphate, magnesium sulfate, initial pH, incubation temperature and inoculum size for the amylase production in submerged fermentation by A. awamori. Considerable enhancement approximately 48% in acid amylase synthesis was observed. The optimized fermentation medium included in (%) soluble starch 3, CSL 0.5, KH2PO4 0.125, casein 1.5 at pH 4 and

38 31C. Shoji et al. (2007) reported a new submerged culture system of A. kawachii NBRC4308 with the help of barley whose surface was wholly or partially covered with husk. Both glucoamylase activity 150.8 U/ml and acid-stable alpha amylase activity 7.7 U/ml were found in the supernatant in the presence of low concentration of glucose. Rao et al. (2007) investigated the formation of spores from B.

amyloliquefaciens B128 in shake flask cultivation. Fermentation media were optimized by applying two steps approach. A rapid identification of an appropriate carbon and nitrogen source was obtained by screening experimentation, and use of response surface methodology (RSM). A five-level four-factor central composite design was used to find out the highest spore yield at optimal level for lactose, tapioca, ammonium sulfate and peptone. A noteworthy linear major effect was observed in the case of topica and peptone, while lactose and ammonium sulfate produced no important linear effect. Lactose-ammonium sulfate and lactose-peptone extensively affected spore production. Optimum conditions for the alpha amylase production were (g/l): lactose 12.7, tapioca 16.7, ammonium sulfate 1.8 and peptone 8. The predicted spore production was 5.93 108 (no/ml). The real experimental results were in concurrence with the prediction. Suganuma et al. (2007) reported that highly humid climate of Japan facilitate the growth of various molds. Among these molds A. oryzae was the most important and popular in Japan, and has been used as yellow-koji in producing many traditional fermented beverages and foods, such as Japanese sake, and soy sauce. The koji molds black-koji and white-koji produce two types of alpha amylase, namely, acid-stable

39 (AA) and common neutral (NA).The latter enzyme was enzymatically genetically similar to Taka amylase. In this review they investigated AA from three molds, A. niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the distinguishable properties between AA and NA. AA has many advantages in industrial applications, such as its acid-stability, thermostability, and raw-starch digesting properties. Bhanja et al. (2007) used Growtek bioreactor as modified solid state fermenter to circumvent many of the problems associated with the conventional tray reactors for solid state fermentation (SSF). A. oryzae IFO-30103 produced very high levels of alpha amylase by modified solid state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High alpha amylase yield of 15,833 U/ g dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6 at 32C for 54 h whereas alpha amylase production in SSF reached its maximal (12,899 U g1 dry solid) at 30C after 66 h of incubation. With the supplementation of 1 % NaNO3, the maximum activity obtained was 19,665 U g1 dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U/ g dry solid in presence of 0.1 % Triton X-100 (20 % higher than the control). Tayeb et al. (2007) conducted the alpha amylase production using amplified variants of B. subtilis (strain SCH) and of B. amyloliquefaciens (strain 267CH) in a bioreactor with multiprotein-mineral media. The time course of fermentation in a bioreactor revealed that the highest yield (about 8 x 104 U/ml within 6 h) by strain SCH was obtained by applying: 3.5 % initial starch, 2 % additional starch after 19 h, 3 vvm aeration and 300 rpm agitation. The highest yield (about 19 x 104 U/ml within

40 100 h) by strain 267CH was obtained by applying: 2.5 % initial starch, 2 % additional starch after 24 h, 3 vvm aeration, and 300 rpm agitation with the productivity after 60 h reaching only about 14 x 104 U/ml. Production occurred in both the logarithmic and post logarithmic phases of growth. Maximum consumption of starch and protein occurred during the first day of incubation. The optical density peak coincided with enzyme production peak in case of strain SCH and preceded that of enzyme production in case of strain 267CH. The alpha amylase produced by the two strains was shown to be the liquefying and not both enzymes liquefied starch to a dextrose equivalent of about 15-17 at 95C hence they are classified among thermostable alpha amylases. They exhibited broad pH and temperature activity profiles. The optimum pH for activity was 4-7 for alpha amylase produced by strain SCH and 4-8 for alpha amylase produced by strain 267CH while the optimum temperatures for their activities were in the range of 37 -75C at 0.5 % starch and in the range of 85 - 95C at 35 % starch. Poornima et al. (2008) isolated different strains of actinomycetes and tested these strain for their ability to synthesize the alpha amylase. Among all the strains, the strain AE-19 showed best alpha amylase production. This strain was identified as Streptomyces aureofasciculus and selected for subsequent studies. The highest alpha amylase production was obtained in the presence of mannose and L-histidine as carbon and nitrogen source, 0.05 % sodium chloride at temperature 45C and pH 9. These results indicated that strain can be successfully used for commercial alpha amylase production after testing strain competence in large scale fermentations. Gupta et al. (2008) studied the nutritional requirements of A. niger and the factors such as

41 incubation temperature, pH of medium, carbon and nitrogen sources, fermentaion period, surfactants and concentration of metal ions. The experimental result showed ideal carbon and nitrogen source for alpha amylase production was 0.5 % starch and 0.3 % peptone. The optimal pH, temperature and fermentation period were 5, 30C and 5th day, respectively. Different surfactant at varying level such as Tween-80, Triton X-100 and Sodium dodecyl sulphate at 0.02, 0.002 and 0.0002 % concentration, respectively exhibited enhanced alpha amylase productivity. The major purpose of the present study was to employ an appropriate fungal strain for extracellular alpha amylase production and find out the fermentation period for the synthesis of alpha amylase and to determine the effects of external substances that might increased the synthesis of alpha amylase. Esfahanibolandbalaie et al. (2008) reported the effect of many chemical and physical factors on alpha amylase production by A. oryzae in shake flasks fermentation via an Adlof-Kuhner orbital shaker. The impact of varying pH of medium ranging from 4-7 was studied. The maximum alpha amylase production was obtained at pH 6.2. Carbon and nitrogen source has discernible effect on the enzyme production. The corn starch at level of 15 g/l proved to be best carbon source for alpha amylase synthesis while glucose represses the alpha amylase production. The medium consist of corn starch, sodium nitrate as inorganic nitrogen resulted in significant enzyme production. Among the organic nitrogen sources yeast extract at the level of 2.5g /l was excellent nitrogen source. The impact of different temperatures and agitation speed from 20 to 40C and 50 to 200 rpm, respectively was observed. The maximum activity was obtained at 35C and 180 rpm. Planchot and Colonna (2008)

42 purified A. fumigatus (Aspergillus sp. K-27) extracellular alpha amylase to homogeneity by using anion-exchange DEAE-cellulose and affinity -cyclodextrinSepharose chromatography. The purified enzyme was glycoprotein in nature which was found to contain 15 % carbohydrate. The purified alpha amylase exhibited an isoelectric point of 3.7, and SDS PAGE estimated that purified enzyme possess a molecular weight of 65,000. A large number of neutral hydrophobic residues were

present in an amino acid. The optimum enzyme activity was obtained at pH 5.5, and the enzyme showed stability at 40C. It hydrolyzed amylose and amylopectin, with respective Km of 0.42 and 7.7 mg mL- 1 and kcat/K m of 3.4 and 2.5 mL mg
-1

min-1.

The main end-products of maltohexaose, hydrolysis were glucose and maltose. While intermediate products were maltotriose, maltotetraose, and maltopentaose having an anomeric configuration. Although its capability to gradually degrade some 1-6 linkages, purified enzyme ought to be classified as an alpha-amylase. Leman et al. (2009) reported alpha amylase from A. oryzae had only very little effect on the side chain segments of the amylopectin molecules and the reason might be enzyme hydrolysis the segments of internal chain. Singh et al., (2009) investigated the effect of various agricultural by products as a substrate such as wheat bran, wheat straw, rye, straw on the alpha amylase production by Humicola lanuginose in solid state fermentation. Wheat bran proved to be good substrates for starch degrading enzymes because highest alpha amylase production was observed when wheat bran was used as a substrate. Various variables such as moisture content, incubation time inoculum size and carbon source has marked effect on the enzyme production. It was noted the optimum condition for the alpha amylase production by Humicola

43 lanuginose in SSF was incubation period 144 h, initial moisture content 90 %, initial pH of medium 6, incubation temperature 50C , size of inoculum 20 % and soluble starch as best carbon source. Shafique et al. (2009) tested the five strains of fungi belonging to two filamentous fungi A. niger and A. flavus for their ability to produce alpha-amylase. The different chosen strains were cultivated on two different typed of media i.e., potato dextrose agar (PDA) and enzyme production medium (EPM), the pH of medium was fixed at 3 level i.e., 4.5, 5.5 and 6.5. The efficiency or ability of strains was estimated on the basis of the formation of hydrolysis zone. EPM medium at pH 4.5 was best for the highest activity of alpha amylase. Strain 74 and strain 198 of A. niger and strain 209 and strain 231 of A. flavus gave best result on solid media; so these strains were selected for the alpha amylase production in submerged fermentation. All the selected strains showed highest activity of alpha amylase after 48 h in shake flasks.

44

Uses of alpha amylase

Starch is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. A large-scale starch processing industry has emerged in the last century. In the past decades, we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production of maltodextrin, modified starches, or glucose and fructose syrups. Currently, these enzymes comprise about 30 % of the world's enzyme production. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-staling agents in baking. A number of these starch-converting enzymes belong to a single family: the alpha amylase family or family13 glycosyl hydrolases. This group of enzymes share a number of common characteristics such as a (/)8 barrel structure, the hydrolysis or formation of glycosidic bonds in the conformation, and a number of conserved amino acid residues in the active site. As many as 21 different reaction and product specificities are found in this family.

Bread and chapatti industry

The quantities, taste, aroma and porosity of the bread are improved by using the enzyme in the flour. More than 70 % bread in U.S.A, Russia and European countries contain alpha amylase. Amylases play important role in bakery products (Goodwin and Mercer, 1972). For decades, enzymes such as malt and fungal alpha-amylases have been used in bread-making. The significance of enzymes is likely to raise as consumers insist more natural products free of chemical additives. For example, enzymes can be employed to

45 replace potassium bromate, a chemical additive that has been prohibited in a number of countries. The dough for bread, rolls, buns and similar products consists of flour, water, yeast, salt and possibly other ingredients such as sugar and fat. Flour consists of gluten, starch, non-starch polysaccharides, lipids and trace amounts of minerals. As soon as the dough is made, the yeast starts to work on the fermentable sugars, transforming them into alcohol and carbon dioxide, which makes the dough rise. The major component of wheat flour is starch. Amylases can degrade starch and produce small dextrins for the yeast to act upon. The alpha-amylases degrade the damaged starch in wheat flour into small dextrins, which allows yeast to work continuously during dough fermentation, proofing and the early stage of baking. The result is improved bread volume and crumb texture. In addition, the small oligosaccharides and sugars such as glucose and maltose produced by these enzymes enhance the Maillard reactions responsible for the browning of the crust and the development of an attractive baked flavour (Lundkvist et al., 2007).

Textile industry
Textile industries are extensively using alpha amylases to hydrolyze and solubilize the starch, which then wash out of the cloth for increasing the stiffness of the finished products. Fabrics are sized with starch. Alpha amylase is used as desizing agent for removing starch from the grey cloth before its further processing in bleaching and dyeing. Many garments especially the ubiquitions Jean are desized after mashing. The desired fabrics are finally laundered and rinsed (Iqbal et al., 1997; Allan et al., 1997).

46

Sugar and Glucose industries

Alpha amylase plays a very important role in the production of starch conversion products of low fermentability. The presence of starch and other polysaccharides in sugar cane creates problem throughout the sugar manufacturing which is minimized or eliminated by the action of alpha amylase. The high quality products depends upon the efficiency of the enzyme which lead to low production, costs for the starch processor has increased (De-cordt et al., 1994; Ensari et al., 1996; Hamilton et al., 1998). Many industries used alpha amylases for the production of glucose. Enzyme hydrolyzed the starch and converted it into glucose. They hydrolyze -1,4 glucosidic linkage in the starch polymer in a random manner to yield glucose and maltose (Akiba et al., 1998). Therefore, alpha amylase is extensively used in many industries for the production of glucose (Shetty and Crab, 1990). This process also gives the water-soluble dextrin.

Alcohol Industry
Alpha amylases convert starch in to fermentable sugars. Starches such as grain; potatoes etc. are used as a raw material that helps to manufacture ethyl alcohol. In the presence of amylases, the starch is first converted in to fermentable sugars. The use of bacterial enzyme partly replaces malt in brewing industry, thus making the process more economically significant. Alpha amylase can also carries out the reactions of alcoholysis by using methanol as a substrate (Santamaria et al., 1999).

47

Paper industry
Starch paste when used as a mounting adhesive modified with additives such as protein glue or alum, frequently, causes damage to paper as a result of its embrittlement. Starch digesting enzymes, e.g. alpha amylase, in immersion or as a gel poultice are applied to facilitate its removal. Alpha amylase hydrolyzed the raw starch that is used for sizing and coating the paper instead of expensive chemically modified starches. So, starch is extensively used for some paper size press publications (Okolo et al., 1996).

Detergent, Building product and Feed industries

In detergent industries, the enzyme alpha amylase plays a vital role. It is widely used for improvement of detergency of laundry bleach composition and bleaching with out color darkening (Borchet et al., 1995; Atsushi and Eiichi, 1998). The addition of enzyme stabilizes the bleach agent and preserves effectiveness of the bleach in laundry detergent bar composition (Onzales, 1997; Mirasol et al., 1997) Modified starch is used in the manufacture of gypsum board for dry wall construction. Enzyme modified the starch for the industry use. Many starches or barely material are present in the feed. So, the nutritional value of the feed can be improved by the addition of alpha amylase.

Chocolate industry
Amylases are treated with cocoa slurries to produce chocolate syrup, in which chocolate starch is dextrinizing and thus syrup does not become thick. Cocoa flavored syrups having a high cocoa content and excellent stability and flow properties at room

48 temperature may be produced by using an amylolytic enzyme and a sufficient proportion of Dutch process cocoa to provide a syrup pH of 5.5 to 7.5. The syrup is made by alternate addition of cocoa and sweetener to sufficient water to achieve a solids content of about 58 to 65 weight percent, adding an amylolytic enzyme, heating to a temperature of about 175 -185F for at least 10 to 15 min, raising the temperature to about 200 F. and cooling. The stabilized cocoa flavored syrups may be added at room temperature to conventional non-acid confection mixes for use in the production of quiescently frozen chocolate flavored confections (Ismail et al., 1992)

49

MATERIALS AND METHODS

3.1: MATERIALS
The chemicals used in this study such as sodium potassium tartarate, 3,5-dinitro salicylic acid, phenol, sodium metabisulphate, dihydrogen phosphate, manganese sulphate, yeast extract, ferrous sulphate, magnesium chloride, diammonium sulphate, starch, ferrous sulphate, acryleamide, bisacryleamide, trizmabase, Tris HCl, SDS, glycine, bromophenol blue, - mercaptoethanol, ammonium per sulphate, coomassie brilliant blue R-250, TEMED, etc were of analytical grade and obtained from Sigma (USA), BDH (UK), E-Merck (Germany), Acros (Belgium) and Fluka (Switzerland). All other chemicals were of the highest possible purity.

3.2: METHODS
3.2.1: Isolation of organism: The isolation of seventy eight Aspergillus oryzae cultures from soil samples collected from different habitats such as textile wastes, garden compost etc was carried out by serial dilution method (Clark et al., 1958). The soil samples were collected in sterile polythene bags. One gram of the soil sample was dissolved in 100 ml of sterilized distilled water. The soil suspension was then diluted up to 105-107 times. Approximately 0.5 ml of this diluted suspension was transferred to the Petri plates containing starch agar medium. The starch agar medium was prepared by dissolving 10 g of starch and 20 g of agar in 900 ml of distilled water and raising the volume up to1000 ml. The pH of medium was adjusted to 4.8 by 0.1N HCl/NaOH. After raising the volume to 1000 ml the medium was heated for 10 min to obtain a homogeneous

50 mixture. Approximately 10 ml of the medium was poured in separate test tubes. The tubes were cotton plugged and sterilized in autoclave at 15 lbs/in2 pressure (121C) for 15 min. After sterilization, the contents of each tube were transferred to the oven sterilized (Model: UM-400 MEMMERT, Germany) Petri plates (at 180C for 2 h) and allowed to solidify at room temperature. The fungal cultures were further purified from bacterial contaminants by using 10 mg/l mixture of penicillin and streptomycin (1:1 ratio) in the plate medium. After the addition of soil suspension, the Petri plates were rotated clockwise and counter clockwise for uniform spreading of suspension on the medium. The plates were placed in an incubator (Model: MIR-153, Sanyo Japan) at 30C for 3-4 days for culture development. The initial colonies forming clear zones of starch hydrolysis were picked up and transferred to potato dextrose starch agar slants for culture maintenance. The cultural and morphological characteristics of A. oryzae isolates were identified according to Onion et al. (1986). The potato dextrose starch agar medium was prepared by dissolving 39 g of PDA and 10 g of starch in 900 ml of distilled water and raising the final volume up to 1000 ml. This was cooked for 10-15 min with constant stirring until a clear solution formed. The pH of the medium was adjusted to 5.6 by 0.1N NaOH/HCl. Approximately 6-8 ml of the medium was poured in different test tube. All the tubes were cotton plugged and sterilized in an autoclave at 15 lbs/in2 pressure (121C) for 15 min. Afterwards, the tubes were kept in a slanting position (at an angle of about 30) to increase the surface area and allowed to solidify. The conidia of isolated fungi were aseptically transferred to the slants containing potato dextrose starch agar medium. The slants were incubated at 30C in an incubator for 3-5 days

51 for maximum growth. The slants were stored in a refrigerator at 4C for culture maintenance. 3.3: Fermentation 3.3.1: Inoculum preparation 3.3.1.1: Conidial inoculum Conidia from 3-4 day old slant cultures were used for inoculation. The conidial suspension was prepared in sterilized 0.005 % dioctyl ester of sodium sulpho succinic acid (Monoxal O.T). Ten milliliter of sterilized Monoxal O.T was transferred to each slant having profuse conidial growth on its surface. An inoculating needle was used to break the clumps of conidia. The test tube was shaken vigorously to make a homogeneous suspension. 3.3.1.2: Conidial count The numbers of conidia were counted with the help of a Haemacytometer. Each milliliter of the suspension contained 2.6 10 6 CFU. 3.3.1.3: Vegetative inoculum Fermentation medium of one hundred milliliter was transferred to a 1.0 L conical flask followed by the addition of approximately 20-25 glass beads (2.0 mm dia.). The flask was cotton plugged and sterilized. The four milliliter of the conidial suspension was transferred aseptically to the flask, which was then incubated at 30C on a orbital shaking incubator (Model: 10X400.XX2.C, SANYO Gallenkamp, PLC, UK) at 200 rpm for 24 h.

52 3.3.2: Fermentation media Different fermentation media (g/l) were evaluated for the alpha amylase production by selected strain of Aspergillus oryzae at pH 6 (Hayashida et al., 1986; Spohr et al., 1998; Nandakumar et al., 1999; Haq et al., 2002). M1: Wheat bran 100, Zn SO4.7H2O 0.062, FeSO4 0.068, Cu SO4.7H2O 0.0008. M2: Starch 10, yeast extract 3.0, MgSO4.7H2O, 0.005, CaCl2.2H2O 0.2, FeSO4 0. 1, Peptone 20, phosphate buffer 1000 ml. M3: Starch 10, MgSO4.7H2O 0.005, CaCl2.2H2O 0.2, FeSO4 0.1, (NH4) 2SO4 2, phosphate buffer 1000 ml. M4: Starch 20, yeast extract 8.5, NH4Cl 1.3, MgSO4.7H2O, 0.12, CaCl2 0.06. M5: Glucose monohydrate 4.84, (NH4)2SO4 4.84, KH2PO4 3.87, MgSO4.7H2O 3.75, NaCl 1.80, CaCl2.2H2O 1.21, trace metal solution 0.12 ml. M6: Glucose 50, NaNO3 3, KH2PO4 1.0, KCl 0.5, MgSO4.7H2O 0.2, FeSO4 0.01. 3.4: Shake flask studies Twenty-five milliliter of fermentation media (M4 optimized) was transferred to separate 250 ml cotton plugged conical flasks. The flasks were sterilized in an autoclave for 15 min and cooled at room temperature. A one milliliter of inoculum was transferred to each flask. The flasks were placed in the orbital shaking incubator for incubation at 30C with shaking speed of 200 rpm. After 72 h of incubation, content of flasks were filtered and filtrate was used for the estimation of enzyme while the residue was used for the estimation of cell mass. All the experiments were run parallel in triplicates.

53 3.5: Fermenter studies Scale up studies were carried out in a 7.5 L glass fermenter (Model: Bioflow-110 Fermenter/Bioreactor, USA) with a working volume of 5.0 L. The fermenter glass vessel containing 4.7 L fermentation medium was sterilized in a stainless steel autoclave (Model: KT-40 L, ALP, Japan) for 20 min at 15 lbs/in2 pressure (121C) and cooled at room temperature. Vegetative inoculum was transferred to the vessel through a hole at the top plate under aseptic conditions. The incubation temperature was kept at 30C, while the aeration and agitation rates were maintained at 1.0 L/L/min (vvm) and 200 rpm, respectively throughout the fermentation period. The air, to be supplied was sterilized by passing through membrane filters (0.45 m pore size). Sterilized solution of 0.1 N HCl/ NaOH was used for pH adjustment. The sterilized silicone oil 10 % (v/v) was used to control foam formed during the fermentation process. 3.6: Nutritional and cultural requirements of Aspergillus oryzae 3.6.1: Fermentation media Fermentation media play a very important role in the alpha amylase production as well as for the growth of organism. Six different media were evaluated for the enzyme production in shake flasks. 3.6.2: Incubation period Incubation period has a vital role for the optimal alpha amylase production by

54 Aspergillus oryzae. The enzyme fermentation was carried out (8-96 h) at 30C and sample was collected every 8 h for the estimation of enzyme and dry cell mass in present study. 3.6.3: Effect of initial pH Maintenance of a favorable pH is one of the most important steps for successful progression and termination of fermentation (Gigras et al., 2002). A range of different pH (4-7) in shake flasks and (4-6.5) in fermenter was tested for alpha amylase production. 3.6.4: Effect of temperature The optimal incubation temperature is a function of the microbial strain and should be determined for each set of conditions (Bhanja et al., 2007). The effect of different temperature (25-50C) on the biomass formation and production of enzyme was investigated in present study. 3.6.5: Effect of volume The effect of different volume of basal medium on the alpha amylase production by Aspergillus oryzae was investigated. The amount of fermentation medium such as 5, 10, 15, 20, and 25 % (w/v) was evaluated in shake flask fermentation. 3.6.6: Effect of inoculum size The size of inoculum is very important for alpha amylase production. Conidial inoculum at varying concentration (2-12 % (v/v) during shake flasks and vegetative inoculum (5.0-12.5 % v/v) during fermenter studies was investigated. The initial pH, temperature, incubation time, agitation intensity, aeration were maintained constant.

55 3.6.7: Effect of agitation and aeration Agitation and aeration are interrelated and had direct influence on the alpha amylase production. Different agitation intensity (120-240 rpm) with air supply from 0.5-2.0 vvm was investigated for optimum alpha amylase production. 3.6.8: Evaluation of carbon sources Carbon sources play a vital role for the growth as well as for the alpha amylase production. Different additional carbon sources such as sucrose, glucose, lactose, xylose, fructose, galactose, glycerol, mannitol and CMC were evaluated for the production of alpha amylase by Aspergillus oryzae (Carlsen and Nielsen, 2001). 3.6.9: Evaluation of nitrogen sources Different organic and inorganic nitrogen sources such as peptone, yeast extract, meat extract, urea, casein, beef extract, corn steep liquor, ammonium nitrate, ammonium sulfate, sodium nitrate, potassium nitrate etc, were evaluated for the enzyme production as well as for the growth of organism. 3.7: Induction of mutation 3.7.1: Minimal inhibitory concentration of 2-deoxy-D-glucose The parental strain was grown on starch agar medium along with 2-deoxy-D-glucose (0.0-0.5 % w/v) at 30C in order to find out the minimal inhibitory concentration (MIC), (Azin and Noroozi, 2001).

56 3.7.2: Ultraviolet (UV) irradiation From the parental fungal isolate (5 day old culture), 1 ml of the conidial suspension was transferred to a cotton wool plugged conical flask containing 25 ml of sterilized M1 medium. The conidia were allowed to grow at 30C on a shaking incubator with 200 rpm for about 6 h to get fresh growing fungal mycelia. Five milliliter of medium containing mycelial suspension was transferred to a sterilized Petri plate and these mycelia were exposed to ultraviolet (UV) irradiation for 15-75 min under the beam (=253 nm and 220 V at 50 c/s) of UV lamp (Model: Mineral Light, UVS-12, California, USA). The radiation dose given to the mycelial suspension was 1.2102 J/m2/s. The distance between lamp and suspension was adjusted at 8 cm for each trial to get more than 95 % death rate (Azin and Noroozi, 2001). 3.7.3: Nitroso guanidine treatment (NG) NG was prepared in four different concentrations from 0.5-2.0 mg/ml. N-methyl-Nnitro-N-nitroso guanidine (NG) was transferred to each sterilized centrifuged tube containing 5 ml of conidial suspension and incubated at 30C using a shaking water bath (Model: WB-14, MEMMERT, Germany) for specific time interval to achieve a death curve with sub-lethal level. After treatment with NG, 1 ml of cystein (1 %, w/v) was added to terminate the reaction. The conidia were treated similarly except replacing NG with sterile distilled water in control experiment. After fixed time interval, the tubes were spun at 6000 g for 15 min. The supernatant was discarded to remove NG from the fungal cells. Traces of NG were removed after three appropriate washings with 0.1 M phosphate citrate buffer (pH 7.5). The treated conidia were resuspended in the same buffer.

57 3.7.4: Nitrous acid treatment A 0.07-0.3M solution of NaNO2 prepared in acetate buffer (0.2 M, pH 4.5) was added to washed and spun conidia of A. oryzae (Carlton and Brown, 1981). The solution was shaken thoroughly for specific time intervals. The one milliliter solution was withdrawn and diluted 5 fold in phosphate buffer (0.2 M, pH 7.1) to stop the reaction. A control was run similarly except replacing NaNO2 in acetate buffer with sterilized saline water. After fixed time interval, the tubes were spun at 6000 g for 15 min. The supernatant was discarded to remove nitrous acid from the fungal conidia and ten milliliter of sterilized phosphate buffer was added to each tube. The tubes were respun for the removal of traces of nitrous acid from conidia and repeated three times. After washing the conidia were resuspended in same buffer. 3.7.5: EMS treatment Different concentrations (25-150 l) of ethyl methane sulphonate (EMS) were added to individual centrifuge tubes containing 4 ml of conidial suspension and shaken to form a homogeneous suspension. After specific time intervals the conidia were spun and washed thrice in phosphate buffer. The EMS treated conidia was resuspended in same buffer. 3.7.6: Selection of mutants After treatment with mutagenic agents, about 100 l of each suspension containing treated conidia was aseptically transferred to the individual Petri plates containing starch agar medium supplemented with (g/L); Triton X-100 (5.0), 2- deoxy-D-glucose (above the MIC of parent strain). The plates were incubated at 30C and were

58 examined regularly after 3-4 day to study the growth pattern. The colonies were selected qualitatively; showing the bigger zone of starch hydrolysis compared to parental strain and was allowed to grow on PDA slants for culture maintenance. These colonies were then tested quantitatively for enzyme production in shake flasks fermentation. 3.8: Analytical techniques After incubation, the fermented broth was filtered. The filtrate was used for the estimation of total protein contents, and alpha amylase activity. 3.8.1: Estimation of alpha amylase The estimation of alpha amylase was carried out according to the method of Rick and Stegbauer (1974). One unit of activity was that amount of enzyme, which in 10 min liberates reducing group from 1 % Lintners soluble starch corresponding to 1mg of maltose hydrate. The enzyme activity was determined by taking 1 ml of diluted filtrate in a test tube. The one milliliter of starch solution (1 % w/v) was also added into it. A blank was run parallel by replacing the filtrate with 1 ml of distilled water. After incubation of 10 min at 40C, the reducing sugar liberated was measured at 546 nm by the DNS method (Miller, 1959) using maltose as a standard. 3.8.2: Estimation of total protein contents Total protein contents were determined by taking 0.1 ml of the filtrate with 5 ml of Bradford reagent in a test tube and vortexes thoroughly. A blank containing 0.1 ml of distilled water with 5 ml of the Bradford reagent was run parallel. The absorbance was taken at 595 nm on a double beam UV/VIS scanning spectrophotometer (Model: CE-

59 7200, CECIL, England, UK) after 15 min of the reaction using bovine serum albumin (BSA) as a standard. Protein contents were determined from the standard curve of BSA (Bradford 1976). 3.8.3: Determination of mycelial morphology Mycelial morphology was determined on an aliquot extended on the Petri plates followed by pellet diameter (Moreira et al., 1996). For rounded pellets, if the diameter was less than 0.5 mm, they were categorized as fine pellets, between 0.5-2 mm as small pellets, between 2-3 as intermediate pellets while those above 3 mm were referred to as large pellets. 3.8.4: Estimation of dry cell mass (DCM) Dry cell mass was determine by filtering the culture broth through preweighed Whatman filter paper No. 44. Mycelia were thoroughly washed with tap water and dry in oven at 105C for 2 h. The dry cell mass was weighed and calculated as g/l by subtracting the initial weight from the final weight. 3.9: Statistical analysis Treatment effects were compared by the method of Snedecor and Cochran (1980). Post-Hoc Multiple comparison tests were applied under one-way ANOVA. Significance has been presented in the form of probability (p<0.05) values. 3.10: Kinetic study Kinetic parameters for batch fermentation were determined according to the method describe by Pirt (1975) and Lawford and Rouseau (1993). The following parameters of kinetics were studied:-

60 Specific growth rate The value of specific growth rate i.e., (h-1) was calculated from plot of In (x) vs time of fermentation. Product yield co efficient Product yield co efficient namely Yp/x was determined by the equation: Yp/x=dP/dx Volumetric rates The volumetric rate of product formation Qp (U/l/h) was determined from the maximum slope of enzyme produced vs time of fermentation. The volumetric rate for biomass formation Qx (g cell mass /l/h) was determined from the maximum slope of cell mass formation vs time of fermentation. Specific rate constant Specific rate constant for product formation was determined by the equation qp = Y p/x 3.11: Purification of alpha amylase 3.11.1: Separation of fungus from fermented broth The fermented broth was spun at 9,000 g for 15 min at 4oC. The clear supernatant was used for enzyme isolation and purification.

61 3.11.2: Ammonium sulfate precipitation Solid ammonium sulfate was added to 1000 milliliter of crude cell free broth of alpha amylase. The suspension was stirred for half an hour at 4C. After sufficient shaking, the precipitates were collected by spining at 14,000 g for 20 min at 4C. To the supernatant, added calculated amount of ammonium sulfate for 20-90 % (w/v) saturation. Same procedure was applied to get the precipitates of all fractions. The pelleted precipitate of each fraction was resuspended in 0.05 M Tris-HCl buffer, pH 7.5. The solution was dialyzed against the same buffer. 3.11.2.1 Anion- exchange chromatography For the purpose of ion exchange chromatography, 0.4 g DEAE-Sephadex A-50 (Sigma, USA) was swollen in 100 milliliter of the 0.05 M Tris-HCl buffer, pH 7.5 in a boiling water bath for 2 h. After cooling poured it into the column and made final bed volume (1.5 10 cm). The dialyzed enzyme solution was applied to column that preequilibrated with five column volumes of the 0.05 M Tris-HCl buffer, pH 7.5. A stepwise NaCl gradient from 0 to 1 M in 150 ml of the same buffer was applied. Fractions of 3 ml were collected at a flow rate 0.5 ml/min. The collected fractions were assayed for protein at 280 nm and alpha amylase activity by performing enzyme assay. The fractions containing enzyme activity were pooled, dialyzed and analyzed on SDS-PAGE. 3.11.2.2: Gel filtration Sephadex G-100 (Phamacia Fine Chemical), 2 g was swollen in 50 ml of 0.05 M TrisHCl buffer, pH 7.5 in a boiling water bath for 2 h. Poured the gel slurry along the side

62 of tilted column by taking care that no air bubble was entrapped. The column (1.5 15.0 cm) was equilibrated with five column volumes of the 0.05 M Tris-HCl buffer, pH 7.5 in order to stabilize the bed. The enzyme sample (5 ml) was eluted with the same buffer; adjusting flow rate at 0.5 ml/min. The collected fractions were assayed for protein and alpha amylase activity. The active enzyme fractions were pooled and used for the determination of main characteristics of the enzyme. 3.11.3: Dialysis The salts were removed by dialyzing precipitates and pooled samples by using 12,000 molecular weight cut off dialyzing bag, which was placed in one liter of the 0.05 M Tris-HCl buffer (pH 7.5) for 3-4 h at 4C. The process was repeated 4-5 times until all salts were removed from the enzyme solution. 3.11.4: Electrophoresis The homogeneity of the purified enzyme was confirmed by sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Hames (1990). 3.11.5: Protein marker The molecular weight of the alpha amylase was estimated by SDS- polyacrylamide gel with protein marker (SMO 313). 3.12: Gel preparation 3.12.1: Separating gel: The separating gel (12 % w/v) was prepared by adding 4 ml acrylamide (30 % w/v); 2.5 ml, 1.5 M Tris HCl (pH 8.8); 0.1 ml SDS (10 % w/v); 0.1 ml ammonium per

63 sulfate(10 % w/v); 6 l TEMED ; 3.3 ml distilled water and poured in the gel assembly leaving one inch vacant space at the top. Almost 100 l of distilled water was layered at the top of the gel to give a flat surface and to remove oxygen which inhibited polymerization. The gel was allowed to polymerize for 30 min. 3.12.2 Stacking gel: The stacking gel was prepared by adding 0.5 ml acrylamide (30 % w/v); 0.38 ml 1M Tris HCl (pH 6.8); 0.03 ml, SDS 10 % (w/v); 0.004 ml APS (10 % w/v); 0.0003 ml TEMED; 2.1 ml Distilled water. The water was removed from top of the separating gel and stacking gel was poured in the gel assembly. Comb was inserted and gel was allowed to polymerize at the room temperature for 10 min. When complete polymerization took place, gel comb was taken out and valves were washed with tank buffer four times by means of a syringe. After removing the bottom spacer the gel assembly was settled in the gel chamber and made contact top and bottom with tank buffer which was previously diluted in the ratio of 1:5 with distilled water. The enzyme solution (6 l) and loading buffer (4 l) were denatured by heating in boiling water bath for 3 min. The samples were loaded along the protein marker and electrophoreses at a constant voltage of 150 v potential difference and 20 mA current supplies for about 4 h. 3.13: Characterization of enzyme Temperature and pH had great influence on the activity of alpha amylase. The enzyme substrate complex was incubated at varying temperatures (25-70C) and pH (3-7) and the effect on the activity of purified enzyme was observed. Metal ions had marked

64 influence on the activity of enzyme so effect of different metal ions such as Mg SO4, MnSO4, NaCl, NiCl2, Zn Cl2, CuCl2, COCl2, and FeSO4 on the activity of alpha amylase was also studied. 3.14: Standard curves 3.14.1: Maltose Anhydrous maltose (100 mg) was dissolved in a small quantity of distilled water and volume was made up to 100 ml in a measuring flask. The stock solution (1 mg/ml) was used to make 10 appropriate dilutions ranging from 0.1 to 1 mg/ml. The one milliliter of each dilution was taken in separate test tubes followed by the addition of 2 ml of DNS reagent. A blank was run in parallel replacing the maltose dilution with 1 ml of distilled water. The tubes were incubated in a boiling water bath for 5 min prior to cooling at room temperature. Absorbance was measured at 546 nm using a spectrophotometer (Model: CECIL CE-7200 Aquarius, UK). A graph was plotted taking the absorbance at the ordinate and sugar concentration at the abscissa (Fig 1). 3.14.2: Bovine serum albumin (BSA) BSA (100 mg) was dissolved in approximately 90 ml of distilled water. The final volume was raised up to 100 ml using a measuring flask. The stock solution (1 mg/ml) was used to make 10 appropriate dilutions ranging from 100-1000 l/ml. Each dilution (0.1 l) was taken in a separate test tube followed by the addition of 5 ml of BSA reagent. A blank was also run in parallel by replacing BSA with distilled water. The mixture was allowed to stand for 5-15 min for maximum coloration and optical density was measured at 595 nm using a spectrophotometer (Model: CECIL CE-7200

65 Aquarius, UK). The curve was plotted by taking BSA concentration along x-axis and optical density along y-axis (Fig 2).

3.15: Preparation of reagents/buffers

3.15.1: Dinitrosalicylic acid (DNS) reagent Dinitrosalicylic acid (10.6 g) and sodium hydroxide (19.5 g) were dissolved in approximately 600-800 ml of distilled water and gently heated in a water bath at 80C until a clear solution was obtained. Sodium potassium tartrate (306 g), phenol melted at 60C (7.5 ml) and sodium metabisulfate (8.3 g) were also added. After dissolving the chemicals, final volume was raised up to 1416 ml with distilled water. The solution was filtered through a large coarse sintered glass filter and stored at room temperature in an amber colored bottle to avoid photo oxidation. It was stable for about six months. 3.15.2: Bradford reagent The coomassie brilliant blue hundred milligrams (G-250) was added in 50 ml of 95 % (v/v) ethanol. This solution was poured into 100 ml of 85 % (w/v) phosphoric acid and the final volume was raised up to 1.0 L with distilled water. After shaking filtered through Whatman filter paper (No. 1) to obtain a clear solution. The reagent was stored in an amber colored bottle to avoid photooxidation. 3.15.3: Starch Solution: The Lintners soluble starch 1g was dissolved in 100 ml of acetate buffer (pH 5) and boiled until solution become transparent.

66 3.15.4: Acetate Buffer (pH 5.0) Sol A: Acetic acid (12.06 ml) was added in approximately 900 ml distilled water and final volume was raised up to 1 L Sol B: Sodium acetate (27.22 g) was dissolved in approximately 900 ml distilled water and final volume was raised up to 1 L. Buffer solution (pH 5) was prepared by adding 148 ml of Sol A and 352 ml of Sol B and raising the final volume up to 1 L. 3.15.5: Phosphate citrate buffer (pH 7.5) The buffer was prepared by dissolving 922.5 g Na2HPO4 and 77.5 g citric acid (0.1 M) in 700-800 ml distilled water and finally volume was raised to 1000 ml to get 0.1 M phosphate citrate buffer having pH 7.5. 3.15.6: Preparation of 0.05 M Tris-HCl buffer (pH 7.5) The Tris HCl buffer was prepared by dissolving 6.25 g of Tris in 700-800 ml of distilled water and adjusted pH 7.5 with 5 N HCl with constant stirring. Finally volume was raised to 1000 ml with distilled water. 3.15.7: Acrylamide bisacrylamide (30 % w/v) The 30 % (w/v) acrylamide bisacrylamide was prepared by dissolving 29 g of acrylamide and 10 g bisacrylamide in 1000 ml of distilled water. The solution was filtered and stored at 4C.

67 3.15.8: Separating buffer (1.5 M Tris HCl, pH 8.8) The 1.5 M Tris HCl buffer was prepared by dissolving 181.5 g Trizma in 900 ml of distilled water with constant stirring to adjust the pH 8.8 by adding concentrated HCl (32 % v/v) drop wise. After pH adjustment raised the final volume upto 1000 ml with distilled water. 3.15.9: Stacking buffer (1 M Tris HCl, pH 6.8) The 1 M Tris HCl, buffer was prepared by dissolving 157.6 g Trizma base in 900 ml of distilled water with constant stirring to adjust the pH at 6.8 by adding concentrated HCl (32 % v/v) drop wise. After pH adjustment, the final volume was raised up to 1000 ml with distilled water. 3.15.10: Tank buffer (10 X, pH 8.3) The Tank buffer was prepared by dissolving 3.94 g of Trizma base, 14.41 g of glycine and 50 g of SDS in distilled water and raising the final volume up to 1000 ml. The solution was stored at 4C. 3.15.11: Gel loading buffer The Gel loading buffer was prepared by mixing 1ml of Tris HCl (pH 6.8) 400g SDS, 200 ml glycerol, 2 g bromophenol blue dye, 1500 l -mercaptoethanol and raised the volume up to 1000 ml and stored at 4C. 3.15.12: SDS solution (10 % w/v) The 10 % w/v SDS solution was prepared by dissolving 100 g of SDS in hot water. The solution was stirred and final volume was made up to 1000 ml.

68 3.15.13: Ammonium per sulfate The Ammonium per sulfate solution was prepared by dissolving 0.1 g of APS in distilled water and raised the final volume up to 1 ml. Freshly prepared solution of APS was used. 3.15.14: Staining and destaining solution Staining solution was prepared by dissolving 2.5 g of Coomassie brilliant blue R-250 in 450 ml of methanol and then added 100 ml of acetic acid and raised the final volume up to 1000 ml with distilled water. Destaining solution was prepared by mixing 300 ml methanol, 100 ml acetic acid and volume was raised up to1000 ml with distilled water.

69 Fig 3.1: Standard curve of maltose

1.2 1.0 0.8 Absorbance 0.6 0.4 0.2 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Maltose conc. (mg/ml)

70

Fig 3.2:

Standard curve of bovine serum albumin (BSA)

0.4

0.3

Absorbance

0.2

0.1

0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 BSA (ug/ml)

71

RESULTS
4.1: IDENTIFICATION, ISOLATION AND SCREENING OF ORGANISM Strains of Aspergillus oryzae were isolated for the of alpha amylase production from different soil samples, by serial dilution method on the plates containing starch agar medium. The strains were identified according to Onion et al. (1986). Colonies of A. oryzae were greenish yellow, olive yellow or with different shades of green, typically with dull brown shades with age. Colonies consisting of long conidiophores often intermixed with aerial mycelium. Conidial heads radiate greenish yellow later becoming light to dull brown. Conidiophores were hyaline, up to 4-5 mm in length, mostly rough-walled. Vesicles were subglobose, 40-80 m in diameter.

Conidiogenous cells uniseriate and biseriate. Phialides often directly borne on the vesicle or on metulae, usually measuring 10-15 x 3-5 m. Metulae 8-12 x 4-5 m. Metulae or phialides covering the entire surface or the upper three-fourths of the vesicle. Conidia ellipsoidal when young, globose to subglobose when mature, 4.5-8.0 m in diameter, green, smooth to finely rough walled. Screening of A. oryzae isolates for alpha amylase production was carried out in shake flasks. The seventy eight isolates were screened out for their enzyme synthesizing ability (Table 4.1). The range of enzyme activity of the wild isolates is given in Table 4.1.1. Of all the isolates examined, the isolate No. 30 gave maximum enzyme production and assigned the code IIB-30. This strain was selected for subsequent studies.

72 Table 4.1: Isolation and screening of Aspergillus oryzae for the alpha amylase production Isolate No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Enzyme activity(U/ml) 1030.3 600.1 200.2 410.5 900.1 290.2 1180.1 130.3 3.00.1 730.1 1000.2 830.4 1110.1 400.75 810.15 110.1 350.1 500.2 960.1 7.00.1 .1.00.1 570.2 330.2 170.3 1090.1 690.3 870.1 260.1 450.1 1300.1 530.2 120.2 600.4 700.1 1200.2 910.1 2.00.2 1110.3 140.1 Dry cell mass (g/l) 9.50.5 6.30.3l 3.50.2 4.50.1 8.50.1 3.90.1 10.00.2 2.90.3 2.00.1 9.00.1 9.50.2 7.30.3 2.70.1 3.90.2 7.50.2 2.90.7 4.00.1 5.70.2 7.80.1 2.00.1 1.50.1 6.00.1 3.00.1 2.70.3 7.00.2 6.30.3l 4.90.1 1.60.1 5.10.1 130.1 3.10.2 1.90.1 4.20.1 7.30.2 10.30.1 5.10.3 1.30.1 5.50.1 2.10.2 Mycelial morphology Large pellets Small pellets Small pellets Large pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets Large pellets Large pellets Small pellets Large pellets Small pellets

73 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 970.5 390.1 9.00.1 790.2 990.2 200.2 470.1 6.00.1 630.2 780.4 130.1 830.3 1140.1 590.1 390.5 1120.1 930.2 150.05 610.4 100.1 1020.1 2.00.5 580.2 270.5 770.2 1100.5 1080.1 110.15 300.5 640.1 870.2 980.1 210.4 6.00.1 510.1 1150.5 160.2 440.2 630.1 9.20.1 5.00.2 2.00.1 7.00.1 7.30.2 2.70.3 6.00.2 2.00.1 11.90.1 5.20. 3.60.2 6.50.1 5.20.1 5.90.1 4.90.2 5.90.2 6.70.1 2.110.05 5.00.4 3.00.1 6.00.1 1.30.5 4.30.2 1.90.5 6.50.2 7.30.5 8.50.1 1.90.1 2.80.5 3.90.1 5.70.2 7.90.1 2.10.4 1.30.1 4.30.1 9.50.5 2.10.2 3.80.2 5.70.1 Small pellets Large pellets Small pellets Large pellets Large pellets Small pellets Small pellets Small pellets Large pellets Large pellets Small pellets Large pellets Large pellets Small pellets Small pellets Large pellets Large pellets Small pellets Large pellets Small pellets Large pellets Small pellets Small pellets Small pellets Large pellets Large pellets Large pellets Small pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets

The mean difference is significant at the level of 0.05, indicates the standard deviation (SD) among the three parallel replicates in each column. Incubation time 72 h, pH 6.0, incubation temperature 30C, agitation rate 160 rpm

74

Table 4.1.1: Sub grouping of alpha amylase producing isolates of A. oryzae Number of isolates 35 30 13 Range of enzyme activity (U/ml) 1-50 51-100 101-150

The one culture gave a maximum of 130U/ml alpha amylase production and it was coded as A. oryzae IIB-30 this culture was selected for mutation through UV radiations.

75

4.2: STRAIN IMPROVEMENT

4.2.1: PHYSICAL MUTAGENESIS 4.2.1.1: SCREENING OF UV TREATED ISOLATES The different isolates were obtained by irradiating the conidia of A. oryzae IIB-30 with different doses of UV light (15-75 min) in order to increase the alpha amylase production. The data of Table (4.2) shows the screening of UV treated isolates for the enzyme production. A total of 32 strains were isolated by observing bigger zones of starch hydrolysis in Petri plate compared to parental strain. Of all the isolates tested, the strain isolated after 45 min of UV irradiation with a zone diameter of 1.5 cm gave maximum enzyme production (1602 U/ml). The mutant strain gave maximum production was assigned the code UV-23 and selected for further studies. The number of survivors and range of enzyme production of the UV treated isolates is given in Table 4.2.1 and 4.2.2, respectively.

76

Table 4.2: Screening of UV isolates of A. oryzae IIB-30 for alpha amylase production* UV irradiated isolates UV-1 UV-2 UV-3 UV-4 UV-5 UV-6 UV-7 UV-8 UV-9 UV-10 UV-11 UV-12 UV-13 UV-14 UV-15 UV-16 UV-17 UV-18 UV-19 UV-20 UV-21 UV-22 UV-23 UV-24 UV-25 UV-26 UV-27 UV-28 UV-29 UV-30 UV-31 UV -32 Exposure time(min) 15 Enzyme activity (U/ml) 1201.0 900.3 700.2 1010.5 930.4 1130.7 1261.5 1401.2 991.0 1060.9 870.4 1001.0 930.8 1171.1 730.9 1291.4 820.6 630.1 1090.8 760.7 1361.3 1231.2 1602.0 280.1 390.1 900.3 780.5 800.6 1151.0 1321.2 530.2 430.1 Dry cell mass (g/l) 12.00.1 10.50.1 9.600.1 11.70.1 10.70.1 11.80.1 12.90.2 13.20.3 11.30.2 11.80.2 10.20.1 11.00.2 10.30.1 11.30.1 9.600.1 13.10.2 10.00.1 8.500.1 12.10.2 9.00.1 13.20.2 12.70.3 14.80.4 5.200.1 6.600.1 10.80.2 13.10.2 10.70.1 12.00.2 13.10.3 7.50.1 7.20.1 Mycelial morphology Large pellets Large pellets Small pellets Large pellets Small pellets Large pellets Large pellets Large pellets Small pellets Large pellets Small pellets Large pellets Small pellets Large pellets Small pellets Large pellets Small pellets Small pellets Large pellets Small pellets Large pellets Large pellets Large pellets Small pellets Small pellets Small pellets Small pellets Small pellets Large pellets Large pellets Small pellets Small pellets

30

45

60

75

The mean difference is significant at the level of 0.05, indicates the standard deviation (SD) among the three parallel replicates in each column. Incubation time 72 h, pH 6.0, incubation temperature 30C, agitation rate 160 rpm

77

Table 4.2.1: UV treated survivors at different exposure time Exposure time 15 30 45 60 75 Total number of survivors 38 25 17 13 2.0

Table 4.2.2: Range of alpha amylase activity of UV isolates Number of strains 3 15 14 Range of enzyme activity (U/ml) 1-50 51-100 101-150

The one isolate gave maximum production of alpha amylase 160 U/ml and it was coded as UV-23. This strain was selected for mutation through NG.

78

4.3: CHEMICAL MUTAGENESIS

4.3.1: SCREENING OF NG TREATED ISOLATES The UV treated mutant was further subjected to chemical treatment in order to increase the alpha amylase production. The A. oryzae UV-23 was exposed to different concentrations of NG (0.5-2.0 mg/ml). The data of Table (4.3) shows the screening of NG treated isolates for the enzyme production. A total of 18 strains were isolated by observing bigger zones of starch hydrolysis in Petri plate compared to parental strain. Of all the isolates tested, the strains isolated after treatment with 1.5 mg/ml NG with a zone diameter of 1.9 cm gave maximum (2700.1 U/ml) enzyme production which is two fold than UV-23. The mutant strain exhibiting highest enzyme production assigns the code NG-15 and selected for further studies. When the concentration of NG increased, the number of survivors decreased as shown in the Table 4.3.1. At 2.0 mg/ml concentration of NG 90 % death rate was observed further increase in concentration cause complete death of organism. The number of survivors and range of enzyme production by NG treated isolates is given in table 4.3.1 and 4.3.2, respectively. 4.3.2: SCREENING OF NITROUS ACID TREATED ISOLATES The NG mutant strain A. oryzae NG-15 were further treated with different concentrations of nitrous acid (0.1-0.4 M). Selective NA treated strains were isolated on the basis of qualitative screening showing bigger zones of starch hydrolysis than the parental strain. The NA treated strains were screened for enzyme production

79 (Table 4.4). Out of which mutant strain NA-17 gave maximum alpha amylase production (2850.1 U/ml). When the concentration of nitrous acid was increased, the number of the survivors was decreased as shown in the Table 4.4.1. Range of enzyme production of the nitrous acid treated isolates is given in Table 4.4.2. 4.3.3: SCREENING OF EMS TREATD ISOLATES The nitrous acid treated mutant NA-17 was subjected to the varying concentration (25150 l/ml) of ethyl methane sulphonate (EMS).A total of twenty-six EMS treated A. oryzae strains were obtained at 90 % death rate, which were screened for alpha amylase production (Table 4.5). The mutant EMS-18 showed 1.5-fold higher enzyme yield (3471.2U/ml) compared to NA-17. As the concentration of EMS was increased, the number of survivors was decreased (Table 4.5.1.) The range of enzyme production of EMS treated isolates is given in Table 4.5.2

80

Table 4.3: Screening of NG treated A. oryzae UV-23 isolates for alpha amylase production *

NG treated isolates NG-1 NG-2 NG-3 NG-4 NG-5 NG-6 NG-7 NG-8 NG-9 NG1-0 NG-11 NG-12 NG-13 NG-14 NG-15 NG-16 NG-17 NG-18

NG concentration (mg/ml) 0.5

1.0

1.5

2.0

Enzyme activity(U/ml) 1600.2 1090.8 1360.3 1500.1 960.2 1110.1 870.6 1230.2 2011.0 1450.4 340.1 2200.3 830.5 1320.5 2700.1 630.4 1030.3 490.1

Dry cell mass(g/l) 14.90.1 11.50.3 12.80.2 13.90.1 12.70.2 11.90.1 10.40.2 12.20.2 15.01 11.60.2 3.600.1 14.30.3 8.300.3 11.60.1 15.60.4 5.600.1 10.50.1 6.200.4

Mycelial morphology Large pellets Small pellets Large pellets Large pellets Small pellets Small pellets Small pellets Large pellets Large pellets Large pellets Small pellets Large pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets

The mean difference is significant at the level of 0.05, indicates the standard deviation (SD) among the three parallel replicates in each column. *Incubation time 72 h, temperature 30C, pH 6.0, agitation rate 160 rpm

81

Table 4.3.1: NG treated survivors of A. oryzae NG concentration(mg/ml) 0.5 1.0 1.5 2.0 Total number of survivors 31 20 11 2.0

Table 4.3.2: Range of alpha amylase activity of NG isolates Number of isolates 2 4 8 1 2 1 Range of enzyme activity(U/ml) 1-50 51-100 101-150 151-200 201-250 251-300

The one culture gave maximum production of alpha amylase 270 U/ml and it was coded as NG 15. This culture was selected for mutation with nitrous acid.

82

Table 4.4: Screening of nitrous acid treated strains of A. oryzae NG-15 for the alpha amylase production* Nitrous acid treated isolates NA-1 NA-2 NA-3 NA-4 NA-5 NA-6 NA-7 NA-8 NA-9 NA-10 NA-11 NA-12 NA-13 NA-14 NA-15 NA-16 NA-17 NA-18 NA-19 NA-20 NA-21 conc. of nitrous acid(M) 0.1 Enzyme activity(U/ml) 2610.2 2560.7 2590.1 2180.6 2470.1 2580.5 2500.7 2620.3 2660.3 2590.0.2 2480.6 2300.6 2560.1 1680.2 2090.5 1960.4 2850.1 390.4 580.7 1090.5 1530.1 Dry cell mass(g/l) 15.00.1 15.20.2 14.00.1 14.30.3 14.90.1 13.80.8 13.70.9 13.90.3 12.70.1 11.90.3 13.00.3 13.80.1 14.90.1 9.900.4 11.60.05 11.30.1 16.10.1 2.600.2 3.800.2 6.800.2 10.60.3 Mycelial morphology Large pellets Large pellets Large pellets Large pellets Large pellets Large pellets Large pellets Large pellets Large pellets Small pellets Large pellets Large pellets Large pellets Small pellets Large pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets

0.2

0.3

0.4

The mean difference is significant at the level of 0.05, indicates the standard deviation (SD) among the three parallel replicates in each column. *Incubation time 72 h, temperature 30C, pH 6.0, agitation rate 160 rpm

83

Table 4.4.1: Nitrous acid treated survivors of A. oryzae Nitrous acid concentrations (M) 0.1 0.2 0.3 0.4 Total number of survivors 39 32 27 24

Table 4.4.2: Range of alpha amylase activity of nitrous acid treated isolates Number of isolates 1 1 1 3 6 9 Range of enzyme activity (U/ml) 1-50 51-100 101-150 151-200 201-250 251-300

The one isolate gave a maximum production of alpha amylase 285 U/ml and it was coded as NA17.This strain was further treated with EMS.

84

Table 4.5: Screening of EMS treated A. oryzae NA17 for the alpha amylase production * EMS treated isolates EMS_1 EMS-2 EMS-3 EMS-4 EMS-5 EMS-6 EMS-7 EMS-8 EMS-9 EMS-10 EMS-11 EMS-12 EMS-13 EMS-14 EMS-15 EMS-16 EMS-17 EMS-18 EMS-19 EMS-20 EMS-21 EMS-22 EMS-23 EMS-24 EMS-25 EMS-26 EMS conc.(l/ml) 25 Enzyme activity (U/ml) 2550.1 1980.2 2500.3 2300.2 2750.2 2001.0 2730.1 2400.5 1680.05 2900.3 1970.2 1000.7 1870.4 3000.8 871 1180.1 1470.5 3470.1 1060.1 47.00.2 930.4 1011 2080.1 2870.1 2010.3 1260.2 Dry cell mass(g/l) 14.70.1 13.90.1 14.20.2 14.10.1 15.60.1 12.31 15.20.1 13.20.2 14.10.1 12.30.3 15.30.1 12.40.4 11.70.2 12.10.1 15.91 7.200.2 10.60.5 16.90.1 11.60.2 5.80.2 2.60.1 5.81 13.30.4 14.10.1 12.60.3 10.20.2 Mycelial morphology Large pellets Large pellets Large pellets Large pellets Large pellets Small pellets Large pellets Large pellets Small pellets Large pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Large pellets Small pellets Small pellets Small pellets Small pellets Small pellets Large pellets Large pellets Small pellets

50

75

100

125

150

The mean difference is significant at the level of 0.05, indicates the standard deviation (SD) among the three parallel replicates. *Incubation time 72h, temperature 30C, pH 6.0, agitation rate 160rpm

85

Table 4.5.1: EMS treated survivors of A. oryzae EMS concentrations (l) 25 50 75 100 125 150 Total number of survivors 45 32 21 5 3 2

Table 4.5.2: Range of -amylase activity of EMS treated isolates Number of isolates 1 2 5 4 6 6 1 Range of enzyme activity (U/ml) 1-50 51-100 101-200 151-200 201-250 251-300 301-350

The one isolate gave a maximum production of alpha amylase 347 U/ml and it was coded as EMS-18. This strain was selected for optimization

86

4.4: OPTIMIZATION OF CULTURAL CONDITIONS IN SHAKE FLASKS

4.4.1: SCREENING OF CULTURE MEDIA The six different fermentation media (cited in literature for the production of alpha amylase by different strains) were evaluated for the alpha amylase production by wild and mutant strains of A. oryzae (Fig 4.1). Of all the media tested, M 4 medium gave highest units of alpha amylase 1682 (wild) and 3852 (mutant). The dry cell mass was 14.92 and 16.82.4 g/l, respectively. The rest of the fermentation media gave not significant enzyme production for both strains as compared to M4 medium. The enzyme production was found to be highly significant (p0.05) in M4 medium so, it was selected in subsequent studies. 4.4.2: RATE OF ALPHA AMYLASE PRODUCTION The effect of incubation period on the alpha amylase production both by wild and mutant strains of A. oryzae was optimized (Fig 4.2). The fermentation was carried out for 96 h and enzyme production was calculated after every 8 h. The production of enzyme was increased with the increase in the incubation period and reached maximum at 72 h after inoculation by both the wild (1682 U/ml) and mutant (3862

U/ml) strains. The dry cell mass was 14.92 and 16.82.4 g/l, respectively. Further
increase in incubation period did not show any increase in the formation of enzyme rather it was decreased. The enzyme production was highly significant (p0.05) after

72 h. So, it was selected in subsequent studies.

87

4.4.3: EFFECT OF INCUBATION TEMPERATURE Figure 4.3 shows the effect of varying incubation temperature (25-50C) on the alpha amylase production by both wild and mutant strains of A. oryzae. Both the wild (1662 U/ml) and mutant (3863 U/ml) strains gave maximum enzyme production at 30C. The dry cell mass was 14.21.0 and17.82.40 g/l, respectively. When temperature of medium was increased from 30C, the enzyme production was reduced. At 50C, the enzyme production became not significant (p0.05) as compared to other temperatures. 4.4.4: EFFECT OF DIFFERENT INITIAL pH The effect of different initial pH of the fermentation medium on the alpha amylase production was investigated (Fig 4.4). The initial pH of the fermentation medium was adjusted at 4-7 in shake flasks. The enzyme production following growth of organism by both wild and mutant strains was found to be significant (p0.05) at pH 5. As the pH of the medium was increased from 5, there was gradual reduction in the enzyme formation by both wild and mutant strains of A. oryzae. At alkaline pH, the enzyme production was extremely low. Thus, pH 5 was selected for of alpha amylase production. 4.4.5: EFECT OF DIFFERENT VOLUMES OF MEDIUM Figure 4.5 depicted the effect of different volumes of the basal medium such as 5, 10, 15, 20 and 25 % (v/v) in 250 ml Erlenmeyer flask on alpha amylase production by both the wild and mutant strains of A. oryzae. The maximum enzyme production by

88 wild (1891) and mutant (4163) strains were observed when 10 % volume (25 ml/250 ml flask) was used. As the volume of fermentation medium was increased, the enzyme production was decreased gradually. The enzyme production was significant at 10 % volume of fermentation medium so it was selected for further studies. 4.4.6: EFFECT OF DIFFERENT INOCULUM SIZES Effect of size of inoculum (2-12 % v/v) on the alpha amylase production by wild and mutant strains of A. oryzae was evaluated (Fig 4.6). The inoculum size of 4 % v/v (1ml=2.6106CFU) yielded maximum enzyme production both by wild (1911.04) and mutant (4183) strains. The dry cell mass was 14.71.0 and 17.92.0 g/l, respectively. Beyond this level the enzyme production was decreased gradually. The enzyme production was found to be significant (p0.05) at 4 % level hence; it was optimized for maximum enzyme production.

89

Fig 4.1: Screening of fermentation media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18*

1000

30

25 800

Enzyme activity (U/ml)

20 600 15 400 10 DCM (g/l)

200 5

0 M1 M2 M3 M4 M5 M6 Fermentation media
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30(g/l)

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strainEMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. *Incubation time 72 h, temperature 30C, pH 6.0, agitation rate 160 rpm

90

Fig 4.2: Rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

25 800

20 Enzyme activity (U/ml) 600 15 400 10 DCM (g/l)

200 5

0 0 8 16 24 32 40 48 Time (h)
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 56 64 72 80 88 96

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. *Incubation temperature 30C, pH 6.0, agitation rate 160 rpm

91 Fig 4.3: Effect of incubation temperature on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 25 30 35 40 45 50 TemperatureC
Enzyme activity of w ild strain IIB-30 (U/ml) DCM of w ild strain IIB-30 (g/l)

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. *Incubation time 72 h, incubation temperature 30C, pH 6.0, agitation rate 160 rpm

92 Fig 4.4: Effect of different initial pH of fermentation medium on the of alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 4 4.5 5 5.5 pH
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 6 6.5 7

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p0.05. *Incubation time 72 h, incubation temperature 30C, agitation rate 160 rpm.

93 Fig 4.5: Effect of different volumes of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 5 10 15 Volume of media (%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)) Enzyme activity of mutant strain EMS-18 DCM of mutant strain EMS-18 (g/l)

0 20 25

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. *Incubation time 72 h, incubation temperature 30C, agitation rate 160 rpm

94 Fig 4.6: Effect of different inoculum sizes on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800 Enzyme activity (U/ml)

25

20 DCM (g/l) 600 15 400 10 200

0 2 4 6 Inoculum (%)
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 8 10 12

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. *Incubation time 72 h, pH 5.0, incubation temperature 30C, agitation rate160 rpm

95

4.5: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS OF A. ORYZAE IN SHAKE FLASKS

4.5.1: EFFECT OF STARCH FROM DIFFERENT SOURCES Figure 4.7 depicted the effect of starch from different sources such as wheat, corn, rice and sweet potato at the concentration of 1 % on the alpha amylase production by both wild and mutant strains of A. oryzae. Of all the starches tested, corn starch gave maximum enzyme production by both the wild (1902.0) and mutant (4193.0) strains. The dry cell mass was 14.62.0 and 16.82.0 g/l, respectively. All other starches gave less significant results (p0.05 as compared to corn starch. Therefore corn starch was selected for subsequent studies. 4.5.2: EFFECT OF DIFFERENT CONCENTRATIONS OF CORN STRACH The effect of different concentration of starch (1.0-5.0 % w/v) was investigated for the alpha amylase production by both the wild and mutant strain of A. oryzae (Fig 4.8). The maximum enzyme production by both strains wild (2011.04) and mutant (4362.0) was obtained at the level of 2 %. (w/v) The dry cell mass was 15.31.0 and 18.11.9 g/l, respectively. Beyond this concentration the enzyme production was decreased. The enzyme production was significant (p0.05) at 2 % (w/v) so; it was selected for further studies. 4.5.3: EVALUATION OF ADDITIONAL CARBON SOURCES The effect of different carbon sources such as glucose, sucrose, xylose, lactose, fructose, galatose, caboxy methyl cellulose, glycerol and mannitol was evaluated for the alpha amylase production by wild and mutant strains of A. oryzae in present course

96 of study. Of all the carbon sources tested lactose showed considerable increase in the enzyme production by both the wild (2351.04) and mutant strains (4602.0) compared to others (Fig 4.9). The dry cell mass was 15.91.0 and 18.82.1 g/l, respectively. These carbon sources were added to the fermentation media at the concentration of 0.5 %.( w/v) Therefore, lactose as additional carbon source was selected and its various concentrations were also tested for the enzyme production (Fig 4.10). The concentration of the lactose was kept from 0.5-2.5 % (w/v). The lactose at the concentration of 1.5 %( w/v) in case of wild (2602.0 U/ml) and 1.0 % (w/v) in case of mutant (4902.3 U/ml) was found to be the best for the enzyme production. Further increase in the concentration of lactose resulted decrease in the enzyme production. At 2.5 % (w/v) concentration of lactose the enzyme production was not significant (p0.05) as compared to other concentrations. 4.5.4: EVALUATION OF INORGANIC NITROGEN SOURCES Various inorganic nitrogen sources such as (NH4)2SO4, NH4NO3, NaNO3 and KNO3 was evaluated for the alpha amylase production by the wild and mutant strains of A. oryzae (Fig 4.11). The nitrogen sources were added to the fermentation media at the level of 0.1 % (w/v). Of all the nitrogen sources examined (NH4)2SO4 gave maximum enzyme production by both the wild (2742 U/ml/) and mutant (5033 U/ml) strains. Therefore, various concentrations of (NH4)2SO4 were also evaluated for the enzyme production (Fig 4.12). The concentration of the (NH4)2SO4 was kept 0.10.5 %.(w/v). The (NH4)2SO4 at the concentration of 0.3 % (w/v) was found to be significant (p0.05) for the enzyme production both by the wild (2841.04) and mutant (5253.0) strains. The dry cell mass was 16.91 and 19.6 2 g/l, respectively.

97 Further increase in the concentration of (NH4)2SO4 was resulted decrease in the enzyme production. 4.5.5: EVALUATION OF ORGANIC NITROGEN SOURCES Different organic nitrogen sources such as peptone, meat extract, Corn steep liquor (CSL), urea, casein and beef extract were evaluated for the alpha amylase production (Fig 4.13). The nitrogen sources were added to the fermentation media at the concentration of 0.1 % (w/v). Of all the nitrogen sources tested, peptone gave maximum enzyme production by both the wild (2981.U/ml) and mutant (5422.9 U/ml) strains. The dry cell mass was 17.51.0 and19.52.0 g/l, respectively. The CSL was proved as the second best nitrogen source for enzyme production by both strains. The least alpha amylase production was observed when casein was used as an organic nitrogen source. Variations in concentrations of peptone were also effective for alpha amylase production. Therefore, various concentrations of peptone (0.1-0.5 % w/v) were also evaluated for the enzyme production by both strains (Fig 4.14). Peptone at the concentration of 0.2 % w/v found to be significant (p0.05) for the enzyme production. Further increase in the concentration of peptone was resulted decrease in the enzyme production. 4.5.6: EFFECT OF SURFACTANTS The effect of various surfactants such as Tween 80, Triton X-100, Sodium dodecyl sulphate (SDS), Di-octyl ester of sodium sulpho succinic acid (Monoxal O.T), Poly ethylene glycol (PEG) and sodium lauryl sulphate was investigated for the alpha amylase production by the A. oryzae IIB-30 and its mutant derivative A. oryzae EMS18 (Fig 4.15). The surfactants were added to the fermentation media at the

98 concentration of 0.05 % (v/v). Of all the surfactants examined, Tween 80 gave maximum enzyme production by both the wild (3122.0 U/ml) and mutant (5732.0 U/ml) strains. Therefore, various concentrations of Tween 80 were also evaluated for the enzyme production (Fig 4.16). The concentrations of the Tween 80 was kept as 0.05 - 0.25 % (v/v). The Tween 80 at the concentration of 0.1 % (v/v) was found to be significant (p0.05) for the enzyme production both by the wild (3202.0 U/ml) and mutant (5893.0 U/ml) strains. The dry cell mass was 16.9 and 19.6 g/l, respectively. Further increase in the amount of Tween 80 was resulted decrease in the enzyme production. Hence, Tween 80 at the concentration of 0.1 % (v/v) was selected for further studies.

99

Fig 4.7: Effect of raw starch from different sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800 Enzyme activity (U/ml)

25

20 DCM (g/l) 600 15 400 10 200

0 Wheat starch Corn starch Rice starch Sweet potato starch

Starch (1%)
Enzyme activity of wild strain IIB-30(U/ml) DCM of wild strain IIB-30 (g/l) Enzyme activity of mutant strainEMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

100 Fig 4.8: Effect of different concentrations of starch on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 1 2 3 Concentration of starch (%)

Enzyme activity of w ild strain IIB-30 (U/ml) DCM of w ild strain IIB-30 (g/l)

0 4 5

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

101 Fig 4.9: Evaluation of additional carbon sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

Xy lo se

G lu co se Su cr os e

La ct os e Fr uc to se G al at os e

G ly

Carbon sources (0.5%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strainEMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

an ni to l Co nt ro l

CM

ce ro l

102 Fig 4.10: Effect of different concentrations of lactose on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 0.5 1 1.5 Lactose concentrations (%)

Enzyme activity of w ild strain IIB-30 (U/ml) DCM of w ild strain IIB-30(g/l)

0 2 2.5

Enzyme activity of mutant strainEMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

103 Fig 4.11: Evaluation of inorganic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800 Enzyme activity (U/ml)

25

20 DCM (g/l) 600 15 400 10 200

0 Ammonium sulfate Ammonium Sodium nitrate Potassium nitrate nitrate Inorganic nitrogen sources (0.1%)
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 Control

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the error from mean value. The values vary significantly at p 0.05.

standard

* Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

104 Fig 4.12: Effect of different concentrations of ammonium sulfate on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

25 800

Enzyme activity (U/ml)

20 600 15 400 10 DCM (g/l)

200 5

0 0.1 0.2 0.3 Ammonium sulfate concentrations (%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strainEMS-18(g/l)

0 0.4 0.5

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate160 rpm

105 Fig: 4.13: Evaluation of organic nitrogen sources on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 Meat extract CSL Urea Casein Beef extract Peptone Control

Organic nitrogen sources (0.1%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate160 rpm

106 Fig 4.14: Effect of different concentrations of peptone on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 0.1 0.2 0.3 Concentrations of peptone

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 0.4 0.5

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate160 rpm

107 Fig 4.15: Effect of different surfactants on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800 Enzyme activity (U/ml)

25 20 DCM (g/l)

600 15 400 10 200

5 0
Tr it o nX -1 do 00 dy cy ls ul ph at e M on ox al Po O .T lye th yl en e So gl di yc um ol la ur y su lp ha te So di um Co nt ro l

0
Tw ee n8 0

Surfactants (0.05%)
Enzyme activity of w ild strain IIB-30 (U/ml) DCM of w ild strain IIB-30 (g/l) Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

108 Fig 4.16: Effect of different concentrations of Tween 80 on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.*

1000

30

800

25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 0.05

0.1

0.15 Concentrations of Tween 80 (%)

0.2

0 0.25

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05. * Incubation time 72 h, incubation temperature 30C, pH 5.0, agitation rate 160 rpm

109

4.6: OPTIMIZATION OF CULTURAL CONDITIONS IN STIRRED FERMENTER

4.6.1: RATE OF ALPHA AMYLASE PRODUCTION The rate of fermentation of both the wild (IIB-30) and mutant (EMS-18) strains of A. oryzae for the alpha amylase production was investigated in stirred fermenter (Fig 4.17). The time course aliquots were withdrawn after every 8 h aseptically and subjected to enzyme estimation (U/ml) and dry cell mass determination (g/l) up to 96 h of fermentation period. It was found that the enzyme production was increased gradually and reached its maximum (335 U/ml) and (608 U/ml) after 64 h for wild and 48 h of fermentation for mutant respectively. The dry cell mass was (18.2) and (19.8), g/l respectively. A significant finding of present experiment was that fermentation period was reduced to 16 h in case of wild and 24 h in case of mutant from 72 h in shake flask studies. Rapid decline in enzyme production was seen in case of wild and mutant strain when incubation period was increased from optimum time period. Data obtained from above experiment was subjected to kinetic analysis for calculations of (h-1)max (specific growth rate), qp (unit product produced/g cell/h), Qp (enzyme produced/l/h), Qx (g cell mass formation/l/h), Yp/x(enzyme produced/g cell mass formation). The kinetic evaluation of results also revealed that optimum fermentation period for enzyme production was 64 h in case of wild and 48 h in case of mutant strains of A. oryzae (Table 4.6).

110 4.6.2: EFFECT OF pH Effect of different initial pH (4-6.5) of fermentation medium by both wild and mutant strains of A. oryzae was investigated in stirred fermenter (Fig 4.18). At pH 5, the maximum enzyme production by both wild (342U/ml) and mutant (626U/ml) strains were observed. The dry cell mass was 18.7 and 22.5 g/l, respectively. With the increase of pH, a decrease in enzyme production was observed. At alkaline pH the production was extremely low. The kinetic parametric study indicated that the yield of the enzyme by biomass formation as well as the rates of enzyme formation was significant at pH 5 (Table 4.7). Thus, pH 5 was selected for the production of enzyme by both wild and mutant strains of A. oryzae. 4.6.3: EFFECT OF AERATION LEVELS Figure 4.19 showed the effect of rate of aeration on the alpha amylase production by wild and mutant strain of A. oryzae. The rate of aeration varied from 0.5-2 vvm in stirred fermenter. Enzyme production by wild strain was found maximum i.e. 350 U/ml when aeration rate was set at 1.0 vvm while mutant strain gave maximum enzyme activity (660 U/ml) at 1.5 vvm. The dry cell mass was 18.9 and 23.1 g/l, respectively. Any variation beyond this optimum level, gave less enzyme production. The kinetic analysis of above parameters revealed that the values of Yp/x, Qp, Qx were significant at an air supply of 1.0 vvm (wild) & 1.5 vvm (mutant). Therefore, 1.5 vvm was optimized for further studies for enhanced enzyme production.

111 4.6.4: EFFECT OF DISSOLVED OXYGEN Figure 4.20 showed the effect of different levels (5-20 % v/v) of dissolved oxygen on alpha amylase production by wild and mutant strains of A. oryzae. Dissolved oxygen at the level of 15 % (v/v) gave the maximum enzyme production by wild (362U/ml) and mutant (687U/ml) strains. The dry cell mass was 19 and 23.6 g/l, respectively. Beyond this level, a decrease in enzyme production was recorded. Data obtained from above experiment was subjected to kinetic analysis for calculations of Qp (enzyme produced/l/h), Qx (g cell mass formation/l/h), Yp/x(enzyme produced/g cell mass formation). The kinetic evaluation of results also revealed that optimum level of dissolved oxygen in fermenter was 15 % (v/v) for enzyme production by A. oryzae IIB-30 and its mutant derivatives A. oryzae EMS-18 (Table 4.9). 4.6.5: EFFECT OF INOCULUM SIZE Effect of different sizes of inoculum was investigated for alpha amylase production by both the wild (IIB-30) and mutant (EMS-18) strains of A. oryzae in stirred fermenter (Fig 4.21). The size of vegetative inoculum was varied from 5-12.5 %, (v/v) and fermentation was carried out. The maximum alpha amylase production by both wild (372 U/ml) and mutant (718 U/ml) strains was observed at the inoculum size of 10 % (v/v). The dry cell mass was 19.2 and 24.1 g/l, respectively. Beyond this concentration the enzyme production decreased gradually.

112 All kinetic parameters showed 10 % (v/v) vegetative inoculum to be optimum for the enzyme production. Thus the inoculum size of 10 % (v/v) was used in further studies for the enzyme production in stirred fermenter. 4.6.6: EFFECT OF AGITATION INTENSITY The effect of rate of agitation on the alpha amylase production by wild and mutant strains of A. oryzae was investigated in stirred fermenter. The rate of agitation was varied from 120-240 rpm (Fig 4.22). Maximum enzyme production was obtained when agitation was maintained at 200 rpm. Further increase or decrease in agitation speed resulted in decrease enzyme production by both the strains. Evaluation of kinetic parameters Yp/x, Qp, Qx indicated that production yield by wild and mutant strains was found optimum when agitation speed of impeller was set at 200 rpm (Table 4.10). Thus agitation speed of 200 rpm was selected for further studies.

113 Fig 4.17: Comparison of rate of alpha amylase production by wild (IIB-30) and mutant strain of A. oryzae (EMS-18) in stirred fermenter*

1000

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Enzyme activity (U/ml)

20 600 15 400 10 DCM (g/l)

200

0 0 8 16 24 32 40 48 Time (h)
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 56 64 72 80 88 96

Enzyme activity of mutant strainEMS-18 (U/ml) DCM of mutant strainEMS-18(g/l)

* Incubation temperature 30C, pH 5.0, agitation rate 160 rpm, aeration 1vvm

114 Table 4.6: Kinetic evaluation of rate of fermentation for the alpha amylase production by A. oryzae IIB-30 and its mutant derivatives in stirred fermenter

Kinetic parameters Yp/x Qp Qx qp

wild 0.2 55000 5583 0.30 11000

Mutant 0.22 185714 10133 0.33 40857

Kinetic parameters: Yp/x= enzyme produced/g cell mass formation. Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h. (h-1)max= specific growth rate.

115

Fig 4.18: Effect of initial pH of media on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

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Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 4 4.5 5 Initial pH
Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 5.5 6 6.5

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18

*Incubation time 48 h, temperature 30C, agitation intensity 160 rpm, aeration 1vvm

116

Table 4.7: Kinetic evaluation of different pH values of media for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter

pH 4 4.5 5.0 5.5 6.0 6.5 Kinetic wild Mutant Wild Mutant Wild Mutant Wild Mutant Wild Mutant Wild Mutant parametes Yp/x 20915 28032 21487 30353 21678 36066 21551 33604 19607 27822 18288 24642 Qp 4333 9016 5166 9633 5700 10433 53333 10016 4166 8550 3333 6900 Qx 0.20 0.25 0.23 0.28 0.31 0.37 0.25 0.33 0.19 0.30 0.17 0.28

Kinetic parameters:

Yp/x= enzyme produced/g cell mass formation.

Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.

117 Fig 4.19: Effect of different aeration levels on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

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25

20 DCM (g/l) 600 15 400 10 200

0 0.5 1 Aeration levels (vvm)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

0 1.5 2

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18(g/l)

*Incubation time 48 h, incubation temperature 30C, agitation intensity 160 rpm

118 Table 4.8: Kinetic evaluation of different aeration for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter

Aeration (vvm) Kinetic parameters Yp/x Qp Qx Wild 18518 4500 0.23

0.5 Mutant 27899 9483 0.31 Wild 19050 5833 0.31

1.0 Mutant 28571 10466 0.36 Wild 18881 5016 0.26

1.5 Mutant 310928 11000 0.38 Wild 18181 4000 0.22

2.0 Mutant 28416 10183 0.35

Kinetic parameters:

Yp/x= enzyme produced/g cell mass formation.

Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.

119 Fig 4.20: Effect of different level of dissolved oxygen on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

1000

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25

600 15 400 10

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0 5 10 15 20 Dissolve oxygen level (%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

*Incubation time 48 h, initial pH 5.0, incubation temperature 30C, agitation intensity 160 rpm, aeration 2.0 vvm.

Dry cell mass (g/l)

20

120 Table 4.9: Kinetic evaluation of different levels of dissolved oxygen for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter

Dissolve oxygen (%) Kinetic parameters Yp/x Qp Qx Wild 19052 4750 0.24

5.0

10

15

20

Mutant 28903 10000 0.31

Wild 19937 5033 0.25

Mutant 31840 10983 0.38

Wild 20133 6033 0.31

Mutant 32258 11450 0.39

Wild 19655 5350 0.26

Mutant 29110 10666 0.33

Kinetic parameters:

Yp/x= enzyme produced/g cell mass formation.

Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.

121 Fig 4.21: Effect of different inoculum size on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

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Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

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0 5 7.5 Inoculum (%)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l)

10

0 12.5

Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

*Incubation time 48 h, incubation temperature 30C, agitation intensity 160 rpm, initial pH 5.0.

122 Table 4.10: Kinetic evaluation of different inoculum sizes for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter

Inoculum (%) Kinetic parameters Yp/x Qp Qx Wild 19375 4800 0.18

5.0 Mutant 29570 10500 0.36 Wild 25396 5333 0.21

7.5 Mutant 29792 11483 0.38 Wild 25486 6200 0.32

10 Mutant 31093 11966 0.40 Wild 21549 5100 0.23

12.5 Mutant 28506 9950 0.32

Kinetic parameters:

Yp/x= enzyme produced/g cell mass formation.

Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.

Fig 4.22: Effect of different agitation intensity on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 *

123

1000

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25

Enzyme activity (U/ml)

20 DCM (g/l) 600 15 400 10

200

0 120

160

200

0 240

Agitation intensities (rpm)

Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l)

*Incubation time 48 h, incubation temperature 30C, pH 5.0.

124 Table 4.11: Kinetic evaluation of different agitation speeds for the alpha amylase production by A. oryzae IIB-30 and its mutant derivative in stirred fermenter

Agitation intensity Kinetic parameters Yp/x Qp Qx

120 rpm Wild 19254 4966 0.17 Mutant 30120 10500 0.26

160rpm Wild 19473 6166 0.31 Mutant 31818 11933 0.39

200 rpm Wild 29215 6416 0.33 Mutant 39622 12500 0.41

240 rpm Wild 19444 5166 0.26 Mutant 30338 11666 0.36

Kinetic parameters:

Yp/x= enzyme produced/g cell mass formation.

Qp=enzyme produced/l/h. Qx= g cell mass formation/l/h.

4.7: Purification of alpha amylase

125

4.7.1: Ammonium sulfate precipitation

Table 4.12 shows the data of ammonium sulfate precipitation at different saturation concentrations (20-90 % w/v) for the alpha amylase recovery from cell free broth. The specific activity of broth was 208.3 U/mg of protein before treating with ammonium sulfate. At 20-40 % (w/v) saturation, no activity was found. At 70 % (w/v) saturation concentration the maximum specific activity (280.9 U/mg of protein) with 1.3 fold purification was obtained. Above 70 % (w/v) both of these values were again decreased and at 90 % (w/v) no pellet was obtained 4.7.2: Stepwise purification: Table 4.13 shows the successive steps of purification of alpha amylase from mutant strain of A. oryzae EMS-18 to homogeneity by ammonium sulfate precipitation followed by using Sephadex DEAE and Sephadex G-100 columns. 4.7.2.1: Ammonium sulfate precipitation The initial specific activity of cell free crude broth was 208.3 U/mg of protein then it increased (280.9 U/mg of protein) with first purification step of ammonium sulfate (70 % w/v). 4.7.2.2: Anion- exchange chromatography The dialyzed enzyme solution was loaded on prepared Sephadex DEAE column. Fig 4.23 shows the stepwise gradient elution pattern of alpha amylase when elution buffer of Tris HCl (0.05 M, pH 7.5) containing NaCl (0-1.0 M) was used. The pooled distinct peak was obtained showing enzyme activity (10113.1U) at the 0.30 M concentration of NaCl. The specific activity (561.8U/mg of protein) was observed as shown in Table

126 4.13. The molecular weight was found to be as 48 kDa by applying the few fractions on SDS-PAGE. Fig 4.25 4.7.2.3: Gel filtration Sephadex G-100 was finally used as finishing step of purification. Upon loading dialyzed sample, twenty five fractions were eluted with Tris HCl (0.05 M, pH 7.5) buffer. Fig 4.24 also shows the elution pattern in the form of distinct peaks. The active fractions containing 5963.1U enzyme activity were pooled up, dialyzed. However, the specific activity (1987.7 U/mg of protein) and fold purification (9.5) were recorded as shown in Table 4.13.

Table 4.12: Purification summary of alpha amylase produced by mutant strain of A. oryzae EMS-18 by using ammonium sulfate

127

Ammonium sulfate fractionation (%) Crude broth 0-20 0-40 0-50 0-60 0-70 0-80 0-90

Total units (U)

Total protein (mg) 120 110 100 90 80 78 -

Specific activity (U/mg) 208.3 185 192.2 280.9 207.6 -

Recovery or % yield 100 74 69.2 89.9 64.8 -

Fold purification

25000 18500 17300 22479 16200 -

1.0 0.8 0.92 1.3 0.99 -

Table 4.13: Step wise purification profile of alpha amylase produced by mutant strain of A. oryzae EMS-18.

128

Purification steps Crude broth Ammonium sulphate fractionation (70%) DEAE Sephadex chromatography Sephadex G100

Enzyme activity (U) 25000 22479

Total protein (mg) 120 80

Specific activity (U/mg) 208.3 280.9

Recovery or % yield 100 89.9

Fold purification 1 1.3

10113.1

18

561.8

40.4

2.6

5963.1

1987.7

23.8

9.5

129 Fig 4.23: Elution pattern on Sephadex DEAE

Fig 4.24:

The elution profile on Sephadex G-100

130

2.5

7000

6000 2 5000 Absorbance ( 280 nm) 1.5 Enzyme activity (U)

4000

3000

2000 0.5 1000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fractions
Absorbance Enzyme activiity (U)

Molecular weight

131 Electrophoretic mobility of purified alpha-amylase with reference to mobilities of protein marker (SMO 313) Fractions was analyzed on SDS-polyacrylamide gel. The mobility of the purified alpha-amylase corresponded to a molecular weight of 48kDa (Fig 31).

116kDa 66.2 45.0 35.0 25.0 18.4 14.4 48kDa

Fig4.26.

SDS-PAGE

analysis

of

pooled

fractions

of

ion

exchange

chromatography and ammonium sulfate fractionation Lane 1; Marker, lane 2; Ammonium sulfate fractionation, lane 3; pooled fractions of ion exchange chromatography.

4.8: CHARACTERIZATION 4.8.1: TEMPERATURE OPTIMA OF PURIFIED ALPHA AMYLASE

132 Figure 4.26 shows the effect of temperature on the activity of purified alpha amylase by mutant strain of A. oryzae. The enzyme substrate complex was incubated at different incubation temperature such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70C. The activity of enzyme was increased with increase in temperature and found optimum at 40C (15603.2 U/ml). Further increase in the incubation temperature resulted decrease in the activity of enzyme. At 70C the activity of purified enzyme is not significant (p 0.05). Thus, the temperature 40C was selected for optimum activity of alpha amylase. 4.8.2: EFFECT OF TIME OF INCUBATION ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE Figure 4.27 shows the effect of incubation time of enzyme substrate complex on the activity of purified alpha amylase. The enzyme substrate complex was incubated for varying time intervals (10-70 min). The enzyme activity was found to be optimum (15682.5) after 30min of incubation. Further increase in the time of incubation resulted decrease in the activity of enzyme. The 30 min incubation time of reaction mixture was highly significant (p0.05) as compared to other temperatures which were tested so; it was selected for further studies. 4.8.3: EFFECT OF DISTILLED WATER AND BUFFER ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE Figure 4.28 shows the effect of distilled water and different buffers on the activity of alpha amylase. Different buffers and distilled water such as citrate, acetate and phosphate were used in reaction mixture. The maximum enzyme activity (15923.2)

133 was obtained in acetate buffer. The other buffers and distilled water show non significant results. Thus, acetate buffer was selected for further studies. 4.8.4: EFFECT OF pH ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE Figure 4.29 shows the effect of acetate buffer pH of reaction mixture (enzyme substrate complex) for the activity of purified alpha amylase. The enzyme substrate complex was incubated at pH 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 and 7. The enzyme activity was found to be optimum at pH 5. Further increase in the pH resulted decrease in the activity of enzyme. At neutral pH, the activity of enzyme was extremely low. The acidic pH 5 of reaction mixture was significant (p0.05) as compared to other pH and selected for subsequent studies. 4.8.4: EFFECT OF METAL IONS ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE The residual activities were determined after incubation of purified enzyme with 5 mM metal ions (Fig 4.30). The result showed CuCl2, Zn Cl2, BaCl2, FeSO4, Mg SO4, NiCl2 and NaCl has inhibitory effect on the activity of enzyme. While CaCl2, COCl2, MnSO4 have stimulatory effect on the activity of enzyme.

Fig 4.26: Effect of temperature on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18

134

2400 2200 2000 1800 Enzyme activity (U/ml) 1600 1400 1200 1000 800 600 400 200 10 20 30 40 50 60 70 80 Temperature C
Enzyme activity (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05.

Fig 4.27: Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18

135

2400 2200 2000 1800 Enzyme activity (U/ml) 1600 1400 1200 1000 800 600 400 200 0 0 10 20 30 40 50 60 70 Time (min)

Enzyme activity (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05.

Fig 4.28: Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18

136

2400

2000

Enzyme activity (U/ml)

1600

1200

800

400

0 Distilled water Citrate buffer Acetate buffer Phosphate Buffer Distilled water & buffers
Enzyme activity (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values in vary significantly at p 0.05.

Fig 4.29: Effect of different pH on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18

137

2400

2000

Enzyme activity (U/ml)

1600

1200

800

400

0 0 1 2 3 4 pH of acetate buffer
Enzyme activity (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05.

Fig 4.30: Effect of metal ions on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18

138

2400

2000 Enzyme activity (U/ml)

1600

1200

800

400

0
ag ne siu m su M lp ag ha ne te se su lp So ha di te um ch lo rid Ni e ck le ch lo rid Zi e nc ch lo cu rid pr e ou s ch Ca lo rid lci e um ch Co lo ba r id lt o e us ch lo Fe rid r ro e us ch lo Ba r id riu e m ch lo rid e M Co nt ro l

Metal ions (5mM)

Enzyme activity (U/ml)

Each value is an average of three parallel replicate. Y error bars indicate the standard error from mean value. The values vary significantly at p 0.05.

Discussion

139 Isolation, identification and screening of a potent strain are the vital steps of alpha amylase production. In this connection, seventy eight strains of A. oryzae were isolated from soils of different habitats by serial dilution method (Clark et al., 1958); identified according to Onion et al. (1986) and tested for enzyme production in submerged fermentation. Of all the isolates tested, strain no. 30 gave maximum alpha amylase production. The strain no.30 was selected for further studies and assigned the code IIB-30. The IIB-30 was subjected to physical (UV radiation) and chemical mutagenesis (NG, HNO2, EMS) to enhanced the enzyme productivity. The isolates obtained after UV irradiation were thirty two in number and screened for enzyme production out of which isolate no.23 showed better enzyme productivity compared to parental strain and was assigned the code UV-23. Perhephs it was due to the fact that UV irradiation possibly changed structure of DNA by photolysis i.e, formation of pyrimidine dimers. The structural alteration in DNA was associated with the activity of the enzyme. Thymidine-thymidine dimer probably promoted mycelial growth and subsequently enzyme activity, which resulted in greater secretion of enzyme from the mycelial cells (Ali et al., 2002). UV mutant of fungi showed more enzyme production compared to parental strain as reported by Azin and Noroozi, (2001); Rubinder et al. (2002); Ellaiah et al. (2002) and Karanam and Medicherla, (2008). The UV-23 mutant was subjected to N-methyl N-nitro N-nitroso guanidine (NG) to induce mutagenesis at various concentrations. The eighteen NG treated colonies were picked up on the basis of starch hydrolysis zones diameter larger than the UV-23 and further screened in shake flasks for enzyme production. Out of which, one mutant NG15 gave 2 fold increase in alpha amylase production. Probably it was due to the

140 fact that treatment with NG resulted alkylation of guanine residues which formed permanent lesions within the structure of DNA and causes mutations (Drazic and Delac, 1970). The NG-15 was further subjected to alternate treatment with nitrous acid and EMS for further improvement in the enzyme production. EMS-18 gave 2.6 fold alpha amylase production than the parental culture. UV, NG and nitrous acid were commonly used for strain improvement as reported by Azin and Noroozi (2001) and Rubinder et al. (2002). The six different media were evaluated for alpha amylase production by both wild and mutant strain of A. oryzae out of which M4 medium (g/l); starch 20, yeast extract, 8.5, NH4Cl 1.3, MgSO4.7H2O 0.12, CaCl2 0.06 was found best for maximum enzyme production. Yeast extract and ammonium chloride serve as inorganic and organic nitrogen source respectively, in M4 medium. Yeast extract is a complex nitrogen source containing free amino acids and peptides and therefore, was considered an ideal source for enzyme production. The production and stability of enzyme are significantly affected by the supplementation of metal ions in the fermentation medium because the metal ions act as activators for enzyme activity (Lin et al., 1997; Noorwez and Satyanarayana, 2000). M4 medium contained the ions such as Ca+2 Cl-, Mg+2 and SO4-which were vital for the growth of fungus and enzyme production. Calcium and chloride ions act as stabilizer, binder, activator and stimulator (Donell et al., 1975; Chambert et al. 1999). All the other media gave less significant results as compared to M4 medium due to the deficiency of any constituent in those media necessary for growth as well as for the enzyme production or it was because of repressor effect of any component of the media on the growth of organism.

141 The alpha amylase production was increased with the increase in the incubation period and found to be maximal after 72 h of inoculation by both strains. The results indicated that enzyme was secreted early in active growth phase and reached maximum towards the end of exponential growth phase. The enzyme activity appreciably decreased after 72 h. However, in fermenter the enzyme production found to be optimum after 64 h (wild) and 48 h (mutant). Probably it was due to denaturation of enzyme because of interaction with other compounds in the fermentation medium and also due to the depletion of the nutrients and formation of other by products such as proteases in the fermentation medium (Ramesh and Lonsane, 1990; Kirshna and Chandrasekaran, 1996). However, in case of fermenter, the reduction in the fermentation period compared to shake flasks perhaps due to the fact that growth factors like pH, temperature, agitation were more accurately controlled which made the environment favourable for growth of organism and enzyme production (Gigras et al., 2002). The kinetic parametric results depicted that the volumetric rate of product and cell mass formation was highly significant after 64 h of inoculation (wild) and 48 h (mutant). The value of Yp/x was highly significant by both wild and mutant strains in fermenter. The maximum enzyme production was obtained after 96 h of inoculation (Kasim 1983; Nguyen et al., 2000; Francis et al., 2002). So, present finding was a significant improvement on that reported by these scientists as there was a reduction in fermentation time that would lead to lower energy requirements for the process and thus make enzyme production more economical.

142 The effect of varying the incubation temperature on the enzyme production was investigated. The enzyme production was found to be optimal at 30C. Higher temperatures resulted decreased enzyme production as reported by Dakhmouche et al. (2006); Bhanja et al. (2007) and Shafique et al. (2009). From shake flask and fermenter experiments investigating the effect of pH, enzyme production was found to be optimum at pH 5. Further increase in pH had an adverse effect on enzyme production which is not un expected as it is known that enzymes are usually very sensitive to small changes in pH; H+ion concentration also has a significant effect on the growth of mycelium and hence enzyme production (Kasim 1983; Stamford et al., 2001 and Gupta et al., 2008). Optimization of the volume of fermentation medium is also necessary for air supply nutrient supply, growth of microorganism and enzyme production. The different volumes of the fermentation medium were evaluated in 250 ml Erlenmeyer flasks by both wild and mutant strains of A. oryzae in present study. The maximum enzyme production was obtained in 10 % of the fermentation medium. As the volume of the medium was increased, the enzyme production was decreased. Probably it was due to the fact that decrease in the agitation speed of medium, reduced air supply and consequently enzyme production. At low volume of fermentation medium, the enzyme production was also decreased. It might be due to nutrient present in the fermentation medium were inadequate for the growth of strains of A. oryzae and hence, for enzyme production (Mimura and Shinichi, 1999; Ivanova et al., 2001). The size of inoculum has direct effect on the growth of organism and enzyme production as reported by Allan et al. (1996) and Shafique et al. (2009). Different

143 inoculum sizes were tested for enzyme production in shake flasks and fermenter. Of all the inoculum size tested, 4 % and 10 % of inoculum containing 2.6106 CFU/ml was found to be optimum for the best enzyme production in shake flasks and fermenter. As the inoculum size was further increased, the enzyme production was decreased. Possibly it was due to the fact that over growth of A. oryzae produced anaerobic conditions during the fermentation and it consumed majority of substrate for growth and metabolic processes, hence enzyme production was reduced. As the inoculum size was decreased, the enzyme production was also decreased. The reason might be inadequate amount of mycelia produced at low amount of conidia which in due course decreased enzyme production. The kinetic parametric study indicated that the yield of the enzyme by biomass formation as well as the rates of enzyme formation was significant at 10 % inoculum size in fermenter. Starches from different sources such as corn, rice, wheat and sweet potato were used in the present study. The corn starch gave maximum enzyme production. The effect of different concentrations of corn starch was evaluated. Of all the concentrations tested starch at the concentration of 2 % gave maximum enzyme production. Beyond this concentration decrease in the enzyme production was take place.Perhephs it was because of that a high starch concentration, when attacked by alpha amylase during fermentation might have undergone degradation resulting into the accumulation of reducing sugars. It might lead to the enhancement in sugar concentration of the substrate and catabolic repression of enzyme synthesis (Dvadtsatova et al., 1976; Gigras et al., 2002; Ajer Dharani, 2004; Krishna and Chandrasekaran, 1996).

144 The effect of addition of different carbon sources such as glucose, sucrose, xylose, lactose, fructose, galactose, caboxy methyl cellulose, glycerol and mannitol were evaluated for enzyme production. Of all the carbon sources tested lactose gave maximum enzyme production. Lactose along with starch was proved to be good carbon source in the present study. Perhaps starch and lactose act as complex carbohydrate sources and were gradually metabolized by a microorganism which enhanced the accumulation of inducible alpha amylase in fermentation media (Nguyen et al., 2000; Calik and Ozdamar, 2001). Thus lactose was selected as additional carbon source for the enzyme production and its various concentrations were tested. Lactose at the concentration of 1 % was found to be best for the enzyme production. Further increase or decrease in the concentration of lactose was resulted decrease in enzyme production. Possibly it was due to the fact that lower level of carbon was inadequate for the growth as well as enzyme production and excess carbon was equally detrimental and cause catabolic repression (Carlsen and Nielsen, 2001; Gupta et al., 2008). Different inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, sodium nitrate and potassium nitrate were evaluated for the enzyme production. Of all the inorganic nitrogen sources tested ammonium sulfate at 0.3 % gave maximum enzyme production. The different additional organic nitrogen sources such as meat extract, corn steep liquor, urea, casein, beef extract and peptone were also evaluated for the enzyme production. Peptone with inorganic nitrogen sources gave the maximum enzyme production as reported by Pedersen and Nielsen (2000) and Gupta et al. (2008). The effect of different surfactants such as Tween 80, Triton

145 X-100, sodium dodecyl sulfate, Monoxal, O.T and poly ethylene glycols were tested for the enzyme production. Of all the surfactants tested Tween 80 gave the maximum enzyme production. There are chances that Tween 80 not only increased the permeability of cell but also have stimulatory effect on the enzyme production compared to other surfactants as reported by Arnesen et al. (1998). A general requirement for a bioreactor is the provision of aeration system that can maintain a high dissolve oxygen level. Optimum supply of oxygen is very essential for aerobic fermentation; in this connection rate of agitation and different volume of air supply was studied for the enzyme production in stirred fermenter. The enzyme production was increased as the agitation intensity was increased and found to be maximal at 200 rpm. Change in the rate of agitation resulted reduction in enzyme production. Probably higher stirring speed above than 200 rpm resulted in mechanical and oxidative stress, excessive foaming, disruption and physiological disturbance of cells, while lower stirring speed seemed to limit oxygen levels along with the lacking of homogeneous suspension of the fermentation medium and breaking of the clumps of cells. The enzyme production increased with the increase of aeration and reached maximum at 1.0 vvm (wild) & 1.5 vvm (mutant). The anaerobic condition available to microorganism greatly disturbed the physiology and metabolism of organism because of this at low level of air supply the productivity of enzyme was greatly inhibited. In addition another toxic by product were produced in the fermentation medium with little titer of enzyme activity, while higher concentration rates have some detrimental effects on the growth of microorganism and subsequently enzyme production during bioprocess time (Ionita et al., 2001).

146 Alpha amylase was purified by ammonium sulfate, and successive chromatography techniques including anion exchange and gel filtration in the present study. Different saturation concentrations of ammonium sulfate were used. The fold purification was 1.3 at 70 % saturation concentration. At this concentration most of the protein having maximum enzyme activity. Possibly it was due to the fact that hydrophobic groups predominate in the interior of protein but some were on the surface. As the concentration of salt increased water was removed and the protein thus exposing the hydrophobic patches on one protein molecule can interact with those on another resulting in the aggregation of desire protein (enzyme). The enzyme solution (dialyzed) was further purified using Sephadex- DEAE column. The positively charged proteins were removed as contaminants. A linear gradient elution pattern indicated that maximum peak was achieved at the 0.30 M concentration of NaCl elution buffer as reported by Kusuda et al. (2003). After anion exchange, the dialyzed active fraction was loaded on Sephadex G-100. The pattern of elution was used to determine the molecular weight of alpha amylase as 48 kDa on SDS-PAGE (Anidyawati et al., 1998; Chang et al., 1995). The comparison of successive purification steps starting from specific activity of crude broth (208.3 U/mg) to final finishing technique of gel filtration (1987.7U/mg) indicated the 9.5-fold increase in specific activity. For characterization of purified alpha amylase, the optimization of temperature, incubation time, different buffers, pH and metal ions were studied. Among different temperature the maximum activity was observed at 40C. Probably it was due to the fact that reaction rate initially increased as the temperature rised, due to increased

147 kinetic energy of reacting molecules. However, as the temperature was increased the kinetic energy of enzyme exceeds the energy barrier. It resulted in the breaking of weak hydrogen bonding and hydrophobic bonds that maintain the structure of enzyme. The inactivation of enzyme at low temperature and thermal denaturation at high temperature might cause decrease in activity. The effect of different buffer and pH were analyzed by carrying out the enzyme assay along with different buffers and pH Acetate buffer at pH 5 gave the maximum enzyme activity. At pH below and above optimal level, a decline in activity was possibly due to the structural unstability of protein (Kusuda et al. 2003). Most of alpha amylase is known to be metalloenzymes; supplementation of metal ion improved the activity of enzyme. Effect of metal ion on the activity of enzyme was observed; in the presence of CaCl2 maximum activity of enzyme was obtained. Perhaphs it may be possible the affinity of Ca+2 to alpha amylase was much stronger than any other ions and Ca+2 stabilize the enzyme activity while the other metals such as CuCl2, ZnCl2, BaCl2, FeSO4, Mg SO4, NiCl2 and NaCl has inhibitory effect on the enzyme activity probably these metal block binding sites of enzyme or enzyme contain number of metals and displacement of these ions by another metal ions, either with some change or similar size result in inhibition of enzyme activity (Abou Zeid 1997).

CONCLUSION

148 In the present study, seventy eight strains of Aspergillus oryzae were isolated for the enzyme production. The improvement in enzyme production was achieved by subjecting parental strain to successive physical (UV) and chemical (NG, NA, EMS) mutagens. The mutant gave 2.6 fold more production compared to parental strain in term of enzyme production. Many factors need to be considered by alpha amylase production to obtain economically most favourable process. The most important among them were physical factors and culture medium. The optimization of process parameters were under taken in shake flasks and fermenter. All fermentation were carried out following growth of organism at 200 rpm (30C) for 72 h in shake flasks, 64 h (wild) and 48 h (mutant) in fermenter.The time required for maximal enzyme production was reduced in fermenter as compared to shake flasks by both wild and mutant strain. Fermentation medium containing (g/l); corn starch 20, lactose 10, ammonium sulfate 3, peptone 2, yeast extract 8, ammonium chloride 1.3, calcium chloride 0.06,magnesium sulphate 0.12 and Tween-80 1.0 at pH 5 was selected. In case of fermenter inoculum size (10 %), aeration (1.5vvm) dissolved oxygen level (15 %) were found optimum for maximum enzyme production. A total of 9.5 fold purification and 23.8 % recovery were obtained. Gel electrophoresis indicated molecular weight of A. oryzae alpha amylase to be 48 kDa.

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149

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