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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
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY GC UNIVERSITY LAHORE
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.
Signature of Deponent
Dr Ikram-ul-Haq (S. Dr Ikram-ul-Haq (S.I) Director. Lahore .I) Supervisor Submitted through ___________________ Prof.iii 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. Institute of Industrial Biotechnology GC University. Lahore _____________________ Controller of Examinations GC University. _________________ _______________________ Date Prof.
Every tiny or massive entity moves with His permission.e. Muhammad Yaqub. Vice . Dr. M. Lahore) and Dr. GCU. SI (Director. Ikram-ul-Haq. Zaheer-ud-Din Khan. Institute of Industrial Biotechnology. I am grateful to Dr. Numan Aftab for their scholarly. cooperation and discussion. His enthusiastic inspiration and fatherly affection enabled me to attain the objectives without any difficulty. Sikander Ali. and Dr. Khalid Aftab. (Chairperson Department of Botany. Dr. Dean. Mohsin Javed.iv ACKNOWLEDGEMENTS All praise for the. A. “KUN FAYAKUN”. Countless thanks to Him for accrediting me to accomplish this important task with in this specified time. Lahore for providing all the necessary facilities through out my research duration. I am thankful to highly esteemed Dr. GCU. 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. during the entire period of my research work. Faculty of Science and Technology. 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. Dr. Lahore for his valuable guidance. I most great fully acknowledge my indebtedness to Dr. scientific discussions and generous advices when needed. Hamid Mukhtar. GCU. encouragement. “ALMIGHTY ALLAH” who is the only supreme Authority and whose presence has been figured on the two words i. Qadeer and Dr. Amin-ul-Haq Khan.
Shazia Malik and Tehreema Iftikhar. Although feelings are deep but unfortunately words are too shallow. Zahid Butt. Fasial. for their moral support in the research work. that cannot follow the depths of my deep gratitude to my loving mother and father Mr. ROHEENA ABDULLAH . Usman . My fortune is due.v Chancellor. Shakeel (Assistant Professor Department of Pathology Punjab University) for helping in the identification of strain. 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. to their prayers. Abdullah. Mr. Mr Ramez and all others for their full cooperation during the whole period of my research. I am also thankful to laboratory staff especially Mr. I feel pleasure to acknowledge Dr. and Mrs. GC University.
3.1. Effect of initial pH 3. Evaluation of carbon sources 3. Induction of mutation 220.127.116.11.18.104.22.168. Vegetative inoculum 3. Effect of inoculum size 3. Effect of agitation 3.6. Fermentation media 3. Isolation of organism 22.214.171.124. Conidial inoculum 3. Inoculum preparation 3.6.5. Methods 3.1.2 Conidial count 126.96.36.199.vi 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 3.6. Effect of temperature 3.6.3. Shake flasks studies 3.7. Materials 3.4.2. Fermentation 188.8.131.52.6.6. Minimal inhibitory concentration of 2-deoxy-D-glucose 3.1. Fermentation media 3.7.6. Evaluation of nitrogen sources 3.3. Effect of volume 3. Nutritional and cultural requirement of Aspergillus oryzae 3.1.6. Fermenter studies 3.9. 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 .6.2.5. Incubation period 184.108.40.206.
5.15. Electrophoresis 220.127.116.11.5) 3.2. Determination of mycelial morphology 3. Statistical analysis 3. isolation and screening of organism 4.5) 3.Gel loading buffer 3.5.10. Phosphate Citrate Buffer (pH 18.104.22.168.2.5 M Tris HCl. Analytical techniques 3. Standard curves 3.11. 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 . Brad ford reagent 3.15. Stacking buffer (1M Tris HCl.1.0) 3. Bovine serum albumin 3.1. Separation of fungus from fermented broth 22.214.171.124) 3. Identification.12.13.8. Selection of mutants 3. Starch Solution 3.11. Kinetic study 3. Acetate Buffer (pH 5. Protein Marker 3.4. Gel Preparation 3. Separating buffer (1.2. DNS reagent 3.11. Anion exchange chromatography 126.96.36.199. Purification of alpha amylase 3. Maltose 3.1.8. Gel filtration 188.8.131.52.15.8. Preparation of reagents/ buffers 3. SDS Solution (10%) 3. Acrylamide bis acrylamide (30%) 3.1.4. Dialysis 3.vii 3.4. Ammonium per sulfate 3. Ammonium sulfate precipitation 3.12.Tank Buffer 184.108.40.206.7.11. Tris HCl buffer (pH 7.11.5. Staining and Destaining solution Chapter # 4: RESULTS AND DISCUSSION 220.127.116.11. Estimation of total protein contents 3.2.2. Stacking gel 3. Characterization of enzyme 18.104.22.168. Separating gel 3.3.11. EMS treatment 3.15.2. Nitrosoguanidine treatment 22.214.171.124. Estimation of alpha amylase 126.96.36.199.3. Nitrous acid treatment 188.8.131.52.7.3. pH 184.108.40.206. Estimation of dry cell mass (DCM) 220.127.116.11.14. pH 18.104.22.168) 3.8.Strain improvement 4.
2.2.8. Effect of inoculum size 4.Effect of time of incubation on the activity of purified alpha amylase 4.6. Rate of alpha amylase production 4. Effect of dissolved oxygen 4. Evaluation of additional carbon sources 4. Rate of alpha amylase production 4. Evaluation of inorganic nitrogen sources 4.1. Screening of Culture media 4.4. Optimization of nutritional requirements of A. Purification of alpha amylase 4.1.6. Effect of different concentrations of corn starch 4. Gel filtration 22.214.171.124.1.2. Anion exchange chromatography 126.96.36.199.6. Effect of agitation intensity 4. Effect of surfactants 4.4. Effect of pH 188.8.131.52. Ammonium sulfate precipitation 184.108.40.206. Effect of starch from different sources 220.127.116.11.1.3. Temperature optima of purified alpha amylase 4.6. Effect of aeration levels 4. Screening of UV treated isolates 4.2.7. Ammonium sulfate precipitation 18.104.22.168.22.214.171.124.1.3.3. Screening of NG treated isolates 4. Step wise purification 126.96.36.199.Screenning of EMS treated isolates 4. Effect of distilled water and buffer on the activity of purified alpha amylase 4.6. Optimization of cultural conditions in stirred fermenter 4.4. Effect of different volume of medium 4.4.5.viii 4.6. oryzae in shake flasks 4.1. Effect of pH on the activity of purified alpha amylase 188.8.131.52.7. 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 .3.4.5. Effect of inoculum size 184.108.40.206.3.2.3. Optimization of cultural conditions in shake flasks 4. Screening of nitrous acid treated isolates 4. Effect of incubation temperature 4. Effect of different initial pH 4.5.2. Characterization 220.127.116.11. Chemical Mutagenesis 18.104.22.168.7.5. Evaluation of organic nitrogen sources 4.
1 4.1. 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 for the alpha amylase 72 production Sub grouping of alpha amylase producing isolates of A.13 Title of Table Page Isolation and screening of A.7 4.2.3. oryzae IIB-30 and its mutant derivative in stirred fermenter Purification summary of alpha amylase produced by mutant 127 strain of A.8 4. oryzae EMS-18 by using ammonium sulfate Step wise purification profile of alpha amylase produced by 128 mutant strain of A.2 4.1 4. oryzae UV-23 isolates for alpha 80 amylase production NG treated survivors of A. oryzae NA17 for the alpha 84 amylase production EMS treated survivors of A. oryzae EMS-18.2 4.10 4.4.5 4. 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.2 4.11 4.4.1 4.6 4.4 4. 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 81 Range of alpha amylase activity of NG isolates 81 Screening of nitrous acid treated strains of A. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different aeration for the alpha amylase 118 production by A.1 4.3.12 4.5. 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 83 Range of alpha amylase activity of nitrous acid treated isolates 83 Screening of EMS treated A.2.2 4. oryzae 74 Screening of UV isolates of A.3 4.1 4. oryzae IIB-30 and its mutant derivative in stirred fermenter Kinetic evaluation of different agitation speeds for the alpha 124 amylase production by A.2 4.9 4. oryzae NG-18 for 82 the alpha amylase production Nitrous acid treated survivors of A. .1 4.ix LIST OF TABLES Table 4.5. 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 EMS-18 Effect of different inoculum sizes on the alpha amylase production by A. oryzae IIB-30 and its mutant derivative A.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 EMS-18. oryzae IIB-30 and its mutant derivative A.4 92 4.10 102 . oryzae IIB-30 and its mutant derivative A. oryzae IIB-30 and its mutant derivative A.3 91 4.8 100 4.6 94 4. oryzae EMS-18.7 99 4. oryzae EMS-18 Effect of different concentrations of lactose on the alpha amylase production by A.2 90 4. oryzae EMS-18 Effect of raw starch from different sources on the alpha amylase production by A.5 93 4. oryzae IIB-30 and its mutant derivative A.1 3.x LIST OF FIGURES Figure 3.9 101 4. oryzae IIB-30 and its mutant derivative A. oryzae IIB-30 and its mutant derivative A. oryzae IIB-30 and its mutant derivative A.2 4. oryzae IIB-30 and its Page 69 70 89 4. 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 volume of media on the alpha amylase production by A. oryzae EMS-18 Effect of incubation temperature on the alpha amylase production by A. Effect of different concentrations of starch on the alpha amylase production by A. oryzae EMS-18 Rate of fermentation for the alpha amylase production by A. oryzae EMS-18 Effect of different initial pH of fermentation medium on the alpha amylase production by A.
16 108 4. oryzae EMS-18 4. oryzae IIB-30 and its mutant derivative A.17 113 4. oryzae IIB-30 and its mutant derivative A. Comparison of rate on the alpha amylase production by wild (IIB-30) and mutant strain of A.19 117 4. Effect of different concentrations of Tween 80 on the alpha amylase production by A. oryzae EMS-18 Effect of different level of dissolved oxygen on the alpha amylase production by A.12 104 4. oryzae IIB-30 and its mutant derivative A.18 115 4. oryzae IIB-30 and its mutant derivative 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 IIB-30 and its mutant derivative A.22 123 . oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. oryzae EMS-18. 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 different inoculum size on the alpha amylase production by A. oryzae EMS-18 Effect of organic nitrogen sources on the alpha amylase production by A.14 106 4.15 107 4. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18 Effect of different aeration levels on the alpha amylase production by A.xi mutant derivative A.20 119 4. oryzae EMS-18 Effect of different surfactants on the alpha amylase production by A. oryzae (EMS-18) in stirred fermenter Effect of initial pH of media on the alpha amylase production by A. oryzae EMS-18 Effect of different agitation intensity on the alpha 103 4.21 121 4.13 105 4.11 Effect of inorganic nitrogen sources on the alpha amylase production by A.
oryzae EMS-18 Effect of metal ions on the activity of purified alpha amylase by mutant strain of A.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.29 4. oryzae EMS-18 Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A.27 4. oryzae IIB-30 and its mutant derivative A.24 4. oryzae EMS-18 Effect of different pH on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18. Effect of temperature on the activity of purified alpha amylase by mutant strain of A.23 4.26 134 4.30 137 138 . oryzae EMS-18 Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A. 129 130 131 4.xii amylase production by A.28 135 136 4. oryzae EMS-18 4.
6 fold increased activity over the parental strain in terms of enzyme production.e. isolates were qualitatively and quantitatively screened. This mutant showed 2. MgSO4.xiii Abstract The present study. initial pH. by both wild and mutant strains. .3.2 U/ml).. NH4Cl 1. screening and selection of Aspergillus oryzae for the alpha amylase production. CaCl2 0.5.12. The rate of fermentation was also studied and the highest yield of enzyme was obtained 72 h after inoculation. pH 5. oryzae in shake flasks fermentation. yeast extract 8. Of all the media. starch 20. oryzae were isolated from different soil samples.05).7H2O 0. 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. EMS-18 exhibited the highest enzyme activity (347±1.1U/ml of enzyme was selected which was assigned the code IIB-30. Six different fermentation media were evaluated for the alpha amylase production by both wild and mutant strains of A.06 gave maximal enzyme production i. During the treatments. Seventy eight isolates of A. The selected strain was subjected to physical and chemical mutagenic treatments in order to improve its amylolytic potential. volume of media and inoculum size was investigated on the 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. volume. 10 % and inoculum size 4 %. The optimal enzyme production was obtained at 30°C. Among these. M4 containing (g/l). The effect of incubation temperature. deals with the isolation. 168±2 (wild) and 385±2 (mutant) which was highly significant (p≤0.
The scale up studies for alpha amylase production was carried out in a 7. respectively. The rate of fermentation for enzyme production by both wild and mutant strains was investigated. inoculum size (10 %) and agitation intensity (200 rpm) were optimized for enzyme production. By using SephadexDEAE column. temperature.2 %) as nitrogen sources were also optimized. A total of 9.xiv Corn starch (2 %) and lactose (1. The fermented broth was subjected to ammonium sulfate precipitation at different saturation levels (20-90 %). Different surfactants were added to the fermentation media and Tween 80 at the level of 0.5 %) as carbon sources while. dissolved oxygen (15 %). .5 L stirred fermenter. the active fractions were eluted using 0.30 M NaCl at pH 7.5 vvm). The kinetic depiction of results showed optimal fermentation period for enzyme production to be 64 h and 48 h.05 M Tris-HCl buffer containing 0.3 %) and peptone (0. The other cultural conditions such as initial pH (5).) 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). pH and metal ions on purified enzyme was also investigated and maximum activity was achieved after 30 min at 40ºC and pH 5 in the presence of Ca+2 ion. ammonium sulfate (0. The effect of time. aeration level (1. The optimum level of ammonium sulfate saturation was found to be 70 % that gave 1.5.5 fold enzyme purification was accomplished.1% was found to be the best for enzyme production.3 fold purification. The molecular weight of alpha amylase was found to be 48 kDa on SDS-PAGE after gel filtration.
microbial source of alpha amylase can be produced in amount meeting the demands of market.product of meat industry. Alpha amylase can be derived from several sources such as plants. textile and paper industries..4 glucan-glucanohydrolase EC 3. 1996. detergent.. animals and microorganisms. This extracellular enzyme hydrolyses α-1. 1) is widely distributed in nature. 2009). glucose and alpha limit dextrin (Omemu et al. Kathiresan and Manivanan.. but production from first two groups is limited for several reasons. 2005..4 glucosidic linkages randomly throughout the starch molecule in an endo-fashion producing oligosaccharides and monosaccharides including maltose. In contrast. . Gupta et al. Different fungal and bacterial strains have been extensively used for the enzyme production (Pandey et al. 2. brewing. 2006. Leman et al. Alpha amylases are one of most important and widely used enzymes whose spectrum of application has widen in many sectors such as clinical.1 INTRODUCTION The starch degrading enzyme alpha amylase (α-1. These are important enzymes used in starch processing industries for hydrolysis of polysaccharides such as starch into simple sugar constituents. Beside their use in starch saccharification they also find applications in food. Ramachandran et al.. Bhanja et al. on the other hand enzyme of animal origin is by. 2008). 2004. medicinal and analytical chemistry... baking. 1. 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. 2007. The concentration of enzymes in the plant material is generally low so the processing of large amount of plant material is necessary.
Ray 2001. However.. Many species of Aspergillus such as A. 1987.. Themomyces lanuginosus. A. 2001. 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. Alpha amylase can be produced both by solid state and submerged fermentation technique (Prescott and Dunn. and it is based on a number of factors such as physiological stability. yield consistency. Morphological variety is a typical feature of filamentous fungi. oryzae have received most attention to obtain many kinds of hydrolytic enzymes like alpha amylase. tamarii. Strain selection is a critical step in the development of a biotechnological process. (Arnesen et al. The ability of filamentous fungi to secrete large amounts of extracellular protein has made them well suited for the industrial enzyme production. non fastidious nutritional requirements and high productivity of alpha amylase (Abe et al. 1988. incubation time required for maximum production as well as the tolerance to temperature. Production of enzyme is greatly effected by the cultivation method. aeration and shear stress etc. The commonly used fungi included Trichoderma sp.2 2000)... (Laluce et al. 2002). and Alternaria sp. A. niger. Poornima et al. Its . awamori and A. 1995. 1981). Fusarium moniliformis. Actinomycetes sp. Archer and Wood. 1995.. Penicillium griseoreseum. 1991). lipase and protease. Agger et al. Yovita et al. Zangirolami et al. 1998. Filamentous fungi have been well known for the starch and cellulose degrading enzymes they naturally secrete. A. 2008).... Nielsen et al. oryzae is the organism of choice because of its ubiquitous nature. 2005).
strain improvement is trial and error process involving laborious procedure. Rational selection procedures are more efficient and usually have a biochemical basis (Elander 1982). 2003). such as ultra violet (UV) radiation.. followed by suitable selection and screening of the survivors (Szafraniec et al. oryzae showed more dextrinizing and saccharogenic activity than the parental strain. ethyl methane sulphonate (EMS) and nitrous acid to obtain superior mutants have been proved successful by subjecting the microorganisms to these mutagens. two extreme types of morphology are generally known. . use of alkylating agents like N-methyl N-nitro N-nitroso guanidine (NG). pellets and free filaments. rational selection is achieved by the use of techniques allowing visual identification of superior mutations. Therefore isolation of improved producers of alpha amylase using starch plate can only be partially selective (Kuek and Kidby. However. 1984). Between these two extremes lies an intermediate aggregated morphology called clumps (Wang et al. 2005). 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. It was studied a mutant strain of A. In primary screening prior to laboratory fermentations. Mutant strains of Aspergillus oryzae were found to be best for enzyme production compared to wild strain. Traditional methods for strain improvement. The morphology of filamentous organism during enzyme production varies from round pellets to free filaments depending upon the cultural conditions and strain genotype.3 morphology has distinct effects both on the enzyme production and rheological nature of a fermentation broth.. In submerged fermentation.
Azin and Noroozi. potassium. better initial improvement can be expected. nitrogen sources and mineral nutrients such as phosphorous. Qirang and Zho. maltose. All these .. 1993.. EMS etc. and calcium are essential for the growth of fungi as well as enzyme production (Hughes and Poole 1991).. Abdullah et al. 2001) Selection of suitable fermentation medium and initial pH is very important for the enhanced alpha amylase production. The influence of different carbon sources such as glucose. The energy for growth generally comes from the oxidation of medium components. maltodextrin..4 In case of mutagenic application to the wild strain. magnesium. oryzae was studied and it was found that starch and maltose strongly increased enzyme productivity by A. 2000. dextrin.. Fungal strains have been grown on starch. 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. (Spohr et al. The carbon sources affect not only the mode of amylase formation but also the rate with which carbohydrates are metabolized (Dubey et al. fructose. A strain of A. All microorganisms require energy and certain minerals for growth and metabolism. Carlsen and Nielsen. The presence of carbon. 1998. oryzae where as glucose led to very low productivity (Lachmund et al. galactose and sucrose on the alpha amylase production by A. 2003). glucose and dextran. amylopectin. 1994. 2001). The enzyme production has been greatly affected by the addition of different carbon sources. oryzae treated with NG gave better enzyme production compared to the parental strain. So it is important to select suitable carbon source for the enhanced enzyme production. maltose.
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 (25-42°C). 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 α-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 30°C 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 30ºC 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 35°C. The purified enzyme had maximum activity at pH 6.2, after 30 h of incubation (El-Safey and Ammar, 2002; Pimpa 2004).
Objectives Specific objectives of present work are as follows 1-Isolation. Fungal alpha amylase was unstable above 45°C but at 25°C attack raw starch granules more efficiently than enzyme from Bacillus amyloliquefaciens. obtained in 11 % yield had optimal temperature and pH 50-55°C and 5.. The alpha amylase is an unusual enzyme which converts starch to maltose in > 75 % yield.5 and temperature 55°C (Kariya et al. The purified enzyme.8 Alpha amylase purified from the cultural broth of A. 2-Random mutagenesis by UV and chemicals to improve the fungal strain as well as enzyme production.6 fold purification and yield being 25..0 and 35oC (Fairbairn et al. The optimum growth conditions for enzyme production by A. 5. 3-Optimization of cultural conditions for the selected strain of A oryzae in shake flasks. The production and stability of the enzyme is very sensitive to pH and temperature. identification and screening of A. respectively.Purification and characterization of alpha amylase. 1998). 2003).. The molecular weight of alpha amylase from A. oryzae was estimated to be 50 kDa..0.3 %. It may be of industrial value in the production of low viscosity corn syrups (Hidaka et al. oryzae was pH 5.0.6. 1986. Jin et al. oryzae indicated 12. . 1980). The purified enzyme was most active at pH 4. oryzae strains. 4-Scale up studies of enzyme production in a laboratory scale stirred fermenter. The enzyme retained 94 % activity in 1 h at 60°C.
bacteria and fungi being the major sources. fermentation. and textile to paper industries etc.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. But different reaction specificities have been observed across the family members.4 linked chains of glucose with 1.9 REVIEW OF LITERATURE Starch degrading amylolytic enzymes is of great importance in biotechnological application ranging from food.4-glucosidic linkages to produce different sizes of oligosaccharides. glycogen and related polysaccharides by randomly cleaving internal α-1. Alpha amylase is ubiquitous in distribution. Amylases are enzymes which hydrolyze the starch molecules in to polymers consists of glucose units. Starch is a carbohydrate source consisting of two molecules amylose and amylopectine. and they contributed numerous common properties. Alpha amylase is a key enzyme in metabolism of spacious diversity of living organisms which utilize starch as carbon and energy sources. Amylose is formed from chains of glucose linked α1. pH and temperature have very vital roles to play. A branched starch chain (which can be . Most of the microbial alpha amylases belong to the family 13 glycosyl hydrolases. It can hydrolyze starch.4 and amylopectine is formed from α1. Stability of alpha amylase has extensively been studied. A single straight chain starch is called an amylose. Structurally alpha amylase possesses barrel structures and is responsible for hydrolysis or formation of glycosidic bonds in the αconformation. with plants. Alpha amylase acts on starch and breaking them up into sugars (hence the term saccharification).
Alpha amylase has been derived from several fungi. yeasts. The main difference between them is that the saccharifying enzyme produces a higher yield of reducing sugar than liquefying enzyme.10 considered as being built from amylose chains) is called an amylopectin. bacteria and actinomycetes. 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. Two types of enzymes have been recognized called as liquefying and saccharifying. animals and microbes. some what and depend on the source of enzyme. 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. 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. Many scientists carried out extensive work on . 2000). 1990). however. These starches are polar molecules and have different ends. properties and product of hydrolysis differ. Alpha amylase can be derived from several sources such as plants.
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. (1975) isolated 86 strains of the Aspergillus producing maximum acid stable alpha-amylase. Generally fungi secrete alpha amylase (dextrinizing enzymes) although a few fungi have been known to secreted alpha amylase and beta amylase (saccharifying enzymes). Yabuki et al. No significant increase was occurred in the weight of mycelia during 2 h of induction.11 alpha amylase production. The enzyme production is dependent on the type of strain. The mycelia were taken from 20 h old cultures and cultivated on the medium containing peptone and glycerol. both enzymes were showed similar properties in all respects. A. oryzae EI 212 secrete alpha and beta amylase or both depending upon the composition of media and fermentation conditions. in this case maltose was added as inducer. oryzae using inducer such as maltose. (1977) studied rapid induction in the alpha amylase production by A. Vallier et al. composition of media and methods of cultivation. Afterwards these cultures were starved for 5 h.5 h remarkable increase in alpha amylase production takes place and enzyme production reached at optimum rate. During first hour of induction. (1977) observed alpha amylase production after the lysis of . After 1. The nature and amount of extracellular amylase produced by Aspergillus species determine the efficiency of conversion of starch to oligosaccharides. both extra and intracellular alpha amylases were produced with the same rate (70-80/µg of cells/h). Tokhadze et al. When the purified samples of these intra and extracellular enzymes were tested by using diethylaminoethylcellulose column and techniques of gel filtration.
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 20°C 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 40°C 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.22.214.171.124) 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 28°C 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
005 M had no effect on the activity in both temperatures. showed three dimensional structures. The fungal alpha amylase had an optimum temperature of 55°C at pH 4. columnaris alpha-amylase was -5°C followed by 5°C. On the other hand. 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. but had 60°C in buffer containing . 0. oryzae. The active site was composed of amino acids which were specifically found in the loop joining the adjacent helix. Repeated freezing (-5°C) and thawing followed by freezing (-5° C) had no effect on stability of alphaamylase. Ali and Abdel-Moneim (1989) reported that the best temperature for the preservation of A.e.6. The molecular weight of purified enzyme was 54. The computerized methodology explained the position of amino acid and gave the predictions about the structure of alpha amylase from different sources. 0000.15 condition i. oryzae. Alpha-amylase produced by A. It was noticed all alpha amylase having known amino acid sequence possess same basic structure. 30°C for 24 h. these alpha amylase possess barrel shape structure which was surrounded by eight helices. 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. 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. 25°C was the lowest preservation temperature without any effect on the stability of alpha-amylase. CaCl2 at 0. flavus var.005 M CaCl2 decreased the activity of alpha amylase and reached a 100 % inhibition at 35th day.
The result of Principal component analysis (PCA) describes the transfer of oxygen at different agitation rate influences enzyme production and carbon dioxide. 6 and 7 resulted in increase of enzyme activity at the temperatures above 50. Whereas. The addition of 0. Statistical analysis was used to explain the behaviour of culture instead of explaining optimization of fermentation conditions. The enzyme was only stable for 4 h at 25°C. The production of carbon dioxide was indirect growth measurement. alpha amylase and glucoamylase by using the submerged fermentation. at pH 6 and 7 optimum temperature was 55°C for the activity of the enzyme only or with 0.01 M CaCl2 showed a loss of 4 % compared to a 22 % loss in the presence of 0.01 M Na2-EDTA in the beginning and a 100 % loss after 4 h at 25°C.01 M CaCl2 or Na2-EDTA 5.16 0. niger for the synthesis of amylolytic enzymes i. 55 and 60°C for 30 min..005 M CaCl2 and 50°C in buffer containing 0.01 M CaCl2. respectively.01 M Na2-EDTA after 4 h at 25°C and 65 % loss in the presence of 0. Optimum pH for alpha amylase was 5. addition of 0. However. The presence of 0. 40 and 25°C.005 M Na2-EDTA.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures below 55 and 45°C.6. 50.01 M CaCl2 together with 0. respectively. but in the presence of 0.01 M CaCl2 greatly increased the thermostability of alpha amylase at 40. The optimum temperature for the activity of alpha-amylase at pH 5 was 50°C for the enzyme only but 55°C in the presence of 0. 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. 0. Rousset and Schlich (1989) screened different species of A. However. . 45.01 M CaCl2.01 M CaCl2 at pH 5.e.
Tsekova et al. Of the entire isolated cultures one was Bacillus coagulans. 48 h incubation period and broth extract starch agar medium. 200 rpm agitation speed. Shah et al.6 % starch broth at 55°C. ambient temperature and 37°C. (1991) optimized the conditions for the synthesis and recovery of alpha amylase from A. (1993) studied the fermentation medium containing all the components which were necessary for the production of acid stable alpha amylase (asAA) by A. When the amount of stored glycogen (CSG) decreased and inducer such as dextrin was present synthesis of acid stable alpha amylase started. (1993) studied the ability of Aspergillus genus for alpha amylase production. Harway (1991) has isolated thermophilic bacteria from the soil which was preliminary enriched with 0. oryzae. 61 % and 58 % respectively when preserved for 12 months at 4°C. When the amount of CGS increased production of acid stable alpha amylase tend to be decreased. The amount of glucose . The maximum production was obtained in optimal condition which consists of incubation temperature 55°C.17 Maximum alpha amylase production was obtained at lower agitation speed while in case of glucoamylase intermediate agitation speed gave maximum alpha amylase production. oryzae alpha amylase retained 100 %. These bacteria had ability to hydrolyze the starch. One hundred and thirty milligram of acid stable alpha amylase per liter of medium was produced after 5 days of inoculation at 30°C in submerged fermentation. Maximum production of as AA was obtained when amount of CGS reaches at zero. Sudo et al. which was best producer of alpha amylase. Glycogen was present as stored polysaccharide. kawachii using submerged fermentation. A. When 3 % soluble starch was used in Czapek-Dox agar and in liquid Czapek-Dox media maximum alpha amylase production was obtained.
The changes in new alpha amylase production was compared with two known alpha amylase represented as A-1 and A-2. Omori et al. Characterization of enzyme showed the maximum activity of purified enzyme was noticed at pH 6 and 55°C. Purification of enzyme was involve the use of ammonium sulfate precipitation. The culture filtrate was subjected to electrophoretic analysis. The enzyme was purified by using the different techniques such as ion exchange chromatography and gel filtration. however. (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. kawachii in barley koji. . Soccol et al. flavus in liquid medium containing topica starch. (1994) isolated acid labile alpha amylase (A-3) from A. The quantity of acid stable alpha amylase was directly proportional to the production of mycelia.18 in the medium and growth of mycelia was strongly influence by the concentration of CGS.5 kDa molar mass with an isoelectric point at pH 3. Cooked cassava showed optimum production in solid state fermentation. ion exchange chromatography and gel electrophoresis. This analysis showed filtrate contains only one type of amylolytic enzyme named alpha amylase. Khoo et al. maltose. oryzae was obtained when raw cassava was used. The purified enzyme showed 52.5 ± 2.5. 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. The protein enrichment and maximum enzyme synthesis were obtained in solid state fermentation. maltotriose and maltotetrose. maximum synthesis of amyloglucosidase by R. (1994) achieved fifty units per milliliter amylolytic activity by using A.
Of all the isolated strains Bacillus subtilis produce 24 U/ml alpha amylase. NBSI (6mM) and NAI (6mM). After passing through these steps the enzyme showed 16 fold increase in the purity and 45 % of enzyme was recovered. However. DEAE-Sepharose ion exchange chromatography and sephacryl S-200 HR gel filtration.3m M). Different carbon sources were added to the fermentation media to check effect of these carbon sources on alpha amylase production. respectively. The heat denaturation constant and molecular weight by gel filtration was 0.000. (1995) reported that alpha amylase produced by A. temperature 50°C and km value for starch hydrolysis was 0. Incubation for 30 min at 50°C result 80 % lose in enzyme activity. The hydrolysis of maltoheptaose by the enzyme resulted in the formation of maltotriose and maltotetraose. The enzyme activity was inhibited by using Mercuric ion (0. oryzae was purified by passing through the different steps in a specific sequence such as amylopectin affinity adsorption. The results indicated production of A3 was influenced both by temperature and initial concentration of citric acid. Chang et al.22 %.19 Sodium dodecyl sulfate poly acrylamide gel eletrophoresis showed A-3 has molecular weight 56. in traditional method formation of A-3 was not detected after 36 h. DNFB# (6mM). The enzyme showed constant production at 40°C approximately for 54 h.024/m and 52 kDa. 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. In the presence of 2 % citric acid in barley A3 was formed upto 36 h. The optimum conditions for purified enzyme was pH range 4-5. The maximum activity of alpha amylase 360 U/ml was obtained in the .
On line FIA system was used and a rate constant was obtained by the empirical expression k = 1. However.99 (h−1) explained the inactivation of enzyme was greatly influenced by pH values. The activity of alpha amylase was greatly inhibited in the presence of manganese. copper and ferric ions. The optimum temperature for purified enzyme was 7 and 30°C. flavus resulted in the formation of glucose and some other oligosaccharides Kajiwara et al. The inactivated enzyme again obtained some of its activity at pH 6 and this reactivation steps also obey the first order kinetics rules. respectively. The use of potassium ions increased the activity of alpha amylase. (1997) studied the production of acid stable alpha amylase from A.000. beyond this pH range a great loss in the activity of enzyme was noticed. flavus.20 presence of dextrin and optimum temperature was 50°C for enzyme production. flavus was 75. oryzae at different pH. if enzyme was incubated for 2 h at 100°C 23 % of this activity was lost. The polyacrylamide gel electrophoresis (PAGE) indicated the molecular weight of A. zinc.19 × 107 [H+]1. Carlsen et al. The enzyme showed highest stability at neutral pH (5-8). (1996) tested the stability of alpha amylase produced by A. The enzyme was purified by using starch adsorption methodology. 000 ± 3. magnesium ions did not extremely influence the enzyme activity. However. The hydrolysis of native starch by A. kawachii during production of shochu-koji. From barley shochu-koji two types of . however. The strain which showed highest ability for alpha amylase production was identified as A. Abou Zeid (1997) isolated filamentous fungi from cereals and screened to test the alpha amylase producing potential. The contamination of protease in the protein sample was not result to the irreversible loss of activity.
while general protein secretion was stimulated after 24 h of inoculation.21 the acid stable alpha amylase (as AA) represented as as A-1 and as A-2 were puified. The production of biomass increased gradually with the . The asA-1 and asA-2 showed different adsorption characteristics on raw starch. The result showed acid stable alpha amylase was found in different form just like the glucoamylase produced by A. Contrary to as A -1 the activity of as A-2 was increased with increase in the incubation time. Spohr et al. The comparison of these strains for morphology and impact of morphology on the protein indicated the mutant strain having denser mycelium. oryzae for the production of recombinant protein and affect of growth on the production of protein. The 2. Temperature affect ratio of as A1 to total as AA activity. (1998) cultivated thermophilic fungus in the presence of dextran (having low molecular weight) along with Tween 80 or Triton X-100. The unknown protein showed all the characteristics which were present in as A-2. The fermentation was carried out in shake flasks for more than 120 h. (1997) examined alpha amylase producer strain of A. produce more alpha amylase compared to other strains. Arnesen et al. A known band was appeared in the place of as A-1 band after 12 h of incubation. The Tweeen 80 also influences the production of biomass. When acid protease and as A-1 were incubated along with each other and this sample was analyzed by SDS-PAGE. The activity of as A-1 slowly increased during the process of shochu-koji production but dropped after incubation of 24 h. awamori during the production of shochu-koji.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.
(1998) purified three forms of alpha amylase to homogeneous state by using the methodology of column chromatography. Bioproduct yield obtained from 12h batch culture was 6. and Amyl 111. The optimum pH for Amyl 11 and Amyl 111 was 5. However Amyl 1 produces no hydrolyzing effects on raw corn starch. The range of soluble starch hydrolysis through Amyl 1. Amyl 11. These forms were designated as Amyl1. oryzae for alpha amylase production and microbial biomass protein (MBP) from starch processing waste water (SPW) in air lift bioreactors. and Amyl 111 have ability to hydrolyze maltotriose. The Tween 80 had no effect both on the hyphal length and diameter. raw corn starch and alpha. It was noticed increase in the amount of Tween 80 resulted greater than 3 fold increase in the total extracelluar protein. Anidyawati et al. Maltose and maltosetriose were formed by the hydrolyzing action of Amyl 1 on malto-tetraose-pentose.-hexaose.1 g/l. isomaltotriose. pH 5 and 35°C.5 while in the case of Amyl 1 the pH was 4. 63. and α-cyclodextrin. isomaltosse. (1998) used A.. Amyl II and Amyl III was 33.e. gamma cyclodextrin resulting in the formation of maltose along with minor products of glucose and maltose. respectively. maltotriose.22 increase in the concentration of Tween 80. Jin et al. maltose. The SDS PAGE indicated these three forms possess 49.000 molecular weight. This yield consists of 55 EU/ml of alpha amylase and 38 % . Unlike Amyl 1 both Amyl 11. These forms of alpha amylase were produced from A. Glycosylation degree was also not effected by the Tween 80.000. beta. Contrary to this Triton X-100 produce reverse effect. awamori.-heptose and β and γ-cyclodextrin.000 and 97. The production of MBP and fungal alpha amylase was carried out under the optimized conditions i. respectively. 35 and 38 %.
The comparison between organic and inorganic nitrogen sources indicated organic nitrogen for example yeast extract or casine hydrolysate was superior to ammonia. tamari formed both alpha amylase and glucoamylase in the mineral medium concomitant with carbon source i. Pedersen and Nielsen (2000) reported the effect of organic and inorganic nitrogen sources on alpha amylase production by A. 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.5 and 6 and stability at pH 4-7. The enzyme showed stability at pH 5-9 and 25-35°C. Spohr et al. oryzae in continuous cultivations. 1 % starch or maltose.05 g/l casine . In the presence of 0. tamari. It was observed the production and growth of organism were correlated.60°C. The formation of alpha amylase and glucoamylase indicated tolerance to wide range of initial fermentation medium pH (4-10) and temperature (25 .. (1998) tested three different strain of A. (1999) isolated a fungal strain from the soil having the ability to produce amylolytic enzymes. Moreira et al.42°C). This strain was identified as A.e. The temperatures at which enzymes showed highest activities was between 50 . Ion exchange chromatography was used for the separation of alpha amylase and glucoamylase.23 protein. In case of inorganic nitrogen source ammonia was better than nitrate. Both nitrogen sources were tested along with glucose. Comparison of production and morphology of these strains indicated the variations in the morphology had direct impact on enzyme production in submerged fermentation. Partially purified alpha amylase and glucoamylase showed maximum activities at pH 4.
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 40°C; however, if alpha amylase was placed at 60°C 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.
It was noticed the optimum amylolytic activity and xylanolytic activity was obtained on 4th and 6th day of fermentation respectively. When the concentration of biomass was increases 2-12 g dry weight/kg the specific rate of enzyme production was decreased. nidulans in which gene creA was removed (which cause carbon catabolite repression) no marked decrease in the specific rate of enzyme formation was observed. oryzae on spent brewing grain (SBG) and corn fiber for alpha amylase production. It was noticed specific rate of alpha amylase production was inversely proportional to the concentration of biomass formation. 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. (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. in fermentation. Bogar et al.27 Agger et al. However in case of recombinant strain of A. in situ enzyme produced in solid substrate fermentation material was economic biocatalytic product for animal feed and for the . A. The results indicated less alpha amylase production at high biomass formation was due to slow mixing rate of vital components in viscous culture medium. The crude enzyme. (2002) tested different strains of A. A Plackett-Burman experimental design was practiced to develop optimized media for alpha amylase production using best producer strain. 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. Ray (2001) isolated Penicillium sp possessing the ability to form alpha amylase and xylanase in the presence of starch and xylan respectively. nidulans in submerged fermentation.
The fermentation was carried out up to 120 h. Maximum alpha amylase production was obtained at 25°C. 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. The alpha amylase production was 133U/ml in shake flasks while in case of bioreactor production was 161 U/ml. (2002) used thermophilic fungus T. . Arnesen et al. 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. Addition of any external carbon source in the spent brewing grain resulted decreased in alpha amylase production.e. and di potassium hydrogen phosphate for alpha amylase production by A. lanuginosus for alpha amylase production in shake flasks. Francis et al. oryzae NRRL 6270 when spent brewing grains utilized as sole carbon source.. Optimum alpha amylase production [6870U/g dry substrate (gds)] was obtained in solid state fermentation at 30°C after 96 h by the use of suspension containing 1×107 spores/ ml. oryzae in shake flasks and bioreactor. (2002) investigated the effect of spent brewing grains on alpha amylase production by A. The fermentation medium contained carbon source in the form of low molecular dextran. (2002) used the central composite design along with 3 variables i.28 production of bio alcohol from starchy substrate. At 30°C almost similar results were obtained. A same pattern was observed in the case of total extra cellular protein. yeast extract. starch. 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. Gigras et al.
there was great difference in the fermentation period of shake flasks and bioreactor. A high concentration of phosphate in the fermentation medium and use of low inoculum size was essential to prevent the unnecessary foaming in bioreactor. A Plackett–Burman design was used to test the influence of nineteen . Thus in present study pH act as sign of commencement or ending of the enzyme production. The model has ability to describe the temporary behavior of B. but managing the pO2 level and agitation rate was not compulsory for alpha amylase production. An age-based population balance model was used to explain the maturity of sporangium in the direction of the formation of spores. oryzae NRRL 6270. substrate consumption. Francis et al. The enzyme production increases with the increase in the pH of medium and reached at its peak at pH above than 7. The segregated model represented three different states of cell and the change from vegetative stage to sporangium and lastly to mature spore. cell differentiation and enzyme formation by Bacillus subtilis in bioreactor. subtilis in both batch and fed-batch cultures. The experimental data was added into a polynomial model to find out alpha amylase production. The fermentation period for the maximum alpha amylase production by A. initial moisture contents and inoculum size by application of Box–Behnken design under the response surface methodology for the highest production of alpha amylase by A.29 However. Parameters in the model were found out by placing the experimental data in the model. (2003) optimized incubation temperature. oryzae in shake flasks was 120 h but in case of bioreactor this time period was reduced to only 48 h. (2003) developed a segregated model to explore the intrinsic associations between growth.5. Huang et al.
maltose did not produce glucose. oryzae MIB 316. and Mono Q column chromatography. The N-terminal sequence of first 10 residues and many other molecular characteristics were similar to Taka-amylase. Incubation temperature 30°C. 4–10. Soybean meal. The sizeexclusion chromatography and SDS-PAGE showed that purified alpha amylase had molecular mass 34 kDa and 46 kDa. Kusuda et al. The mercuric ion did not inhibit the activity of enzyme. oryzae NRRL 6270 on SBG.. gel filtration. The alpha amylase showed 3580 fold purity and 10. A Box–Behnken design was used to select the best condition for alpha amylase production. respectively. SDS-PAGE analysis resulted in a single protein band. Under selected conditions of solid state fermentation SSF. TLC and HPLC analysis indicated .5 % recovery.. The experimental results also indicated the alpha amylase was somewhat thermostable and showed thermostability at 50°C while the optimal temperature was 60°C. oryzae. The enzyme had ability to efficiently hydrolyzed amylopectin. about 20 % enhancement in enzyme production was found. However. The characterization of purified alpha amylase showed that it was most active at pH 5–6 and having stability between wide range pH i. Kariya et al.30 nutrient components on alpha amylase production by the A. (2003) purified alpha amylase from culture broth of A. (2003) isolated alpha amylase from an immobile culture filtrate of Tricholoma matsutak.e. The enzyme was purified to homogeneity by sequential steps of Toyopearl-DEAE. CaCl2 and Mg SO4 were chosen on the basis of their positive affect on alpha amylase production. initial moisture contents 70 % and an inoculum size of 1×107 spores/g dry substrate were the optimum conditions for the alpha amylase formation by A. Measurement of viscosity. amylose and starch and break down maltopentose to produce a maltotriose and maltose.
0x10-3 g/ml. rice and wheat. The enzyme was further purified by gel filtration chromatography (Sephadex G-150). the enzyme was purified by passing sequential steps of purification. The specificity of alpha amylase was tested by using amylose along with various polysaccharides. Apar and Ozbek (2004) studied the effects of temperature on the enzymatic hydrolysis of starch from different sources such as corn. This partially purified enzyme produces 4. Kanwal et al. Molecular weight of alpha amylase was 51. the concentration of residual starch and the residual activity of alpha amylase in percentage were determined at 50 and 60°C temperature based upon the processing .09 U/ml alpha amylase activity was subjected to ammonium sulfate precipitation.95 U/ml along with 20-fold purification.6 linkage and cyclic polysaccharides e. After gel filtration chromatograph it produces of 5. After extraction.31 amylases from T. A oryzae and B.76 U/ml and showed 5. Km value 2. but was not able to hydrolyze the α-1. matsutake was of endo type. Amylase exhibited optimal pH 6.4 glucoside linkage in soluble starch and amylose A (MW. incubation temperature 37°C. The crude extract exhibited 3.and β-cyclodextrin. λmax 540nm and incubation time for enzyme assay was ten min. licheniformis were employed for hydrolysis of starch. Three commercial alpha amylases produced from Bacillus sp. In every starch hydrolysis process. This alpha amylase rapidly hydrolyzed the α-1.8.2900).01 U/mg specific activity. SDS-PAGE of enzyme removed the undesirable proteins and single band of enzyme was appeared.025 U/ml and specific activity 38. (2004) extracted alpha amylase from Malus pumila (apple) by homogenizing the apple in buffer for alpha amylase.g α.180 D which was finding out by Sephadex G-150 column.
defatted soyabean 10 g/l.6 (units/mg port/ml) specific activity. Mathematical models were developed for using experimental data of residual starch concentration from each source. The alpha amylase produced by Aspergillus sp. potassium di hydrogen phosphate 10g/l. The purified alpha amylase showed maximum activity at pH 6. Pimpa (2004) reported that the highest alpha amylase production by Aspergillus sp. magnesium sulfate 5 g/l. Addition of suitable nitrogen sources and inorganic salts to the medium appreciably increased the enzyme production. The purified enzyme showed 9. The optimal pH and temperature of partially purified enzyme was 5 and 50°C. while the optimal temperature was 35°C.1 g/l. El-Safey and Ammar (2004) reported that the amylolytic family has great importance due to its wide spectrum of application.5 U/ml was obtained in the media containing wastewater. soluble potato. The maximum enzyme yield 36. zinc chloride 0.2% (w/v) starch.32 time in a stirred batch reactor. Alpha amylase produced from Aspegillus flavus var. The optimum condition for the production of alpha amylase was 0. Chavez et al. showed catabolic repression.columinaris was isolated and characterized. The enzyme was purified by using ammonium sulfate precipitation and Sephadex G200 filtration method.2 after 30 h of incubation. Some inactivation models were used to understand the relation between temperature and stability of enzyme during hydrolysis of starch from enzymes having different origins.97 fold purification and 6471. The enzyme was partially purified by subjecting into 60 % ammonium sulfate. (2004) screened different carbon sources namely sorghum. corn and cassava starches as well as maltose for the concurrent cultivation and . The alpha amylase activity amplified with the enhancement of enzyme concentration. respectively. was obtained after 24 h.
The use of the clear supernatant as enzyme source was highly advantageous mainly because it decreases the cost of the hydrolysis.5.000 U/l) and production of enzyme was almost similar to those obtained with maltose (about 100 U/l/h). the Trichoderma sp used in this study proved to be beneficial in a direct process of raw starch saccharification with no preliminary gelatinization. The medium containing low starch concentration showed maximum alpha amylase production at 40°C. This thermostable alpha amylase was employed for the hydrolysis of different starches.33 production of both alpha amylase and glucoamylase by a novel Trichoderma sp.00018. from fresh milk of sheep possess the ability to produce extracellular thermostable alpha amylase. The enzyme exhibited highest activity at 135°C and pH 6. cassava and corn starches showed maximum glucoamylase activities (17. When the reaction temperature increased up to 70°C. in case of corn and oat starch alpha amylase showed somewhat less affinity.000 U/l) and alpha amylase production (about 390 U/l/h). Ammonium sulfate crude enzyme preparations as well as the cell-free supernatant actively break down the starches. Because of its capability to produce both alpha amylase and glucoamylase. However. Potato starch upon hydrolysis produced higher concentration of reducing sugars compared to other starches at all tested temperatures. The thermostability of alpha amylase increased in the presence of calcium or starch. all of the substrate showed higher rates of hydrolysis. Konsula and Kyriakides (2004) isolated a somewhat thermophilic Bacillus subtilis strain. . with respect to reducing sugar liberating ability. Soluble and rice starch came at second and third position respectively. even though maltose gave better results compared to other carbon sources with respect to activity of alpha amylase (about 28.
Addition of glucose and 0.05 ml/Kg with an estimated alpha amylase activity of 5.5 % starch further increased the alpha amylase production (1911 U/gds). .The substrate used was coconut oil cake (COC). The best calculated values of these variables for optimal amylase production were soluble starch 71. (2004) investigated alpha amylase production by A. 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 1.10 g/Kg. Raw COC was a good substrate and 1372 U/gds alpha amylase was produced in 24 h. Kunamneni et al. peptone 91.085 ´ 105 U/Kg of wheat bran. was very near to the calculated value. As a result of optimization of media component alpha amylase production was increased (1827 U/gds) when solid state fermentation was carried out at 30°C for 72 h along with media contained 68 % moisture contents. The activity became maximal when the fungal biomass was at its peak at 72 h. nitrogen source (peptone) and a concentrated mineral medium.7-fold increase in enzyme activity (3388 U/gds) was observed. A twenty three factorial central composite design using response surface methodology was used to optimize the above three variables.946 ´ 105 U/Kg of wheat bran. However. These parameters were checked in the laboratory and ultimate alpha amylase activity obtained. lanuginosus. oryzae in solid state fermentation (SSF). (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. The enzyme production and growth were correlated. These nutritional parameters consist of carbon source (soluble starch). 4.34 Ramachandran et al.97 g/Kg and mineral salts solution 175. maltose repressed the alpha amylase production.
K-12 from soil samples having the ability to produced amylolytic enzyme.0 % (w/v) were screened for synthesis of enzyme. liquid soap.35 Kiran et al.5 % pear millet and 0.. Tween 80. Haq et al. Various surfactants (laundry soap. gram and wheat starch were screened for the alpha amylase production by parental and its mutant derivative. sulphonic acid. acyle benzene sulphonic acid. Among all the surfactants. The use of agriculture by products made the medium economic. However. sodium silicate. (2005a) reported the choice of the appropriate surfactant for alpha amylase production by Bacillus subtilis GCBM-25 during shake flasks. corn. rice. Soluble starch.2 % (w/v) in the medium. The mutant strain B. licheniformis GCUCM-30 exhibited 10 fold more enzyme production compared to parental strain when1.0 % inoculum. addition of surfactants in the medium reduced the thermostability from 70 to 50°C. tested laundry soap proved to be superior with respect to alpha amylase production (605 U/ml/min) after 44 h of fermentation using 4. bath soap. (2005) isolated the thermophilic Bacillus sp. Haq et al.25 % of nutrient broth was added to fermentation medium. Medium consist of 1 % starch showed maximum alpha amylase activity after 60 h of fermentation. detergent powder. hordium. However manganese sulfate. . pearl millet. The enzyme production was enhanced (857 U/ml/min) with the addition of Millon soap at rate 3. zinc sulfate and EDTA showed inhibitory effect on alpha amylase production by Bacillus sp. (2005) reported the use of agricultural starchy substrate for alpha amylase production by Bacillus licheniformis. sodium lauryl ether sulphate or sodium lauryl sulphate) at rate 2. sodium tripolyphosphate. Effects of different carbon sources and chemicals on production of alpha amylase were checked in the laboratory.
casein. Tween 40. respectively. The optimum temperature. malt extract. There were no significant variations in enzyme yield between shake flask and laboratory fermenter cultures. subtilis isolated earlier from cow dung microflora. wheat flour. Optimal alpha amylase activity was observed at 55°C and pH 5.36 Anto et al. potato starch. and sodium lauryl sulphate) at 0.0 ± 1 kDa in native SDS-PAGE.0 ± 1 and 43. (2006) reported the alpha amylase production by B. Maximum alpha amylase activity (94±2) U/g was obtained when wheat bran was used as a substrate. Enzyme secretion was very similar in the presence of any of the carbon sources tested (soluble starch. beef extract) whereas ammonium chloride. Yeast extract and ammonium acetate (1 %) as nitrogen sources gave higher yield compared to other nitrogen sources (peptone. (2006) reported the alpha amylase production by B. (0. cereus MTCC 1305 in solid state fermentation using wheat bran and rice flake manufacturing waste as substrates. glucose. Swain et al. 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. Addition of different nitrogen sources (0. fructose.04 g/g).). ammonium sulfate and urea inhibited the enzyme activity. 5-9 and 36 h. etc. glycine. The purified enzyme was in two forms with molecular mass of 18.02 % resulted in 2-15 % increase in enzyme production. Kathireasan and Manivannan (2006) isolated Penicillium fellutanum from coastal mangrove soil and screened out the sound effects of different variables such . pH and incubation period for amylase production were 5070°C. Tween 80.02 g/g) showed decrease in enzyme production. asparagine. Optimal conditions for alpha amylase production were inoculum size 10 % substrate moisture ratio 1:1 and glucose. cassava starch.
carbon and nitrogen sources in shake flasks fermentation for alpha amylase production. at pH 6. temperature. fellutanum. awamori.5 at pH 4 and . gave maximum alpha amylase production by P. magnesium sulfate. casein. KH2PO4 0. CSL 0. initial pH. niger ATCC 16404 . salinity. potassium dihydrogen phosphate. incubation temperature and inoculum size for the amylase production in submerged fermentation by A. The fermentation medium with no addition of seawater and supplemented with maltose and peptone as carbon and nitrogen source was incubated for 96 h. Considerable enhancement approximately 48% in acid amylase synthesis was observed. Djekrif-Dakhmouche et al.37 as pH. casein 1. (2006) studied the alpha amylase production and optimization from A. 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.5 and temperature 30°C. Calcium chloride at 1 g/l (a structural and stabilizing element for the alpha amylase) solely affect the enzyme production.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.5. The use of other salts (manganese. (2007) reported fractional factorial design of Taguchi methodology for the optimization of medium along with eight variables soluble starch. corn steep liquor. The optimized fermentation medium included in (%) soluble starch 3. 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. Prakasham et al. 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. incubation time.125.
Suganuma et al. The koji molds black-koji and white-koji produce two types of alpha amylase. oryzae was the most important and popular in Japan. and use of response surface methodology (RSM). Among these molds A. such as Japanese sake. Lactose-ammonium sulfate and lactose-peptone extensively affected spore production. tapioca 16. The predicted spore production was 5.8 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. while lactose and ammonium sulfate produced no important linear effect.7. The real experimental results were in concurrence with the prediction. and has been used as yellow-koji in producing many traditional fermented beverages and foods. A five-level four-factor central composite design was used to find out the highest spore yield at optimal level for lactose.38 31°C. namely. Shoji et al. A noteworthy linear major effect was observed in the case of topica and peptone.8 and peptone 8. (2007) reported that highly humid climate of Japan facilitate the growth of various molds. amyloliquefaciens B128 in shake flask cultivation. (2007) investigated the formation of spores from B. ammonium sulfate 1.93 × 108 (no/ml). Both glucoamylase activity 150.7. Fermentation media were optimized by applying two steps approach. (2007) reported a new submerged culture system of A. ammonium sulfate and peptone. tapioca. Optimum conditions for the alpha amylase production were (g/l): lactose 12. and soy sauce. A rapid identification of an appropriate carbon and nitrogen source was obtained by screening experimentation. Rao et al. acid-stable . kawachii NBRC4308 with the help of barley whose surface was wholly or partially covered with husk.
The highest yield (about 19 x 104 U/ml within . 2 % additional starch after 19 h. regarding the distinguishable properties between AA and NA. 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. (2007) conducted the alpha amylase production using amplified variants of B. Tayeb et al. A. A.665 U g–1 dry solid (24% higher than control) in mSSF. niger. in SSF maximum activity was 15. 3 vvm aeration and 300 rpm agitation. High alpha amylase yield of 15. and raw-starch digesting properties. 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. the maximum activity obtained was 19. AA has many advantages in industrial applications. awamori. In this review they investigated AA from three molds.39 (AA) and common neutral (NA). such as its acid-stability. Bhanja et al.5 % initial starch. and the yeast Cryptococcus sp.899 U g–1 dry solid) at 30°C after 66 h of incubation. whereas. subtilis (strain SCH) and of B.The latter enzyme was enzymatically genetically similar to Taka amylase.480 U/ g dry solid in presence of 0. thermostability. (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). With the supplementation of 1 % NaNO3. A. kawachii and A. amyloliquefaciens (strain 267CH) in a bioreactor with multiprotein-mineral media.1 % Triton X-100 (20 % higher than the control).833 U/ g dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6 at 32°C for 54 h whereas alpha amylase production in SSF reached its maximal (12.
the strain AE-19 showed best alpha amylase production. (2008) isolated different strains of actinomycetes and tested these strain for their ability to synthesize the alpha amylase.95°C at 35 % starch. Gupta et al. 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 95°C hence they are classified among thermostable alpha amylases. This strain was identified as Streptomyces aureofasciculus and selected for subsequent studies. 2 % additional starch after 24 h. Maximum consumption of starch and protein occurred during the first day of incubation. 3 vvm aeration. These results indicated that strain can be successfully used for commercial alpha amylase production after testing strain competence in large scale fermentations.05 % sodium chloride at temperature 45°C and pH 9.5 % starch and in the range of 85 . 0. They exhibited broad pH and temperature activity profiles. and 300 rpm agitation with the productivity after 60 h reaching only about 14 x 104 U/ml. The highest alpha amylase production was obtained in the presence of mannose and L-histidine as carbon and nitrogen source.5 % initial starch. Poornima et al. 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 -75°C at 0. Among all the strains. Production occurred in both the logarithmic and post logarithmic phases of growth. 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. (2008) studied the nutritional requirements of A. niger and the factors such as .40 100 h) by strain 267CH was obtained by applying: 2.
Esfahanibolandbalaie et al. 0.0002 % concentration. sodium nitrate as inorganic nitrogen resulted in significant enzyme production. 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. Triton X-100 and Sodium dodecyl sulphate at 0. The maximum activity was obtained at 35°C and 180 rpm. The impact of different temperatures and agitation speed from 20 to 40°C and 50 to 200 rpm.002 and 0.02. 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.3 % peptone. 30°C and 5th day. Among the organic nitrogen sources yeast extract at the level of 2.2.5g /l was excellent nitrogen source. pH of medium. fermentaion period. Carbon and nitrogen source has discernible effect on the enzyme production.41 incubation temperature. (2008) reported the effect of many chemical and physical factors on alpha amylase production by A. Different surfactant at varying level such as Tween-80. The optimal pH. The experimental result showed ideal carbon and nitrogen source for alpha amylase production was 0. surfactants and concentration of metal ions. respectively. respectively was observed. The impact of varying pH of medium ranging from 4-7 was studied.5 % starch and 0. Planchot and Colonna (2008) . temperature and fermentation period were 5. The medium consist of corn starch. The maximum alpha amylase production was obtained at pH 6. oryzae in shake flasks fermentation via an Adlof-Kuhner orbital shaker. carbon and nitrogen sources. respectively exhibited enhanced alpha amylase productivity.
5 mL mg -1 min-1.7 mg mL. It was noted the optimum condition for the alpha amylase production by Humicola . The purified enzyme was glycoprotein in nature which was found to contain 15 % carbohydrate. 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.000.1 and kcat/K m of 3. While intermediate products were maltotriose. wheat straw. and SDS PAGE estimated that purified enzyme possess a molecular weight of 65. Leman et al.42 and 7.. purified enzyme ought to be classified as an alpha-amylase. Singh et al. incubation time inoculum size and carbon source has marked effect on the enzyme production. Although its capability to gradually degrade some α1-6 linkages.7. maltotetraose. and the enzyme showed stability at 40°C. rye. The main end-products of maltohexaose.5. The purified alpha amylase exhibited an isoelectric point of 3. K-27) extracellular alpha amylase to homogeneity by using anion-exchange DEAE-cellulose and affinity α-cyclodextrinSepharose chromatography. 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. hydrolysis were glucose and maltose. (2009) reported alpha amylase from A. fumigatus (Aspergillus sp.42 purified A. A large number of neutral hydrophobic residues were present in an amino acid. with respective Km of 0. The optimum enzyme activity was obtained at pH 5. (2009) investigated the effect of various agricultural by products as a substrate such as wheat bran. and maltopentaose having an αanomeric configuration. It hydrolyzed amylose and amylopectin. straw on the alpha amylase production by Humicola lanuginose in solid state fermentation.4 and 2.
potato dextrose agar (PDA) and enzyme production medium (EPM).5 was best for the highest activity of alpha amylase. incubation temperature 50ºC . (2009) tested the five strains of fungi belonging to two filamentous fungi A. initial moisture content 90 %. flavus for their ability to produce alpha-amylase. flavus gave best result on solid media. 4. initial pH of medium 6.e..5 and 6.5. so these strains were selected for the alpha amylase production in submerged fermentation. Strain 74 and strain 198 of A. size of inoculum 20 % and soluble starch as best carbon source.5. niger and A. 5. All the selected strains showed highest activity of alpha amylase after 48 h in shake flasks.43 lanuginose in SSF was incubation period 144 h. The efficiency or ability of strains was estimated on the basis of the formation of hydrolysis zone.e. The different chosen strains were cultivated on two different typed of media i. . EPM medium at pH 4.. niger and strain 209 and strain 231 of A. Shafique et al. the pH of medium was fixed at 3 level i.
Bread and chapatti industry The quantities. For example. In the past decades. enzymes can be employed to . 1972). A number of these starch-converting enzymes belong to a single family: the alpha amylase family or family13 glycosyl hydrolases. the hydrolysis or formation of glycosidic bonds in the α conformation.44 Uses of alpha amylase Starch is a major storage product of many economically important crops such as wheat. modified starches. rice. maize. Amylases play important role in bakery products (Goodwin and Mercer. Russia and European countries contain alpha amylase.S. these enzymes comprise about 30 % of the world's enzyme production. aroma and porosity of the bread are improved by using the enzyme in the flour. Besides the use in starch hydrolysis. and potato. Currently. starch-converting enzymes are also used in a number of other industrial applications. As many as 21 different reaction and product specificities are found in this family. such as laundry and porcelain detergents or as anti-staling agents in baking. A large-scale starch processing industry has emerged in the last century. we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production of maltodextrin. tapioca. and a number of conserved amino acid residues in the active site. The significance of enzymes is likely to raise as consumers insist more natural products free of chemical additives. For decades. enzymes such as malt and fungal alpha-amylases have been used in bread-making. This group of enzymes share a number of common characteristics such as a (β/α)8 barrel structure. More than 70 % bread in U.A. taste. or glucose and fructose syrups.
45 replace potassium bromate. The dough for bread. which allows yeast to work continuously during dough fermentation. The alpha-amylases degrade the damaged starch in wheat flour into small dextrins.. Many garments especially the ubiquitions‛ Jean ’ are desized after mashing. a chemical additive that has been prohibited in a number of countries. which makes the dough rise. The result is improved bread volume and crumb texture. . Fabrics are sized with starch. salt and possibly other ingredients such as sugar and fat. In addition. The desired fabrics are finally laundered and rinsed (Iqbal et al. Textile industry Textile industries are extensively using alpha amylases to hydrolyze and solubilize the starch. starch. rolls. Allan et al. water. Amylases can degrade starch and produce small dextrins for the yeast to act upon. the yeast starts to work on the fermentable sugars. As soon as the dough is made. 1997).. non-starch polysaccharides. 2007).. yeast. which then wash out of the cloth for increasing the stiffness of the finished products. 1997. Alpha amylase is used as desizing agent for removing starch from the grey cloth before its further processing in bleaching and dyeing. Flour consists of gluten. 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. transforming them into alcohol and carbon dioxide. proofing and the early stage of baking. The major component of wheat flour is starch. lipids and trace amounts of minerals. buns and similar products consists of flour.
potatoes etc. Enzyme hydrolyzed the starch and converted it into glucose. 1998). thus making the process more economically significant. 1996. The high quality products depends upon the efficiency of the enzyme which lead to low production. the starch is first converted in to fermentable sugars. Starches such as grain.. This process also gives the water-soluble dextrin. 1998). 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. 1994. . Therefore. alpha amylase is extensively used in many industries for the production of glucose (Shetty and Crab. In the presence of amylases.4 glucosidic linkage in the starch polymer in a random manner to yield glucose and maltose (Akiba et al.. Alcohol Industry Alpha amylases convert starch in to fermentable sugars. costs for the starch processor has increased (De-cordt et al. Hamilton et al. 1999). Ensari et al. are used as a raw material that helps to manufacture ethyl alcohol. They hydrolyze α-1.. Alpha amylase can also carries out the reactions of alcoholysis by using methanol as a substrate (Santamaria et al. 1990). Many industries used alpha amylases for the production of glucose...46 Sugar and Glucose industries Alpha amylase plays a very important role in the production of starch conversion products of low fermentability. The use of bacterial enzyme partly replaces malt in brewing industry.
1996). Mirasol et al. alpha amylase. It is widely used for improvement of detergency of laundry bleach composition and bleaching with out color darkening (Borchet et al. 1997. starch is extensively used for some paper size press publications (Okolo et al. Chocolate industry Amylases are treated with cocoa slurries to produce chocolate syrup. Alpha amylase hydrolyzed the raw starch that is used for sizing and coating the paper instead of expensive chemically modified starches.. 1997) Modified starch is used in the manufacture of gypsum board for dry wall construction. Many starches or barely material are present in the feed. causes damage to paper as a result of its embrittlement. Detergent.g. Building product and Feed industries In detergent industries. frequently. Starch digesting enzymes. the enzyme alpha amylase plays a vital role. So. Cocoa flavored syrups having a high cocoa content and excellent stability and flow properties at room . So. in immersion or as a gel poultice are applied to facilitate its removal.47 Paper industry Starch paste when used as a mounting adhesive modified with additives such as protein glue or alum. 1995. the nutritional value of the feed can be improved by the addition of alpha amylase. Atsushi and Eiichi. in which chocolate starch is dextrinizing and thus syrup does not become thick. The addition of enzyme stabilizes the bleach agent and preserves effectiveness of the bleach in laundry detergent bar composition (Onzales. e.. 1998).. Enzyme modified the starch for the industry use.
heating to a temperature of about 175 -185°F for at least 10 to 15 min. 1992) . 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.5 to 7.48 temperature may be produced by using an amylolytic enzyme and a sufficient proportion of Dutch process cocoa to provide a syrup pH of 5. 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..5. raising the temperature to about 200° F. and cooling.
. sodium metabisulphate. TEMED.mercaptoethanol. bisacryleamide.1: Isolation of organism: The isolation of seventy eight Aspergillus oryzae cultures from soil samples collected from different habitats such as textile wastes. coomassie brilliant blue R-250. Approximately 0. diammonium sulphate.1N HCl/NaOH. trizmabase. 3. yeast extract.8 by 0. manganese sulphate. After raising the volume to 1000 ml the medium was heated for 10 min to obtain a homogeneous . phenol. etc were of analytical grade and obtained from Sigma (USA). Acros (Belgium) and Fluka (Switzerland). ferrous sulphate. magnesium chloride. Tris HCl. BDH (UK).2. starch. The soil samples were collected in sterile polythene bags. All other chemicals were of the highest possible purity. bromophenol blue.5-dinitro salicylic acid. garden compost etc was carried out by serial dilution method (Clark et al. 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. ferrous sulphate. dihydrogen phosphate. 1958). ammonium per sulphate.49 MATERIALS AND METHODS 3.1: MATERIALS The chemicals used in this study such as sodium potassium tartarate. β. 3.5 ml of this diluted suspension was transferred to the Petri plates containing starch agar medium. 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. acryleamide. E-Merck (Germany). The pH of medium was adjusted to 4. glycine.2: METHODS 3. SDS.
(1986). Sanyo Japan) at 30°C for 3-4 days for culture development. The slants were incubated at 30°C in an incubator for 3-5 days . The initial colonies forming clear zones of starch hydrolysis were picked up and transferred to potato dextrose starch agar slants for culture maintenance. Approximately 10 ml of the medium was poured in separate test tubes.1N NaOH/HCl. the tubes were kept in a slanting position (at an angle of about 30°) to increase the surface area and allowed to solidify.50 mixture. The pH of the medium was adjusted to 5. Germany) Petri plates (at 180ºC for 2 h) and allowed to solidify at room temperature. The conidia of isolated fungi were aseptically transferred to the slants containing potato dextrose starch agar medium. After sterilization. This was cooked for 10-15 min with constant stirring until a clear solution formed. The cultural and morphological characteristics of A. 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. The tubes were cotton plugged and sterilized in autoclave at 15 lbs/in2 pressure (121°C) for 15 min. All the tubes were cotton plugged and sterilized in an autoclave at 15 lbs/in2 pressure (121°C) for 15 min. the contents of each tube were transferred to the oven sterilized (Model: UM-400 MEMMERT.6 by 0. oryzae isolates were identified according to Onion et al. After the addition of soil suspension. Afterwards. The plates were placed in an incubator (Model: MIR-153. the Petri plates were rotated clockwise and counter clockwise for uniform spreading of suspension on the medium. 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. Approximately 6-8 ml of the medium was poured in different test tube.
T was transferred to each slant having profuse conidial growth on its surface.3: Fermentation 3.3.51 for maximum growth. 3.005 % dioctyl ester of sodium sulpho succinic acid (Monoxal O. SANYO Gallenkamp. .XX2. The slants were stored in a refrigerator at 4°C for culture maintenance.0 mm dia. PLC.1.3.C.0 L conical flask followed by the addition of approximately 20-25 glass beads (2.3: Vegetative inoculum Fermentation medium of one hundred milliliter was transferred to a 1.2: Conidial count The numbers of conidia were counted with the help of a Haemacytometer. The flask was cotton plugged and sterilized.3. An inoculating needle was used to break the clumps of conidia.1: Conidial inoculum Conidia from 3-4 day old slant cultures were used for inoculation. 3.3. UK) at 200 rpm for 24 h.6 ×10 6 CFU.1.T). which was then incubated at 30°C on a orbital shaking incubator (Model: 10X400. Each milliliter of the suspension contained 2. Ten milliliter of sterilized Monoxal O. The conidial suspension was prepared in sterilized 0. The test tube was shaken vigorously to make a homogeneous suspension.1.1: Inoculum preparation 3.). 3. The four milliliter of the conidial suspension was transferred aseptically to the flask.
1.0.2H2O 1.2. After 72 h of incubation. (NH4) 2SO4 2.84. CaCl2. Haq et al.75.005. (NH4)2SO4 4. M1: Wheat bran 100.. MgSO4. NaCl 1.7H2O 0.7H2O 0. 0. The flasks were placed in the orbital shaking incubator for incubation at 30°C with shaking speed of 200 rpm. MgSO4. M5: Glucose monohydrate 4.068.12. A one milliliter of inoculum was transferred to each flask. yeast extract 3.. Nandakumar et al. FeSO4 0. M4: Starch 20.. trace metal solution 0. 1986.01. 1. M3: Starch 10.005.7H2O 0.7H2O 3. MgSO4.0.21. M2: Starch 10.7H2O 0. 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.4: Shake flask studies Twenty-five milliliter of fermentation media (M4 optimized) was transferred to separate 250 ml cotton plugged conical flasks.7H2O. FeSO4 0..2H2O 0. . KH2PO4 3. M6: Glucose 50. NaNO3 3. 1999. Cu SO4. phosphate buffer 1000 ml. Spohr et al. All the experiments were run parallel in triplicates.84.2.2: 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. 0. NH4Cl 1. Zn SO4.3. CaCl2.5.12 ml. MgSO4.2H2O 0. 3. MgSO4.52 3.0008.80. KH2PO4 1.7H2O. CaCl2 0. The flasks were sterilized in an autoclave for 15 min and cooled at room temperature. yeast extract 8. phosphate buffer 1000 ml. CaCl2. FeSO4 0. Peptone 20.5. 2002). FeSO4 0.87.3. KCl 0.062.2. 1998.06.
Sterilized solution of 0.1: Fermentation media Fermentation media play a very important role in the alpha amylase production as well as for the growth of organism.6. The air.0 L/L/min (vvm) and 200 rpm.1 N HCl/ NaOH was used for pH adjustment. to be supplied was sterilized by passing through membrane filters (0.45 µm pore size). while the aeration and agitation rates were maintained at 1. The sterilized silicone oil 10 % (v/v) was used to control foam formed during the fermentation process.53 3. The fermenter glass vessel containing 4. USA) with a working volume of 5. Six different media were evaluated for the enzyme production in shake flasks. ALP. Vegetative inoculum was transferred to the vessel through a hole at the top plate under aseptic conditions.2: Incubation period Incubation period has a vital role for the optimal alpha amylase production by .7 L fermentation medium was sterilized in a stainless steel autoclave (Model: KT-40 L.5 L glass fermenter (Model: Bioflow-110 Fermenter/Bioreactor.0 L.6: Nutritional and cultural requirements of Aspergillus oryzae 3. 3.5: Fermenter studies Scale up studies were carried out in a 7. Japan) for 20 min at 15 lbs/in2 pressure (121ºC) and cooled at room temperature. The incubation temperature was kept at 30°C.6. respectively throughout the fermentation period. 3.
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. 3..54 Aspergillus oryzae.5) in fermenter was tested for alpha amylase production. The amount of fermentation medium such as 5. Conidial inoculum at varying concentration (2-12 % (v/v) during shake flasks and vegetative inoculum (5.4: 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. 3. The initial pH.6..6: Effect of inoculum size The size of inoculum is very important for alpha amylase production. 2002). 10. 2007). 3.0-12.6. . agitation intensity. incubation time. temperature. and 25 % (w/v) was evaluated in shake flask fermentation. aeration were maintained constant. 126.96.36.199: Effect of volume The effect of different volume of basal medium on the alpha amylase production by Aspergillus oryzae was investigated. 15. The effect of different temperature (25-50°C) on the biomass formation and production of enzyme was investigated in present study. A range of different pH (4-7) in shake flasks and (4-6. The enzyme fermentation was carried out (8-96 h) at 30°C and sample was collected every 8 h for the estimation of enzyme and dry cell mass in present study.5 % v/v) during fermenter studies was investigated. 3.
3. .6. potassium nitrate etc. xylose.7. 2001). Different additional carbon sources such as sucrose.7: Effect of agitation and aeration Agitation and aeration are interrelated and had direct influence on the alpha amylase production. ammonium sulfate. glucose.55 3.1: Minimal inhibitory concentration of 2-deoxy-D-glucose The parental strain was grown on starch agar medium along with 2-deoxy-D-glucose (0. ammonium nitrate.5 % w/v) at 30ºC in order to find out the minimal inhibitory concentration (MIC).6. urea.9: Evaluation of nitrogen sources Different organic and inorganic nitrogen sources such as peptone. (Azin and Noroozi. 2001). were evaluated for the enzyme production as well as for the growth of organism. yeast extract. beef extract. 3. 3. glycerol.0 vvm was investigated for optimum alpha amylase production. fructose.7: Induction of mutation 3. galactose. meat extract. Different agitation intensity (120-240 rpm) with air supply from 0.5-2. corn steep liquor.8: Evaluation of carbon sources Carbon sources play a vital role for the growth as well as for the alpha amylase production. lactose.0-0. mannitol and CMC were evaluated for the production of alpha amylase by Aspergillus oryzae (Carlsen and Nielsen. sodium nitrate. casein.6.
7. MEMMERT.7.1 M phosphate citrate buffer (pH 7. California. 3.56 3. 2001). N-methyl-Nnitro-N-nitroso guanidine (NG) was transferred to each sterilized centrifuged tube containing 5 ml of conidial suspension and incubated at 30°C using a shaking water bath (Model: WB-14.5). Germany) for specific time interval to achieve a death curve with sub-lethal level. After treatment with NG. UVS-12. The radiation dose given to the mycelial suspension was 1.0 mg/ml. .2×102 J/m2/s. w/v) was added to terminate the reaction. Traces of NG were removed after three appropriate washings with 0. The distance between lamp and suspension was adjusted at 8 cm for each trial to get more than 95 % death rate (Azin and Noroozi.2: Ultraviolet (UV) irradiation From the parental fungal isolate (5 day old culture).3: Nitroso guanidine treatment (NG) NG was prepared in four different concentrations from 0. The treated conidia were resuspended in the same buffer. USA). After fixed time interval. The conidia were treated similarly except replacing NG with sterile distilled water in control experiment. 1 ml of the conidial suspension was transferred to a cotton wool plugged conical flask containing 25 ml of sterilized M1 medium. the tubes were spun at 6000 g for 15 min. The conidia were allowed to grow at 30°C on a shaking incubator with 200 rpm for about 6 h to get fresh growing fungal mycelia. The supernatant was discarded to remove NG from the fungal cells.5-2. 1 ml of cystein (1 %. 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 (λ=253 nm and 220 V at 50 c/s) of UV lamp (Model: Mineral Light.
3M solution of NaNO2 prepared in acetate buffer (0.57 3. After fixed time interval. After specific time intervals the conidia were spun and washed thrice in phosphate buffer. 2.1) to stop the reaction.5: EMS treatment Different concentrations (25-150 µl) of ethyl methane sulphonate (EMS) were added to individual centrifuge tubes containing 4 ml of conidial suspension and shaken to form a homogeneous suspension. about 100 µl of each suspension containing treated conidia was aseptically transferred to the individual Petri plates containing starch agar medium supplemented with (g/L). oryzae (Carlton and Brown. the tubes were spun at 6000 g for 15 min. 1981). Triton X-100 (5.7.4: Nitrous acid treatment A 0.7. A control was run similarly except replacing NaNO2 in acetate buffer with sterilized saline water.6: Selection of mutants After treatment with mutagenic agents. After washing the conidia were resuspended in same buffer.07-0.7.0). 3. pH 4. pH 7. The supernatant was discarded to remove nitrous acid from the fungal conidia and ten milliliter of sterilized phosphate buffer was added to each tube.2 M.5) was added to washed and spun conidia of A. 3. The tubes were respun for the removal of traces of nitrous acid from conidia and repeated three times. The EMS treated conidia was resuspended in same buffer.2 M. The one milliliter solution was withdrawn and diluted 5 fold in phosphate buffer (0.deoxy-D-glucose (above the MIC of parent strain). The solution was shaken thoroughly for specific time intervals. The plates were incubated at 30ºC and were .
1 ml of the filtrate with 5 ml of Bradford reagent in a test tube and vortexes thoroughly.2: Estimation of total protein contents Total protein contents were determined by taking 0.1 ml of distilled water with 5 ml of the Bradford reagent was run parallel. 1959) using maltose as a standard.” The enzyme activity was determined by taking 1 ml of diluted filtrate in a test tube. After incubation of 10 min at 40°C. These colonies were then tested quantitatively for enzyme production in shake flasks fermentation. 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. 3. The one milliliter of starch solution (1 % w/v) was also added into it. the fermented broth was filtered.58 examined regularly after 3-4 day to study the growth pattern. The filtrate was used for the estimation of total protein contents. 3.8.8: Analytical techniques After incubation. A blank containing 0.1: Estimation of alpha amylase The estimation of alpha amylase was carried out according to the method of Rick and Stegbauer (1974). the reducing sugar liberated was measured at 546 nm by the DNS method (Miller. “One unit of activity was that amount of enzyme. which in 10 min liberates reducing group from 1 % Lintner‘s soluble starch corresponding to 1mg of maltose hydrate. A blank was run parallel by replacing the filtrate with 1 ml of distilled water. The absorbance was taken at 595 nm on a double beam UV/VIS scanning spectrophotometer (Model: CE- . and alpha amylase activity. 3.8.
59 7200.3: Determination of mycelial morphology Mycelial morphology was determined on an aliquot extended on the Petri plates followed by pellet diameter (Moreira et al.05) values.8.8. 3. CECIL. between 2-3 as intermediate pellets while those above 3 mm were referred to as large pellets. 44. The following parameters of kinetics were studied:- . Significance has been presented in the form of probability (p<0.4: Estimation of dry cell mass (DCM) Dry cell mass was determine by filtering the culture broth through preweighed Whatman filter paper No. 3.. The dry cell mass was weighed and calculated as g/l by subtracting the initial weight from the final weight. 3. they were categorized as fine pellets. UK) after 15 min of the reaction using bovine serum albumin (BSA) as a standard. if the diameter was less than 0. England. 1996). 3.9: Statistical analysis Treatment effects were compared by the method of Snedecor and Cochran (1980).5-2 mm as small pellets. Protein contents were determined from the standard curve of BSA (Bradford 1976). For rounded pellets. between 0.10: Kinetic study Kinetic parameters for batch fermentation were determined according to the method describe by Pirt (1975) and Lawford and Rouseau (1993). Mycelia were thoroughly washed with tap water and dry in oven at 105°C for 2 h. Post-Hoc Multiple comparison tests were applied under one-way ANOVA.5 mm.
11: Purification of alpha amylase 3.11. The clear supernatant was used for enzyme isolation and purification.1: Separation of fungus from fermented broth The fermented broth was spun at 9.e. 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 =µ × Y p/x 3. 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.000 g for 15 min at 4oC. .60 Specific growth rate The value of specific growth rate i. µ (h-1) was calculated from plot of In (x) vs time of fermentation..
After cooling poured it into the column and made final bed volume (1. USA) was swollen in 100 milliliter of the 0. The fractions containing enzyme activity were pooled.5 in a boiling water bath for 2 h.11.2. The pelleted precipitate of each fraction was resuspended in 0.5 in a boiling water bath for 2 h. 3.exchange chromatography For the purpose of ion exchange chromatography.5. The solution was dialyzed against the same buffer.11.5 ml/min.11. Fractions of 3 ml were collected at a flow rate 0. To the supernatant. pH 7. Poured the gel slurry along the side . 0. pH 7. pH 7.05 M Tris-HCl buffer.05 M Tris-HCl buffer.4 g DEAE-Sephadex A-50 (Sigma. The collected fractions were assayed for protein at 280 nm and alpha amylase activity by performing enzyme assay. 2 g was swollen in 50 ml of 0. The suspension was stirred for half an hour at 4ºC.2: Gel filtration Sephadex G-100 (Phamacia Fine Chemical).2.5 × 10 cm). pH 7. After sufficient shaking. 3.2: Ammonium sulfate precipitation Solid ammonium sulfate was added to 1000 milliliter of crude cell free broth of alpha amylase.1 Anion. Same procedure was applied to get the precipitates of all fractions.05 M Tris-HCl buffer.05 M TrisHCl buffer. the precipitates were collected by spining at 14.000 g for 20 min at 4ºC. dialyzed and analyzed on SDS-PAGE.5. added calculated amount of ammonium sulfate for 20-90 % (w/v) saturation. A stepwise NaCl gradient from 0 to 1 M in 150 ml of the same buffer was applied. The dialyzed enzyme solution was applied to column that preequilibrated with five column volumes of the 0.61 3.
0. pH 7.5 M Tris HCl (pH 8.4: 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.polyacrylamide gel with protein marker (SMO 313).0 cm) was equilibrated with five column volumes of the 0. 0.1 ml SDS (10 % w/v).05 M Tris-HCl buffer. The collected fractions were assayed for protein and alpha amylase activity.3: Dialysis The salts were removed by dialyzing precipitates and pooled samples by using 12.05 M Tris-HCl buffer (pH 7. The process was repeated 4-5 times until all salts were removed from the enzyme solution. The column (1.11.5 ml/min.12: Gel preparation 3.5 ml.11.000 molecular weight cut off dialyzing bag.1 ml ammonium per . 1.12.62 of tilted column by taking care that no air bubble was entrapped.5 in order to stabilize the bed.5: Protein marker The molecular weight of the alpha amylase was estimated by SDS. adjusting flow rate at 0. The active enzyme fractions were pooled and used for the determination of main characteristics of the enzyme.5 × 15. 3. 2.1: Separating gel: The separating gel (12 % w/v) was prepared by adding 4 ml acrylamide (30 % w/v).5) for 3-4 h at 4ºC.8).11. The enzyme sample (5 ml) was eluted with the same buffer. which was placed in one liter of the 0. 3. 3.
gel comb was taken out and valves were washed with tank buffer four times by means of a syringe.03 ml. 2.12. When complete polymerization took place.13: Characterization of enzyme Temperature and pH had great influence on the activity of alpha amylase. 0.5 ml acrylamide (30 % w/v). 0.38 ml 1M Tris HCl (pH 6.0003 ml TEMED.1 ml Distilled water. 6 µl TEMED . The water was removed from top of the separating gel and stacking gel was poured in the gel assembly. 0. 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. 3.63 sulfate(10 % w/v). 3. 3.8). The enzyme substrate complex was incubated at varying temperatures (25-70°C) and pH (3-7) and the effect on the activity of purified enzyme was observed. 0. 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.004 ml APS (10 % w/v).2 Stacking gel: The stacking gel was prepared by adding 0. SDS 10 % (w/v). The gel was allowed to polymerize for 30 min. The enzyme solution (6 µl) and loading buffer (4 µl) were denatured by heating in boiling water bath for 3 min. Metal ions had marked . Comb was inserted and gel was allowed to polymerize at the room temperature for 10 min. Almost 100 µl of distilled water was layered at the top of the gel to give a flat surface and to remove oxygen which inhibited polymerization.3 ml distilled water and poured in the gel assembly leaving one inch vacant space at the top.
and FeSO4 on the activity of alpha amylase was also studied. CuCl2. NaCl.64 influence on the activity of enzyme so effect of different metal ions such as Mg SO4.1: 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.1 to 1 mg/ml. Absorbance was measured at 546 nm using a spectrophotometer (Model: CECIL CE-7200 Aquarius. The stock solution (1 mg/ml) was used to make 10 appropriate dilutions ranging from 100-1000 µl/ml. The stock solution (1 mg/ml) was used to make 10 appropriate dilutions ranging from 0.1 µl) was taken in a separate test tube followed by the addition of 5 ml of BSA reagent.14. The one milliliter of each dilution was taken in separate test tubes followed by the addition of 2 ml of DNS reagent. COCl2. The tubes were incubated in a boiling water bath for 5 min prior to cooling at room temperature. A blank was also run in parallel by replacing BSA with distilled water. 3. The final volume was raised up to 100 ml using a measuring flask.2: Bovine serum albumin (BSA) BSA (100 mg) was dissolved in approximately 90 ml of distilled water. A graph was plotted taking the absorbance at the ordinate and sugar concentration at the abscissa (Fig 1).14: Standard curves 3. NiCl2.14. 3. UK). A blank was run in parallel replacing the maltose dilution with 1 ml of distilled water. Zn Cl2. 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 . Each dilution (0. MnSO4.
3.0 L with distilled water.15. The curve was plotted by taking BSA concentration along x-axis and optical density along y-axis (Fig 2). The reagent was stored in an amber colored bottle to avoid photooxidation. UK). 3. 3. After dissolving the chemicals. It was stable for about six months. 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. phenol melted at 60°C (7. . This solution was poured into 100 ml of 85 % (w/v) phosphoric acid and the final volume was raised up to 1. After shaking filtered through Whatman filter paper (No.65 Aquarius.15. Sodium potassium tartrate (306 g).3 g) were also added. final volume was raised up to 1416 ml with distilled water.3: Starch Solution: The Lintner’s soluble starch 1g was dissolved in 100 ml of acetate buffer (pH 5) and boiled until solution become transparent.15.1: Dinitrosalicylic acid (DNS) reagent Dinitrosalicylic acid (10.15: Preparation of reagents/buffers 3. 1) to obtain a clear solution.5 ml) and sodium metabisulfate (8.2: Bradford reagent The coomassie brilliant blue hundred milligrams (G-250) was added in 50 ml of 95 % (v/v) ethanol.5 g) were dissolved in approximately 600-800 ml of distilled water and gently heated in a water bath at 80°C until a clear solution was obtained.6 g) and sodium hydroxide (19.
Finally volume was raised to 1000 ml with distilled water. 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.66 3. 3.6: Preparation of 0. The solution was filtered and stored at 4оC.5.1 M) in 700-800 ml distilled water and finally volume was raised to 1000 ml to get 0.06 ml) was added in approximately 900 ml distilled water and final volume was raised up to 1 L Sol B: Sodium acetate (27.25 g of Tris in 700-800 ml of distilled water and adjusted pH 7.5 with 5 N HCl with constant stirring.5 g citric acid (0.15.15.15.05 M Tris-HCl buffer (pH 7. 3.4: Acetate Buffer (pH 5. 3.5: Phosphate citrate buffer (pH 7. .15.5 g Na2HPO4 and 77.5) The Tris HCl buffer was prepared by dissolving 6.7: 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.5) The buffer was prepared by dissolving 922.1 M phosphate citrate buffer having pH 7.0) Sol A: Acetic acid (12.22 g) was dissolved in approximately 900 ml distilled water and final volume was raised up to 1 L.
8 by adding concentrated HCl (32 % v/v) drop wise.5 M Tris HCl.8) The 1 M Tris HCl.15.3) The Tank buffer was prepared by dissolving 3. 2 g bromophenol blue dye.15. buffer was prepared by dissolving 157. 3. 3.15. The solution was stirred and final volume was made up to 1000 ml. 3. 1500 µl β-mercaptoethanol and raised the volume up to 1000 ml and stored at 4оC.8: Separating buffer (1. After pH adjustment raised the final volume upto 1000 ml with distilled water. 3.6 g Trizma base in 900 ml of distilled water with constant stirring to adjust the pH at 6.94 g of Trizma base.5 M Tris HCl buffer was prepared by dissolving 181.41 g of glycine and 50 g of SDS in distilled water and raising the final volume up to 1000 ml.9: Stacking buffer (1 M Tris HCl.8) 400g SDS.8) The 1.12: SDS solution (10 % w/v) The 10 % w/v SDS solution was prepared by dissolving 100 g of SDS in hot water. After pH adjustment. pH 8.67 3. the final volume was raised up to 1000 ml with distilled water. pH 6. pH 8.15. The solution was stored at 4оC. 200 ml glycerol.11: Gel loading buffer The Gel loading buffer was prepared by mixing 1ml of Tris HCl (pH 6.8 by adding concentrated HCl (32 % v/v) drop wise.15.5 g Trizma in 900 ml of distilled water with constant stirring to adjust the pH 8.10: Tank buffer (10 X. 14. .
14: Staining and destaining solution Staining solution was prepared by dissolving 2.13: Ammonium per sulfate The Ammonium per sulfate solution was prepared by dissolving 0. 100 ml acetic acid and volume was raised up to1000 ml with distilled water.68 188.8.131.52 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. Freshly prepared solution of APS was used.1 g of APS in distilled water and raised the final volume up to 1 ml. 3. . Destaining solution was prepared by mixing 300 ml methanol.
8 0.69 Fig 3.7 0.2 0.9 1.1 0.3 0.0 0.1: Standard curve of maltose 1.6 0.1 1.2 0.2 1.4 0.0 0.0 1.8 Absorbance 0. (mg/ml) .6 0.4 0.0 0.2 Maltose conc.5 0.
70 Fig 3.3 0.1 0.6 0.5 0.7 BSA (ug/ml) .2 0.4 0.0 0.2: Standard curve of bovine serum albumin (BSA) 0.4 0.0 0.3 Absorbance 0.2 0.1 0.
green.71 RESULTS 4.5-8. by serial dilution method on the plates containing starch agar medium. Metulae 8-12 x 4-5 µm.1). The strains were identified according to Onion et al. This strain was selected for subsequent studies. Conidiogenous cells uniseriate and biseriate. Colonies of A. Colonies consisting of long conidiophores often intermixed with aerial mycelium. 4. (1986). oryzae were greenish yellow. up to 4-5 mm in length. ISOLATION AND SCREENING OF ORGANISM Strains of Aspergillus oryzae were isolated for the of alpha amylase production from different soil samples. typically with dull brown shades with age. olive yellow or with different shades of green. Conidiophores were hyaline. Phialides often directly borne on the vesicle or on metulae. Screening of A. 30 gave maximum enzyme production and assigned the code IIB-30. Vesicles were subglobose.1.0 µm in diameter.1: IDENTIFICATION. The seventy eight isolates were screened out for their enzyme synthesizing ability (Table 4. Of all the isolates examined. smooth to finely rough walled. the isolate No. Conidia ellipsoidal when young. oryzae isolates for alpha amylase production was carried out in shake flasks. mostly rough-walled. usually measuring 10-15 x 3-5 µm. The range of enzyme activity of the wild isolates is given in Table 4.1. Conidial heads radiate greenish yellow later becoming light to dull brown. . 40-80 µm in diameter. globose to subglobose when mature. Metulae or phialides covering the entire surface or the upper three-fourths of the vesicle.
9±0.3 60±0.3 14±0.8±0.1 69±0.1 5.3l 4.2 7.15 11±0.2 33±0.1 53±0.1 50±0.2 2.9±0.2 41±0.3 87±0.0±0.2 4.1±0.4 111±0.1 2.3±0.1 29±0.0±0.1 45±0.3±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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Enzyme activity(U/ml) 103±0.1 .7±0.0±0.2 1.7±0.0±0.2±0.3±0.3l 3.9±0.9±0.1 20±0.1 1.5 6.1±0.1 3.1 9.2 118±0.1 13±0.1: Isolation and screening of Aspergillus oryzae for the alpha amylase production Isolate No.72 Table 4.9±0.1 2.1 130±0.1 13±0.3 1.2 17±0.1 40±0.5 90±0.1 3.1 9.1 7.3±0.1 Dry cell mass (g/l) 9.5±0.2 7.3±0.5±0.1 4.2 10.1 26±0.2 7.7±0.3 2.1 1.2 111±0.1 100±0.2 12±0.5±0.1±0.5±0.2 83±0.1.4 70±0.5±0.1 5.1 3.3±0.3 2.1 2.1 5.1 35±0.0±0.1 57±0.5±0.0±0.3 109±0.3 3.0±0.0±0.2 91±0.2 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 .0±0.1 10.1 8.1 3.3 7.1±0.1 2.1 73±0.2 2.75 81±0.5±0.5±0.0±0.9±0.2 6.6±0.2 60±0.0±0.1 6.0±0.1 5.2 96±0.1 7.7 4.1 120±0.
2 5.1 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 0.2 1.1 1.1 9.11±0.1±0.1 2.3±0.1 2.8±0.2 47±0.2±0.1 5.0±0.73 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 97±0.2 110±0.4 13±0.1 2.2±0.1 102±0.4 6.3 114±0.1 9.1 11.1 5. agitation rate 160 rpm .5 16±0.3±0.05 61±0.05 5.3±0.8±0.5 77±0.1 87±0.1 6.0±0.2 5.5 4.1 11±0.1 7.2 20±0.0±0.4 1.2 7.5 108±0.2 44±0.5 112±0.2 2.5 39±0.0±0.5 58±0.5 6.9±0.2 6.0±0.1 39±0.15 30±0.1 93±0.9±0.4 3.1 51±0.0±0.2±0. incubation temperature 30°C.1 5.2 6.1 21±0.2 27±0.1 2.0±0.2 2.1 7. Incubation time 72 h.05.5±0.1 59±0.6±0.7±0.2 63±0.9±0.2 98±0.2 7. pH 6.9±0.9±0.2 15±0.0±0.3±0.5±0.1±0.0±0.1 4.2 99±0.1 5.0±0.3±0.9±0.3±0.4 10±0. 3.1 9.5 64±0. ± indicates the standard deviation (SD) among the three parallel replicates in each column.5±0.0±0.3 6.1 6.2 78±0.1 1.1 79±0.7±0.1 115±0.5 2.1 4.1 5.5 3.0.9±0.5±0.2 2.5 8.1 63±0.2 3.7±0.9±0.1 83±0.7±0.0±0.
1. .1: Sub grouping of alpha amylase producing isolates of A. oryzae IIB-30 this culture was selected for mutation through UV radiations.74 Table 4. 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.
.2. oryzae IIB-30 with different doses of UV light (15-75 min) in order to increase the alpha amylase production.75 184.108.40.206. The number of survivors and range of enzyme production of the UV treated isolates is given in Table 4.2) shows the screening of UV treated isolates for the enzyme production.5 cm gave maximum enzyme production (160±2 U/ml).2. Of all the isolates tested.2. The data of Table (4. The mutant strain gave maximum production was assigned the code UV-23 and selected for further studies.1: PHYSICAL MUTAGENESIS 4. the strain isolated after 45 min of UV irradiation with a zone diameter of 1.1: SCREENING OF UV TREATED ISOLATES The different isolates were obtained by irradiating the conidia of A. respectively.2: STRAIN IMPROVEMENT 4.1 and 4. A total of 32 strains were isolated by observing bigger zones of starch hydrolysis in Petri plate compared to parental strain.
1 90±0.9 87±0.2 53±0.1 12.8±0.2±0.1±0.1 11.7 126±1.60±0.1 10.8±0.1 73±0.0 93±0.1 109±0.0±0.4 100±1. incubation temperature 30°C.0±0.1 13.1 12.50±0.3 11.4 113±0.8±0.2 10.2±0.3±0.0 28±0. agitation rate 160 rpm .7±0.1 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 0.2 160±2.6 115±1.4 82±0.1±0.0 132±1.1 9.8±0.5 93±0.1 10.1 11.2 10.5±0.1 9.60±0.2: Screening of UV isolates of A.1 13.2±0.2 12.1 10.7±0.2±0.2 13.3 70±0. ± indicates the standard deviation (SD) among the three parallel replicates in each column.7±0.1 8.3 14.1 11.0 90±0.0±0.1±0.1 11.2 43±0.2 9.4 5.3±0.6 63±0.2 101±0.0±0.0±0.5 80±0.1 12.3 78±0.5±0.8 117±1.2 99±1.1 39±0. 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) 120±1.20±0.5 140±1.1 Dry cell mass (g/l) 12.1±0.1 7.2 13.2 13. pH 6.3 7.3±0.9 129±1.60±0.0.2 11.7±0.7 136±1.76 Table 4.9±0.1 6.0 106±0.8 76±0.3 123±1.05.2 10.2 10. Incubation time 72 h.
1: UV treated survivors at different exposure time Exposure time 15 30 45 60 75 Total number of survivors 38 25 17 13 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-23.2.77 Table 4.0 Table 4.2. This strain was selected for mutation through NG. .
78 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. A total of 18 strains were isolated by observing bigger zones of starch hydrolysis in Petri plate compared to parental strain.0 mg/ml).3. the number of survivors decreased as shown in the Table 4.2. The A.3) shows the screening of NG treated isolates for the enzyme production. respectively.5-2.3. The mutant strain exhibiting highest enzyme production assigns the code NG-15 and selected for further studies.0 mg/ml concentration of NG 90 % death rate was observed further increase in concentration cause complete death of organism. 4.2: SCREENING OF NITROUS ACID TREATED ISOLATES The NG mutant strain A.1.1 and 4.9 cm gave maximum (270±0. oryzae UV-23 was exposed to different concentrations of NG (0. At 2.3: CHEMICAL MUTAGENESIS 4.4 M). oryzae NG-15 were further treated with different concentrations of nitrous acid (0.1 U/ml) enzyme production which is two fold than UV-23.3. When the concentration of NG increased. the strains isolated after treatment with 1. Of all the isolates tested. The number of survivors and range of enzyme production by NG treated isolates is given in table 4. Selective NA treated strains were isolated on the basis of qualitative screening showing bigger zones of starch hydrolysis than the parental strain.3.5 mg/ml NG with a zone diameter of 1.1-0. The data of Table (4. The NA treated strains were screened for enzyme production .
3: SCREENING OF EMS TREATD ISOLATES The nitrous acid treated mutant NA-17 was subjected to the varying concentration (25150 µl/ml) of ethyl methane sulphonate (EMS).A total of twenty-six EMS treated A. Out of which mutant strain NA-17 gave maximum alpha amylase production (285±0.1. which were screened for alpha amylase production (Table 4.2. the number of survivors was decreased (Table 4. oryzae strains were obtained at 90 % death rate. 4. As the concentration of EMS was increased.5-fold higher enzyme yield (347±1. The mutant EMS-18 showed 1.1.5).5.1 U/ml).) The range of enzyme production of EMS treated isolates is given in Table 4.5.2 .4.2U/ml) compared to NA-17. When the concentration of nitrous acid was increased. Range of enzyme production of the nitrous acid treated isolates is given in Table 4.79 (Table 4.4.4).3. the number of the survivors was decreased as shown in the Table 4.
1 10.2 3.2 11.05.2 12.6±0.3 83±0.80 Table 4.4±0.1 10.5 2.9±0. pH 6.6±0.4 103±0.1 87±0.9±0.1 12.3±0.2 15.3 150±0.7±0. temperature 30°C.6±0.3 49±0.4 34±0.1 220±0.60±0.1 15.0 1.2 109±0. agitation rate 160 rpm . 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) 0.0±1 11.3: Screening of NG treated A.60±0.9±0.1 11.0 145±0.2±0.8±0.20±0.5±0.4 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 0.5±0. *Incubation time 72 h. ± indicates the standard deviation (SD) among the three parallel replicates in each column.3 8.6 123±0.2 201±1.8 136±0.1 6.2 111±0.3 12.1 14.1 96±0.3 11.30±0.0 Enzyme activity(U/ml) 160±0.1 Dry cell mass(g/l) 14.0.5 1.4 5.5 270±0.1 63±0.5 132±0.2 13.
3. oryzae NG concentration(mg/ml) 0.81 Table 4.1: NG treated survivors of A. .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.0 Table 4.5 1.0 Total number of survivors 31 20 11 2.3.5 2.0 1.
60±0.8±0.3 14.2 0.0±0.2 14.3 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 0.1 14. of nitrous acid(M) 0.7±0.1 15.3 12.5 250±0.1 Enzyme activity(U/ml) 261±0. agitation rate 160 rpm .4 285±0.0.1 168±0.5 153±0.1 39±0.6 247±0.3 266±0.7±0. *Incubation time 72 h.2 248±0.4: Screening of nitrous acid treated strains of A.9±0.1±0.2 6.1 Dry cell mass(g/l) 15.1 14.2±0.1 13.2 3.7 259±0.3 259±0.2 10.6±0. ± indicates the standard deviation (SD) among the three parallel replicates in each column.1 16.1 218±0.5 196±0.3±0.9±0.1 2.4 11.9±0.3±0.05.1 9.9 13.82 Table 4. 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.6 256±0.6±0.3 0.0±0.4 The mean difference is significant at the level of 0.3 13.9±0.0.80±0.0±0.2 209±0. temperature 30°C.3 13.4 58±0.1 258±0.8 13.1 11.7 262±0.2 256±0.05 11. pH 6.7 109±0.6 230±0.8±0.90±0.80±0.
83 Table 4.2 0.1: Nitrous acid treated survivors of A.4.3 0. .This strain was further treated with EMS.4. oryzae Nitrous acid concentrations (M) 0.1 0.4 Total number of survivors 39 32 27 24 Table 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.
2 93±0.4 300±0. temperature 30°C.8±1 13. *Incubation time 72h.5 16.8 87±1 118±0.9±1 7.2 14.6±0.3±1 15.1 287±0.2 14.2 10.2 275±0.1 15.0 273±0.4 101±1 208±0.05 290±0.(µl/ml) 25 Enzyme activity (U/ml) 255±0.3 197±0.2 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 0.1±0.1 15.1±0.20±0.7±0.1 11.1 106±0.7 187±0.1 147±0.4±0. 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.1 198±0.3 230±0.3 10.2±0.1±0.2 100±0.3±0.8±0.1 201±0.3 15.1 12.6±0. pH 6.3 126±0.2 250±0.2±0.2±0.1±0.05.4 14.1 12.5: Screening of EMS treated A.6±0.1 47.2 Dry cell mass(g/l) 14.9±0.1 12.4 11.3±0.6±0.2 5.2±0.6±0. agitation rate 160rpm .5 347±0.7±0.1 14.1 5.9±0.2 2.1 13.2 12. ± indicates the standard deviation (SD) among the three parallel replicates.1 12.1 13.5 168±0.2 200±1.1 240±0.0±0.3±0.0.84 Table 4.
5.1: EMS treated survivors of A.85 Table 4. This strain was selected for optimization .5.2: Range of α-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. oryzae EMS concentrations (µl) 25 50 75 100 125 150 Total number of survivors 45 32 21 5 3 2 Table 4.
86 4. The dry cell mass was 14.2). So.4 g/l.8±2.05) after 72 h. 4. respectively. . The fermentation was carried out for 96 h and enzyme production was calculated after every 8 h. Of all the media tested.05) in M4 medium so.1). it was selected in subsequent studies. The enzyme production was found to be highly significant (p≤0. oryzae (Fig 4. The dry cell mass was 14.8±2. oryzae was optimized (Fig 4.4 g/l.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. 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 (168±2 U/ml) and mutant (386±2 U/ml) strains. M 4 medium gave highest units of alpha amylase 168±2 (wild) and 385±2 (mutant).9±2 and 16. The enzyme production was highly significant (p≤0. The rest of the fermentation media gave not significant enzyme production for both strains as compared to M4 medium.4: OPTIMIZATION OF CULTURAL CONDITIONS IN SHAKE FLASKS 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.4. respectively. it was selected in subsequent studies. Further increase in incubation period did not show any increase in the formation of enzyme rather it was decreased.9±2 and 16.
20 and 25 % (v/v) in 250 ml Erlenmeyer flask on alpha amylase production by both the wild and mutant strains of A.3: EFFECT OF INCUBATION TEMPERATURE Figure 4. At 50°C.05) as compared to other temperatures. At alkaline pH. respectively. Both the wild (166±2 U/ml) and mutant (386±3 U/ml) strains gave maximum enzyme production at 30°C.4). oryzae. there was gradual reduction in the enzyme formation by both wild and mutant strains of A. As the pH of the medium was increased from 5. 4. The initial pH of the fermentation medium was adjusted at 4-7 in shake flasks. the enzyme production was extremely low.4.2±1. oryzae.4. the enzyme production was reduced.3 shows the effect of varying incubation temperature (25-50°C) on the alpha amylase production by both wild and mutant strains of A. The dry cell mass was 14.5 depicted the effect of different volumes of the basal medium such as 5. When temperature of medium was increased from 30°C.40 g/l. oryzae. 15. The enzyme production following growth of organism by both wild and mutant strains was found to be significant (p≤0.4: EFFECT OF DIFFERENT INITIAL pH The effect of different initial pH of the fermentation medium on the alpha amylase production was investigated (Fig 4. 10. Thus. the enzyme production became not significant (p≤0. 4.8±2.87 4.0 and17.4.05) at pH 5. The maximum enzyme production by .5: EFECT OF DIFFERENT VOLUMES OF MEDIUM Figure 4. pH 5 was selected for of alpha amylase production.
0 and 17.6Χ106CFU) yielded maximum enzyme production both by wild (191±1.04) and mutant (418±3) strains. it was optimized for maximum enzyme production.05) at 4 % level hence. 4. The enzyme production was significant at 10 % volume of fermentation medium so it was selected for further studies.88 wild (189±1) and mutant (416±3) strains were observed when 10 % volume (25 ml/250 ml flask) was used. respectively.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.6). The enzyme production was found to be significant (p≤0. As the volume of fermentation medium was increased. The inoculum size of 4 % v/v (1ml=2. Beyond this level the enzyme production was decreased gradually. The dry cell mass was 14. . the enzyme production was decreased gradually.0 g/l.7±1.9±2. oryzae was evaluated (Fig 4.4.
05. pH 6. Y error bars indicate the standard error from mean value.1: Screening of fermentation media for the alpha amylase production by A. The values vary significantly at p≤ 0.89 Fig 4. agitation rate 160 rpm .0. 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) 0 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. *Incubation time 72 h. oryzae IIB-30 and its mutant derivative A. temperature 30°C.
pH 6. agitation rate 160 rpm .2: Rate of fermentation for the alpha amylase production by A. Y error bars indicate the standard error from mean value. 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.0.05. oryzae IIB-30 and its mutant derivative A.90 Fig 4. *Incubation temperature 30°C. The values vary significantly at p≤ 0.
3: Effect of incubation temperature on the alpha amylase production by A. incubation temperature 30°C.91 Fig 4. pH 6. Y error bars indicate the standard error from mean value.05. The values vary significantly at p≤ 0. *Incubation time 72 h. agitation rate 160 rpm . oryzae IIB-30 and its mutant derivative A.0. oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 25 30 35 40 45 50 Temperature°C Enzyme activity of w ild strain IIB-30 (U/ml) DCM of w ild strain IIB-30 (g/l) 0 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.
oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 4 4.5 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. oryzae IIB-30 and its mutant derivative A. The values vary significantly at p≤0.92 Fig 4.4: Effect of different initial pH of fermentation medium on the of alpha amylase production by A. *Incubation time 72 h. Y error bars indicate the standard error from mean value.5 pH Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) 0 6 6. agitation rate 160 rpm. . incubation temperature 30°C.05.5 5 5.
05. incubation temperature 30°C. oryzae IIB-30 and its mutant derivative A. agitation rate 160 rpm .93 Fig 4. Y error bars indicate the standard error from mean value. *Incubation time 72 h. The values vary significantly at p≤ 0.5: Effect of different volumes of media on the alpha amylase production by A. oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 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.
0. oryzae EMS-18 * 1000 30 800 Enzyme activity (U/ml) 25 20 DCM (g/l) 600 15 400 10 200 5 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. *Incubation time 72 h. oryzae IIB-30 and its mutant derivative A.05. The values vary significantly at p≤ 0. pH 5. Y error bars indicate the standard error from mean value. agitation rate160 rpm . incubation temperature 30°C.94 Fig 4.6: Effect of different inoculum sizes on the alpha amylase production by A.
0) strains.5: OPTIMIZATION OF NUTRITIONAL REQUIREMENTS OF A.3: EVALUATION OF ADDITIONAL CARBON SOURCES The effect of different carbon sources such as glucose. Beyond this concentration the enzyme production was decreased.95 4.7 depicted the effect of starch from different sources such as wheat.1±1. 4.5. respectively.2: EFFECT OF DIFFERENT CONCENTRATIONS OF CORN STRACH The effect of different concentration of starch (1.05 as compared to corn starch. corn.3±1. galatose.8±2. rice and sweet potato at the concentration of 1 % on the alpha amylase production by both wild and mutant strains of A.0 % w/v) was investigated for the alpha amylase production by both the wild and mutant strain of A.0) and mutant (419±3.0 g/l.5. Of all the starches tested.1: EFFECT OF STARCH FROM DIFFERENT SOURCES Figure 4. lactose.0 and 16.04) and mutant (436±2. oryzae (Fig 4. fructose. oryzae. ORYZAE IN SHAKE FLASKS 4. glycerol and mannitol was evaluated for the alpha amylase production by wild and mutant strains of A.5. caboxy methyl cellulose.9 g/l.0-5. All other starches gave less significant results (p≤0. Therefore corn starch was selected for subsequent studies. respectively. it was selected for further studies.6±2. The enzyme production was significant (p≤0.0 and 18. corn starch gave maximum enzyme production by both the wild (190±2. xylose. The dry cell mass was 14. 4. (w/v) The dry cell mass was 15. oryzae in present course . The maximum enzyme production by both strains wild (201±1.0) was obtained at the level of 2 %.8). sucrose.05) at 2 % (w/v) so.
6± 2 g/l. respectively. NaNO3 and KNO3 was evaluated for the alpha amylase production by the wild and mutant strains of A.9). The dry cell mass was 16.05) as compared to other concentrations.(w/v). The (NH4)2SO4 at the concentration of 0.12). Therefore.5 %. 4. lactose as additional carbon source was selected and its various concentrations were also tested for the enzyme production (Fig 4. Further increase in the concentration of lactose resulted decrease in the enzyme production. The dry cell mass was 15.0) compared to others (Fig 4. oryzae (Fig 4.11).9±1 and 19.5. Of all the nitrogen sources examined (NH4)2SO4 gave maximum enzyme production by both the wild (274±2 U/ml/) and mutant (503±3 U/ml) strains.0 and 18. NH4NO3.0 % (w/v) in case of mutant (490±2.1 g/l.10).9±1.5 %( w/v) in case of wild (260±2. The lactose at the concentration of 1.0 U/ml) and 1. The concentration of the (NH4)2SO4 was kept 0.04) and mutant strains (460±2.3 U/ml) was found to be the best for the enzyme production.4: EVALUATION OF INORGANIC NITROGEN SOURCES Various inorganic nitrogen sources such as (NH4)2SO4. Of all the carbon sources tested lactose showed considerable increase in the enzyme production by both the wild (235±1.5-2.( w/v) Therefore.8±2.0) strains.05) for the enzyme production both by the wild (284±1. The nitrogen sources were added to the fermentation media at the level of 0.5 % (w/v). These carbon sources were added to the fermentation media at the concentration of 0.5 % (w/v) concentration of lactose the enzyme production was not significant (p≤0. various concentrations of (NH4)2SO4 were also evaluated for the enzyme production (Fig 4.3 % (w/v) was found to be significant (p≤0. .1 % (w/v).96 of study.10.5 %. The concentration of the lactose was kept from 0. respectively.04) and mutant (525±3. At 2.
U/ml) and mutant (542±2. oryzae IIB-30 and its mutant derivative A. various concentrations of peptone (0.5: EVALUATION OF ORGANIC NITROGEN SOURCES Different organic nitrogen sources such as peptone.13). respectively.05) for the enzyme production.1-0. The CSL was proved as the second best nitrogen source for enzyme production by both strains.0 and19.5±1. Peptone at the concentration of 0. Sodium dodecyl sulphate (SDS). The surfactants were added to the fermentation media at the .T). Poly ethylene glycol (PEG) and sodium lauryl sulphate was investigated for the alpha amylase production by the A. The dry cell mass was 17. urea. Of all the nitrogen sources tested. Therefore. Variations in concentrations of peptone were also effective for alpha amylase production. peptone gave maximum enzyme production by both the wild (298±1.97 Further increase in the concentration of (NH4)2SO4 was resulted decrease in the enzyme production. meat extract.6: EFFECT OF SURFACTANTS The effect of various surfactants such as Tween 80.14). casein and beef extract were evaluated for the alpha amylase production (Fig 4. 4. Di-octyl ester of sodium sulpho succinic acid (Monoxal O.1 % (w/v). Further increase in the concentration of peptone was resulted decrease in the enzyme production.2 % w/v found to be significant (p≤0. Corn steep liquor (CSL).0 g/l.9 U/ml) strains.5 % w/v) were also evaluated for the enzyme production by both strains (Fig 4. The least alpha amylase production was observed when casein was used as an organic nitrogen source.5±2. The nitrogen sources were added to the fermentation media at the concentration of 0.5. oryzae EMS18 (Fig 4. Triton X-100.15).5. 4.
05 . The concentrations of the Tween 80 was kept as 0.0 U/ml) and mutant (573±2. various concentrations of Tween 80 were also evaluated for the enzyme production (Fig 4. Of all the surfactants examined.16).0 U/ml) strains. Tween 80 gave maximum enzyme production by both the wild (312±2.1 % (v/v) was found to be significant (p≤0. Hence.0.0 U/ml) and mutant (589±3.05) for the enzyme production both by the wild (320±2. .25 % (v/v). The dry cell mass was 16. The Tween 80 at the concentration of 0.6 g/l. Further increase in the amount of Tween 80 was resulted decrease in the enzyme production.1 % (v/v) was selected for further studies.9 and 19.98 concentration of 0. Tween 80 at the concentration of 0. respectively.05 % (v/v).0 U/ml) strains. Therefore.
oryzae EMS-18. * Incubation time 72 h. agitation rate 160 rpm . Y error bars indicate the standard error from mean value. pH 5.* 1000 30 800 Enzyme activity (U/ml) 25 20 DCM (g/l) 600 15 400 10 200 5 0 Wheat starch Corn starch Rice starch Sweet potato starch 0 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.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.0.05. The values vary significantly at p≤ 0. incubation temperature 30°C.
oryzae IIB-30 and its mutant derivative A. Y error bars indicate the standard error from mean value. oryzae EMS-18.8: Effect of different concentrations of starch on the alpha amylase production by A. The values vary significantly at p≤ 0.100 Fig 4. pH 5.0. agitation rate 160 rpm .05. * Incubation time 72 h.* 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 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. incubation temperature 30°C.
9: Evaluation of additional carbon sources on the alpha amylase production by A. incubation temperature 30°C.05. pH 5. * Incubation time 72 h. oryzae EMS-18.0. oryzae IIB-30 and its mutant derivative A.* 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 0 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.101 Fig 4. The values vary significantly at p≤ 0. agitation rate 160 rpm M an ni to l Co nt ro l CM ce ro l C .
102 Fig 4. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18.05.5 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. agitation rate 160 rpm . The values vary significantly at p≤ 0. * Incubation time 72 h. pH 5.5 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. incubation temperature 30°C.0.* 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 0. Y error bars indicate the standard error from mean value.10: Effect of different concentrations of lactose on the alpha amylase production by A.
pH 5.* 1000 30 800 Enzyme activity (U/ml) 25 20 DCM (g/l) 600 15 400 10 200 5 0 Ammonium sulfate Ammonium Sodium nitrate Potassium nitrate nitrate Inorganic nitrogen sources (0. The values vary significantly at p≤ 0. Y error bars indicate the error from mean value. oryzae IIB-30 and its mutant derivative A. agitation rate 160 rpm .103 Fig 4. standard * Incubation time 72 h.05. incubation temperature 30°C. oryzae EMS-18.0.11: Evaluation of inorganic nitrogen sources on the alpha amylase production by A.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.
The values vary significantly at p≤ 0.4 0.05.12: Effect of different concentrations of ammonium sulfate on the alpha amylase production by A. Y error bars indicate the standard error from mean value.0. incubation temperature 30°C.104 Fig 4.1 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. agitation rate160 rpm . * Incubation time 72 h.* 1000 30 25 800 Enzyme activity (U/ml) 20 600 15 400 10 DCM (g/l) 200 5 0 0.5 Each value is an average of three parallel replicate.2 0. oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. pH 5.
* Incubation time 72 h. oryzae IIB-30 and its mutant derivative A. The values vary significantly at p≤0. pH 5. incubation temperature 30°C. Y error bars indicate the standard error from mean value. agitation rate160 rpm .0.105 Fig: 4.1%) 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.05.13: Evaluation of organic nitrogen sources on the alpha amylase production by A. oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 Meat extract CSL Urea Casein Beef extract Peptone Control 0 Organic nitrogen sources (0.
106 Fig 4.14: Effect of different concentrations of peptone on the alpha amylase production by A. Y error bars indicate the standard error from mean value.1 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 0.0. agitation rate160 rpm . * Incubation time 72 h. oryzae IIB-30 and its mutant derivative A.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. incubation temperature 30°C. oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 0. The values vary significantly at p≤0.2 0. pH 5.05.
* 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 . Y error bars indicate the standard error from mean value. oryzae IIB-30 and its mutant derivative A. The values vary significantly at p≤ 0. incubation temperature 30°C.107 Fig 4. * Incubation time 72 h.05. agitation rate 160 rpm .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.0.15: Effect of different surfactants on the alpha amylase production by A. pH 5.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. oryzae EMS-18.
oryzae IIB-30 and its mutant derivative A. oryzae EMS-18. incubation temperature 30°C.15 Concentrations of Tween 80 (%) 0.05. Y error bars indicate the standard error from mean value.108 Fig 4.* 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 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.1 0.0. pH 5.05 0. The values vary significantly at p≤ 0.2 0 0. * Incubation time 72 h. agitation rate 160 rpm .16: Effect of different concentrations of Tween 80 on the alpha amylase production by A.
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. .2) and (19.6: OPTIMIZATION OF CULTURAL CONDITIONS IN STIRRED FERMENTER 4. qp (unit product produced/g cell/h). The dry cell mass was (18.8).6). Data obtained from above experiment was subjected to kinetic analysis for calculations of µ (h-1)max (specific growth rate).17). oryzae for the alpha amylase production was investigated in stirred fermenter (Fig 4. 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. Qx (g cell mass formation/l/h). g/l respectively.109 4. Qp (enzyme produced/l/h). Rapid decline in enzyme production was seen in case of wild and mutant strain when incubation period was increased from optimum time period. Yp/x(enzyme produced/g cell mass formation).1: RATE OF ALPHA AMYLASE PRODUCTION The rate of fermentation of both the wild (IIB-30) and mutant (EMS-18) strains of A. 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. 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.6. oryzae (Table 4.
At alkaline pH the production was extremely low.9 and 23. 350 U/ml when aeration rate was set at 1. Enzyme production by wild strain was found maximum i. 1. 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 4.0 vvm while mutant strain gave maximum enzyme activity (660 U/ml) at 1.18).7). The dry cell mass was 18.110 4. Qp.e. At pH 5.5 vvm.19 showed the effect of rate of aeration on the alpha amylase production by wild and mutant strain of A. Thus. The kinetic analysis of above parameters revealed that the values of Yp/x.2: EFFECT OF pH Effect of different initial pH (4-6. oryzae. Therefore. a decrease in enzyme production was observed. the maximum enzyme production by both wild (342U/ml) and mutant (626U/ml) strains were observed. oryzae.1 g/l.6. The rate of aeration varied from 0.0 vvm (wild) & 1. gave less enzyme production. 4.6. With the increase of pH.5 vvm (mutant). Qx were significant at an air supply of 1. oryzae was investigated in stirred fermenter (Fig 4. Any variation beyond this optimum level. respectively. respectively.7 and 22.5-2 vvm in stirred fermenter.3: EFFECT OF AERATION LEVELS Figure 4. .5 g/l. The dry cell mass was 18.5 vvm was optimized for further studies for enhanced enzyme production. pH 5 was selected for the production of enzyme by both wild and mutant strains of A.5) of fermentation medium by both wild and mutant strains of A.
4: EFFECT OF DISSOLVED OXYGEN Figure 4. Dissolved oxygen at the level of 15 % (v/v) gave the maximum enzyme production by wild (362U/ml) and mutant (687U/ml) strains. . respectively. The dry cell mass was 19. Beyond this concentration the enzyme production decreased gradually.9). Beyond this level. Data obtained from above experiment was subjected to kinetic analysis for calculations of Qp (enzyme produced/l/h).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. Yp/x(enzyme produced/g cell mass formation). oryzae EMS-18 (Table 4. oryzae in stirred fermenter (Fig 4.21). 4. The dry cell mass was 19 and 23.1 g/l.111 4.5 %.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. 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). oryzae IIB-30 and its mutant derivatives A. respectively.6 g/l.2 and 24. a decrease in enzyme production was recorded. oryzae. The size of vegetative inoculum was varied from 5-12.6. (v/v) and fermentation was carried out. The kinetic evaluation of results also revealed that optimum level of dissolved oxygen in fermenter was 15 % (v/v) for enzyme production by A. Qx (g cell mass formation/l/h).
.6. 4.112 All kinetic parameters showed 10 % (v/v) vegetative inoculum to be optimum for the enzyme production. Maximum enzyme production was obtained when agitation was maintained at 200 rpm. The rate of agitation was varied from 120-240 rpm (Fig 4.22).10). 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. Thus the inoculum size of 10 % (v/v) was used in further studies for the enzyme production in stirred fermenter. Qp.6: EFFECT OF AGITATION INTENSITY The effect of rate of agitation on the alpha amylase production by wild and mutant strains of A. Thus agitation speed of 200 rpm was selected for further studies. Further increase or decrease in agitation speed resulted in decrease enzyme production by both the strains. oryzae was investigated in stirred fermenter. Evaluation of kinetic parameters Yp/x.
0. aeration 1vvm .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 30 800 25 Enzyme activity (U/ml) 20 600 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 strainEMS-18 (U/ml) DCM of mutant strainEMS-18(g/l) * Incubation temperature 30°C. agitation rate 160 rpm. pH 5.
6: Kinetic evaluation of rate of fermentation for the alpha amylase production by A. Qx= g cell mass formation/l/h. Qp=enzyme produced/l/h. µ(h-1)max= specific growth rate.33 40857 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.22 185714 10133 0. .30 11000 Mutant 0.2 55000 5583 0. oryzae IIB-30 and its mutant derivatives in stirred fermenter Kinetic parameters µ Yp/x Qp Qx qp wild 0.114 Table 4.
oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 4 4. temperature 30°C.5 Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 *Incubation time 48 h.18: Effect of initial pH of media on the alpha amylase production by A.115 Fig 4. oryzae IIB-30 and its mutant derivative A.5 5 Initial pH Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) 0 5. agitation intensity 160 rpm.5 6 6. aeration 1vvm .
37 0.116 Table 4.25 0.5 5.33 0.31 0. Qp=enzyme produced/l/h. .30 0.25 0. Qx= g cell mass formation/l/h.20 0.0 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. oryzae IIB-30 and its mutant derivative in stirred fermenter pH 4 4.0 5.5 6.28 0.7: Kinetic evaluation of different pH values of media for the alpha amylase production by A.19 0.28 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.17 0.23 0.
117 Fig 4. oryzae EMS-18 * 1000 30 800 Enzyme activity (U/ml) 25 20 DCM (g/l) 600 15 400 10 200 5 0 0. agitation intensity 160 rpm . incubation temperature 30°C.5 1 Aeration levels (vvm) Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) 0 1. oryzae IIB-30 and its mutant derivative A.5 2 Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18(g/l) *Incubation time 48 h.19: Effect of different aeration levels on the alpha amylase production by A.
5 Mutant 310928 11000 0. Qx= g cell mass formation/l/h.38 Wild 18181 4000 0.22 2.31 Wild 19050 5833 0.8: 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 0.35 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation. Qp=enzyme produced/l/h.0 Mutant 28416 10183 0.0 Mutant 28571 10466 0.36 Wild 18881 5016 0.31 1.5 Mutant 27899 9483 0.26 1.118 Table 4.
oryzae EMS-18 * 1000 30 800 Enzyme activity (U/ml) 25 600 15 400 10 200 5 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) 0 Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l) *Incubation time 48 h. agitation intensity 160 rpm.0. initial pH 5. Dry cell mass (g/l) 20 .20: Effect of different level of dissolved oxygen on the alpha amylase production by A.0 vvm. oryzae IIB-30 and its mutant derivative A.119 Fig 4. incubation temperature 30°C. aeration 2.
Qx= g cell mass formation/l/h. . oryzae IIB-30 and its mutant derivative in stirred fermenter Dissolve oxygen (%) Kinetic parameters Yp/x Qp Qx Wild 19052 4750 0. Qp=enzyme produced/l/h.38 Wild 20133 6033 0.120 Table 4.24 5.31 Mutant 32258 11450 0.31 Wild 19937 5033 0.33 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.0 10 15 20 Mutant 28903 10000 0.25 Mutant 31840 10983 0.9: Kinetic evaluation of different levels of dissolved oxygen for the alpha amylase production by A.26 Mutant 29110 10666 0.39 Wild 19655 5350 0.
oryzae IIB-30 and its mutant derivative A. .5 Enzyme activity of mutant strain EMS-18 (U/ml) DCM of mutant strain EMS-18 (g/l) *Incubation time 48 h.5 Inoculum (%) Enzyme activity of wild strain IIB-30 (U/ml) DCM of wild strain IIB-30 (g/l) 10 0 12. initial pH 5.121 Fig 4. agitation intensity 160 rpm.0. incubation temperature 30°C. oryzae EMS-18 * 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 0 5 7.21: Effect of different inoculum size on the alpha amylase production by A.
oryzae EMS-18 * .32 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.40 Wild 21549 5100 0. Qx= g cell mass formation/l/h.32 10 Mutant 31093 11966 0.38 Wild 25486 6200 0. Fig 4.122 Table 4.36 Wild 25396 5333 0.23 12.21 7.5 Mutant 28506 9950 0.18 5. oryzae IIB-30 and its mutant derivative A. Qp=enzyme produced/l/h.10: Kinetic evaluation of different inoculum sizes for the alpha amylase production by A.5 Mutant 29792 11483 0. oryzae IIB-30 and its mutant derivative in stirred fermenter Inoculum (%) Kinetic parameters Yp/x Qp Qx Wild 19375 4800 0.0 Mutant 29570 10500 0.22: Effect of different agitation intensity on the alpha amylase production by A.
0. pH 5.123 1000 30 800 25 Enzyme activity (U/ml) 20 DCM (g/l) 600 15 400 10 200 5 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 30°C. .
Qx= g cell mass formation/l/h.7: Purification of alpha amylase . 4.36 Kinetic parameters: Yp/x= enzyme produced/g cell mass formation.26 160rpm Wild 19473 6166 0.39 200 rpm Wild 29215 6416 0. oryzae IIB-30 and its mutant derivative in stirred fermenter Agitation intensity Kinetic parameters Yp/x Qp Qx 120 rpm Wild 19254 4966 0.124 Table 4.41 240 rpm Wild 19444 5166 0.33 Mutant 39622 12500 0.31 Mutant 31818 11933 0.26 Mutant 30338 11666 0.17 Mutant 30120 10500 0. Qp=enzyme produced/l/h.11: Kinetic evaluation of different agitation speeds for the alpha amylase production by A.
05 M.7.125 4.30 M concentration of NaCl. At 20-40 % (w/v) saturation.13 shows the successive steps of purification of alpha amylase from mutant strain of A. At 70 % (w/v) saturation concentration the maximum specific activity (280.7. no activity was found.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. pH 7. The specific activity of broth was 208. The specific activity (561.9 U/mg of protein) with 1. Fig 4.3 U/mg of protein before treating with ammonium sulfate.2. The pooled distinct peak was obtained showing enzyme activity (10113.2.0 M) was used.3 fold purification was obtained.8U/mg of protein) was observed as shown in Table . oryzae EMS-18 to homogeneity by ammonium sulfate precipitation followed by using Sephadex DEAE and Sephadex G-100 columns. Above 70 % (w/v) both of these values were again decreased and at 90 % (w/v) no pellet was obtained 4.exchange chromatography The dialyzed enzyme solution was loaded on prepared Sephadex DEAE column.1U) at the 0.3 U/mg of protein then it increased (280.1: Ammonium sulfate precipitation The initial specific activity of cell free crude broth was 208.9 U/mg of protein) with first purification step of ammonium sulfate (70 % w/v).2: Stepwise purification: Table 4.1: Ammonium sulfate precipitation Table 4.7.23 shows the stepwise gradient elution pattern of alpha amylase when elution buffer of Tris HCl (0. 4.2: Anion.7. 4.5) containing NaCl (0-1.
3: Gel filtration Sephadex G-100 was finally used as finishing step of purification.5) were recorded as shown in Table 4.2. twenty five fractions were eluted with Tris HCl (0.7. However. dialyzed.13.7 U/mg of protein) and fold purification (9.05 M. Fig 4. The active fractions containing 5963. pH 7.1U enzyme activity were pooled up.24 also shows the elution pattern in the form of distinct peaks. Fig 4. Upon loading dialyzed sample.12: Purification summary of alpha amylase produced by mutant strain of A. The molecular weight was found to be as 48 kDa by applying the few fractions on SDS-PAGE. oryzae EMS-18 by using ammonium sulfate .13.5) buffer.126 4.25 4. Table 4. the specific activity (1987.
99 - Table 4.2 280.9 207. .3 0.92 1.9 64.127 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) 208.6 - Recovery or % yield 100 74 69.3 185 192.2 89.13: Step wise purification profile of alpha amylase produced by mutant strain of A.8 - Fold purification 25000 18500 17300 22479 16200 - 1.8 0.0 0. 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) 208.5 .7 23.9 Fold purification 1 1.1 3 1987.8 40.3 280.9 Recovery or % yield 100 89.6 5963.1 18 561.8 9.4 2.3 10113.
23: Elution pattern on Sephadex – DEAE Fig 4.129 Fig 4.24: The elution profile on Sephadex G-100 .
5 Enzyme activity (U) 4000 1 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) 0 Molecular weight .130 2.5 7000 6000 2 5000 Absorbance ( 280 nm) 1.
1: TEMPERATURE OPTIMA OF PURIFIED ALPHA AMYLASE .0 18. Marker. pooled fractions of ion exchange chromatography.8: CHARACTERIZATION 4. The mobility of the purified alpha-amylase corresponded to a molecular weight of 48kDa (Fig 31).0 35.0 25. 1 2 3 116kDa 66. 4. Ammonium sulfate fractionation. lane 3.8.4 14.4 48kDa Fig4. lane 2.131 Electrophoretic mobility of purified alpha-amylase with reference to mobilities of protein marker (SMO 313) Fractions was analyzed on SDS-polyacrylamide gel.2 45. SDS-PAGE analysis of pooled fractions of ion exchange chromatography and ammonium sulfate fractionation Lane 1.26.
it was selected for further studies. The enzyme substrate complex was incubated for varying time intervals (10-70 min). 50. 40. The enzyme substrate complex was incubated at different incubation temperature such as 25. the temperature 40°C was selected for optimum activity of alpha amylase.28 shows the effect of distilled water and different buffers on the activity of alpha amylase. 55. Different buffers and distilled water such as citrate. 60. At 70°C the activity of purified enzyme is not significant (p ≤0.2: EFFECT OF TIME OF INCUBATION ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE Figure 4. 35.26 shows the effect of temperature on the activity of purified alpha amylase by mutant strain of A.05). 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 (p≤0.2) . Thus.5) after 30min of incubation.05) as compared to other temperatures which were tested so.8. 65.132 Figure 4. 70°C. 30. oryzae.2 U/ml). Further increase in the incubation temperature resulted decrease in the activity of enzyme. The maximum enzyme activity (1592±3. acetate and phosphate were used in reaction mixture. 45. 4.3: EFFECT OF DISTILLED WATER AND BUFFER 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. 4. The activity of enzyme was increased with increase in temperature and found optimum at 40°C (1560±3.8. The enzyme activity was found to be optimum (1568±2.
The result showed CuCl2. The enzyme substrate complex was incubated at pH 3. Further increase in the pH resulted decrease in the activity of enzyme. FeSO4.30).5.8. oryzae EMS-18 .5.5. 3. Fig 4. 4.4: 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 4.5 and 7. While CaCl2.26: Effect of temperature on the activity of purified alpha amylase by mutant strain of A. At neutral pH. 5. 4. The enzyme activity was found to be optimum at pH 5. Zn Cl2. NiCl2 and NaCl has inhibitory effect on the activity of enzyme. The other buffers and distilled water show non significant results.05) as compared to other pH and selected for subsequent studies. 6. 4. 6. Mg SO4. MnSO4 have stimulatory effect on the activity of enzyme. acetate buffer was selected for further studies. 4. The acidic pH 5 of reaction mixture was significant (p≤0. the activity of enzyme was extremely low. 5.133 was obtained in acetate buffer. COCl2.8.4: EFFECT OF pH ON THE ACTIVITY OF PURIFIED ALPHA AMYLASE Figure 4. Thus. BaCl2.29 shows the effect of acetate buffer pH of reaction mixture (enzyme substrate complex) for the activity of purified alpha amylase.
The values vary significantly at p≤ 0.05. Y error bars indicate the standard error from mean value. Fig 4.134 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. oryzae EMS-18 .27: Effect of time of incubation on the activity of purified alpha amylase by mutant strain of A.
Y error bars indicate the standard error from mean value. The values vary significantly at p≤ 0.135 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.28: Effect of different buffers and distilled water on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 . Fig 4.05.
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. Fig 4. oryzae EMS-18 .29: Effect of different pH on the activity of purified alpha amylase by mutant strain of A.05.
30: Effect of metal ions on the activity of purified alpha amylase by mutant strain of A. oryzae EMS-18 . The values vary significantly at p≤ 0. Y error bars indicate the standard error from mean value.05.137 2400 2000 Enzyme activity (U/ml) 1600 1200 800 400 0 0 1 2 3 4 pH of acetate buffer Enzyme activity (U/ml) 5 6 7 8 Each value is an average of three parallel replicate. Fig 4.
Y error bars indicate the standard error from mean value.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. Discussion .05. The values vary significantly at p≤ 0.
e. HNO2. seventy eight strains of A. (2002) and Karanam and Medicherla. The strain no.30 was selected for further studies and assigned the code IIB-30. 30 gave maximum alpha amylase production. oryzae were isolated from soils of different habitats by serial dilution method (Clark et al. The UV-23 mutant was subjected to N-methyl N-nitro N-nitroso guanidine (NG) to induce mutagenesis at various concentrations. Out of which. one mutant NG15 gave 2 fold increase in alpha amylase production. 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. Of all the isolates tested. 1958).. Ellaiah et al. formation of pyrimidine dimers.23 showed better enzyme productivity compared to parental strain and was assigned the code UV-23. EMS) to enhanced the enzyme productivity. UV mutant of fungi showed more enzyme production compared to parental strain as reported by Azin and Noroozi. which resulted in greater secretion of enzyme from the mycelial cells (Ali et al. Perhephs it was due to the fact that UV irradiation possibly changed structure of DNA by photolysis i. The isolates obtained after UV irradiation were thirty two in number and screened for enzyme production out of which isolate no. (2002). In this connection. Rubinder et al. The IIB-30 was subjected to physical (UV radiation) and chemical mutagenesis (NG.. strain no. 2002). (2001). identified according to Onion et al. (1986) and tested for enzyme production in submerged fermentation. Thymidine-thymidine dimer probably promoted mycelial growth and subsequently enzyme activity.139 Isolation. (2008). identification and screening of a potent strain are the vital steps of alpha amylase production. The structural alteration in DNA was associated with the activity of the enzyme. Probably it was due to the .
oryzae out of which M4 medium (g/l).5. MgSO4. .. The six different media were evaluated for alpha amylase production by both wild and mutant strain of A. UV. The NG-15 was further subjected to alternate treatment with nitrous acid and EMS for further improvement in the enzyme production.12.3. starch 20. EMS-18 gave 2. NG and nitrous acid were commonly used for strain improvement as reported by Azin and Noroozi (2001) and Rubinder et al.6 fold alpha amylase production than the parental culture. was considered an ideal source for enzyme production. 8.7H2O 0. CaCl2 0. 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. in M4 medium. yeast extract. (2002).. 1970). Mg+2 and SO4-which were vital for the growth of fungus and enzyme production. activator and stimulator (Donell et al.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. binder. M4 medium contained the ions such as Ca+2 Cl-. Noorwez and Satyanarayana. 1997. Yeast extract and ammonium chloride serve as inorganic and organic nitrogen source respectively. Calcium and chloride ions act as stabilizer. Yeast extract is a complex nitrogen source containing free amino acids and peptides and therefore. Chambert et al. 1975. 1999). NH4Cl 1.06 was found best for maximum enzyme production. 2000). 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.
Francis et al. So. Nguyen et al. The enzyme activity appreciably decreased after 72 h. 2000. 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. The results indicated that enzyme was secreted early in active growth phase and reached maximum towards the end of exponential growth phase. 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. The maximum enzyme production was obtained after 96 h of inoculation (Kasim 1983.. .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. agitation were more accurately controlled which made the environment favourable for growth of organism and enzyme production (Gigras et al. temperature.. in case of fermenter. 1990. 1996). 2002).. The value of Yp/x was highly significant by both wild and mutant strains in fermenter. Kirshna and Chandrasekaran. 2002). the reduction in the fermentation period compared to shake flasks perhaps due to the fact that growth factors like pH. 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). in fermenter the enzyme production found to be optimum after 64 h (wild) and 48 h (mutant). However. However.
reduced air supply and consequently enzyme production. (1996) and Shafique et al. Higher temperatures resulted decreased enzyme production as reported by Dakhmouche et al. 2008). (2009). Probably it was due to the fact that decrease in the agitation speed of medium. The different volumes of the fermentation medium were evaluated in 250 ml Erlenmeyer flasks by both wild and mutant strains of A.. H+ion concentration also has a significant effect on the growth of mycelium and hence enzyme production (Kasim 1983. 2001 and Gupta et al. Optimization of the volume of fermentation medium is also necessary for air supply nutrient supply. The enzyme production was found to be optimal at 30ºC...142 The effect of varying the incubation temperature on the enzyme production was investigated. Bhanja et al. The size of inoculum has direct effect on the growth of organism and enzyme production as reported by Allan et al. At low volume of fermentation medium. growth of microorganism and enzyme production. 1999. The maximum enzyme production was obtained in 10 % of the fermentation medium. enzyme production was found to be optimum at pH 5. It might be due to nutrient present in the fermentation medium were inadequate for the growth of strains of A. 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. for enzyme production (Mimura and Shinichi. Stamford et al. From shake flask and fermenter experiments investigating the effect of pH. the enzyme production was also decreased. oryzae and hence. Different . As the volume of the medium was increased. the enzyme production was decreased. (2009). (2007) and Shafique et al. 2001). oryzae in present study. (2006). Ivanova et al.
. 2002. 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.Perhephs it was because of that a high starch concentration. hence enzyme production was reduced. rice. Of all the concentrations tested starch at the concentration of 2 % gave maximum enzyme production. Gigras et al. Of all the inoculum size tested. wheat and sweet potato were used in the present study. the enzyme production was also decreased. The corn starch gave maximum enzyme production. when attacked by alpha amylase during fermentation might have undergone degradation resulting into the accumulation of reducing sugars.. 2004.143 inoculum sizes were tested for enzyme production in shake flasks and fermenter. Beyond this concentration decrease in the enzyme production was take place. It might lead to the enhancement in sugar concentration of the substrate and catabolic repression of enzyme synthesis (Dvadtsatova et al. The effect of different concentrations of corn starch was evaluated. 4 % and 10 % of inoculum containing 2. Starches from different sources such as corn. As the inoculum size was further increased. The reason might be inadequate amount of mycelia produced at low amount of conidia which in due course decreased enzyme production. 1976. 1996). Krishna and Chandrasekaran. As the inoculum size 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. the enzyme production was decreased.6×106 CFU/ml was found to be optimum for the best enzyme production in shake flasks and fermenter. Ajer Dharani.
Thus lactose was selected as additional carbon source for the enzyme production and its various concentrations were tested. 2001. galactose. 2001). 2008). urea. Of all the inorganic nitrogen sources tested ammonium sulfate at 0.3 % gave maximum enzyme production. Triton . sodium nitrate and potassium nitrate were evaluated for the enzyme production.. fructose. Calik and Ozdamar. Further increase or decrease in the concentration of lactose was resulted decrease in enzyme production. sucrose. Of all the carbon sources tested lactose gave maximum enzyme production. corn steep liquor. ammonium nitrate. 2000. caboxy methyl cellulose.. 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. beef extract and peptone were also evaluated for the enzyme production. lactose.144 The effect of addition of different carbon sources such as glucose. (2008). Lactose at the concentration of 1 % was found to be best for the enzyme production. The effect of different surfactants such as Tween 80. Lactose along with starch was proved to be good carbon source in the present study. The different additional organic nitrogen sources such as meat extract. xylose. Peptone with inorganic nitrogen sources gave the maximum enzyme production as reported by Pedersen and Nielsen (2000) and Gupta et al. Different inorganic nitrogen sources such as ammonium sulfate. 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. casein. Gupta et al. glycerol and mannitol were evaluated for enzyme production.
Change in the rate of agitation resulted reduction in enzyme production. 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 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.. Optimum supply of oxygen is very essential for aerobic fermentation. Of all the surfactants tested Tween 80 gave the maximum enzyme production.T and poly ethylene glycols were tested for the enzyme production. excessive foaming. sodium dodecyl sulfate. The enzyme production increased with the increase of aeration and reached maximum at 1. A general requirement for a bioreactor is the provision of aeration system that can maintain a high dissolve oxygen level. 2001). O. . Probably higher stirring speed above than 200 rpm resulted in mechanical and oxidative stress. while higher concentration rates have some detrimental effects on the growth of microorganism and subsequently enzyme production during bioprocess time (Ionita et al. Monoxal. in this connection rate of agitation and different volume of air supply was studied for the enzyme production in stirred fermenter. 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.145 X-100.5 vvm (mutant). In addition another toxic by product were produced in the fermentation medium with little titer of enzyme activity. The enzyme production was increased as the agitation intensity was increased and found to be maximal at 200 rpm.0 vvm (wild) & 1. (1998).
After anion exchange.5-fold increase in specific activity.3 U/mg) to final finishing technique of gel filtration (1987. The comparison of successive purification steps starting from specific activity of crude broth (208.30 M concentration of NaCl elution buffer as reported by Kusuda et al.DEAE column. Chang et al. Among different temperature the maximum activity was observed at 40°C. 1995).. A linear gradient elution pattern indicated that maximum peak was achieved at the 0.3 at 70 % saturation concentration. At this concentration most of the protein having maximum enzyme activity. The enzyme solution (dialyzed) was further purified using Sephadex. the optimization of temperature. incubation time. The pattern of elution was used to determine the molecular weight of alpha amylase as 48 kDa on SDS-PAGE (Anidyawati et al.7U/mg) indicated the 9. due to increased . the dialyzed active fraction was loaded on Sephadex G-100. The positively charged proteins were removed as contaminants. 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).. Different saturation concentrations of ammonium sulfate were used. Possibly it was due to the fact that hydrophobic groups predominate in the interior of protein but some were on the surface. pH and metal ions were studied. different buffers. 1998.146 Alpha amylase was purified by ammonium sulfate. and successive chromatography techniques including anion exchange and gel filtration in the present study. For characterization of purified alpha amylase. (2003). The fold purification was 1. Probably it was due to the fact that reaction rate initially increased as the temperature rised.
BaCl2. At pH below and above optimal level. either with some change or similar size result in inhibition of enzyme activity (Abou Zeid 1997). as the temperature was increased the kinetic energy of enzyme exceeds the energy barrier. 2003). Most of alpha amylase is known to be metalloenzymes. CONCLUSION . The inactivation of enzyme at low temperature and thermal denaturation at high temperature might cause decrease in activity. Mg SO4. 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. However. 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. Effect of metal ion on the activity of enzyme was observed. 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. ZnCl2.147 kinetic energy of reacting molecules. It resulted in the breaking of weak hydrogen bonding and hydrophobic bonds that maintain the structure of enzyme. a decline in activity was possibly due to the structural unstability of protein (Kusuda et al. supplementation of metal ion improved the activity of enzyme. FeSO4. in the presence of CaCl2 maximum activity of enzyme was obtained.
148 In the present study. calcium chloride 0. NA. oryzae alpha amylase to be 48 kDa. yeast extract 8. seventy eight strains of Aspergillus oryzae were isolated for the enzyme production. All fermentation were carried out following growth of organism at 200 rpm (30°C) for 72 h in shake flasks.5 fold purification and 23. Many factors need to be considered by alpha amylase production to obtain economically most favourable process. A total of 9. corn starch 20.3. 64 h (wild) and 48 h (mutant) in fermenter. Gel electrophoresis indicated molecular weight of A. In case of fermenter inoculum size (10 %). peptone 2.6 fold more production compared to parental strain in term of enzyme production. ammonium sulfate 3. The most important among them were physical factors and culture medium. EMS) mutagens.12 and Tween-80 1. ammonium chloride 1. aeration (1.magnesium sulphate 0. REFERENCES .0 at pH 5 was selected.5vvm) dissolved oxygen level (15 %) were found optimum for maximum enzyme production. The mutant gave 2. The improvement in enzyme production was achieved by subjecting parental strain to successive physical (UV) and chemical (NG. The optimization of process parameters were under taken in shake flasks and fermenter. lactose 10.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).8 % recovery were obtained.06.
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