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Molecular Analysis of the Α-Amylase Production in Streptomyces Griseus (MTCC 3756) and Development of a Low Cost Maltose Processing Technology

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Molecular Analysis of the Α-Amylase Production in Streptomyces Griseus (MTCC 3756) and Development of a Low Cost Maltose Processing Technology Molecular analysis of the α-amylase production in Streptomyces griseus (MTCC 3756) and development of a low cost maltose processing technology FINAL TECHNICAL REPORT BACK TO LAB PROGRAMME (Sanction No: 1367/2014/KSCSTE dated 20/02/2014) WOMEN SCIENTISTS DIVISION KERALA STATE COUNCIL FOR SCIENCE TECHNOLOGY AND ENVIRONMENT GOVT. OF KERALA Project Period : 03/03/2014 to 02/03/2017 Principal Investigator: Dr. Divya Balakrishnan Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala, India JANUARY 2017 AUTHORIZATION The work entitled “Molecular analysis of the α-amylase production in Streptomyces griseus (MTCC 3756) and development of a low cost maltose processing technology” by Dr. Divya Balakrishnan, was carried out under the “Back to lab programme” of Women Scientists Division, Kerala State Council for Science Technology and Environment, Govt. of Kerala. The work was carried out at Microbiology Department, Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode. The project was initiated wide sanction No: 1367/2014/KSCSTE dated 02/03/2014, with scheduled completion by 02/03/2017. The field and laboratory works were completed by March 2017. The project was completed on 02/03/2017 with a financial expenditure of Rs. 15,96,000/-lakhs. ACKNOWLEDGMENTS I am deeply indebted to Kerala State Council for Science, Technology and Environment for the financial aid through “Back to Lab program for Women” which helped me to get an independent project with great exposure leading to career development. I am grateful to Dr. K.R. Lekha, Head, Women Scientists Division for the encouragement and support throughout the tenure of the project. I am grateful to Dr. S. Shiburaj, Scientist mentor, JNTBGRI for giving consent to become the mentor of the Project . Words are not enough to express my heartfelt gratitude to Dr. N.S. Pradeep for the support given during the tenure of the project for the support to make this endeavor a success. I am highly obliged to Jawaharlal Nehru Tropical Botanic Garden and Research Institute for providing me necessary facilities and logistical support for carrying out the a laboratory experiments during the course of the project. I would like to express my deep gratitude to Dr. P. G. Latha, Former Director, for the facilities, support and encouragement provided. I like to manifest my sincere gratitude to Dr. A. G. Pandurangan, Director, Head, Division of Plant Systematics and Evolutionary Science for their facilities provided. I extend my deep gratitude to Prof K. Dharmalingam, AMRF, Madurai for providing LC-MS analysis. I am also most grateful that he had taken time and patience from a very busy schedule to contribute the very important part of this thesis. I also express my thanks to Dr. Kesavan Namboothiri, Scientific Officer, Central Plantation Crop Research Institute, Kayamkulam for his help in statistical analysis of the data. I wish to record my bountiful thanks to all my labmates of microbiology Division. I owe my greatest debt of gratitude to the God Almighty for strengthening me with his love, grace and peace. Dr. Divya Balakrishnan CONTENTS 1. ABSTRACT 1 2. INTRODUCTION 3 3. REVIEW OF LITERATURE 11 4. OBJECTIVES 17 5. MATERIALS AND METHODS 18 6. RESULT 30 7. DISCUSSION 63 8. SUMMARY 70 9. OUT COME OF THE PROJECT 73 10. SCOPE OF FUTURE WORK 74 11. BIBLIOGRAPHY 75 Abstract A mesophilic isolate of Streptomyces griseus TBG19NRA1 (synonym: S. setonii) from the forest soil of Neyyar WLS, Kerala, which produces extracellular thermo stable α- amylase. The strain was grown in a medium (pH 7) containing starch as the sole carbon source and showed maximum α-amylase activity at the end of 48 h of incubation at room temperature. The amylase activity of the TBG19NRA1 was demonstrated on starch-casein agar plate containing 1% soluble starch. The cells were grown in a medium containing starch as the sole carbon source. Optimum production of the enzyme was observed when inoculated medium of pH 7.0 was incubated for 48 h of incubation. It was observed that enzyme activity was highest with 1% sucrose and 2% starch as carbon source. Among different organic nitrogen sources, 0.5% of Casein resulted maximum activity. Supplementation of 0.5% Valine and 0.025% CaCl2 enhanced the enzyme production. It was found that α-amylase yield were also improved by the addition of 0.1% of Maleic acid and 0.05% of Triton X-100 in the medium. The enzyme was purified using ammonium sulfate precipitation, dialysis and followed by gel filtration using sephadex G-100. SDS-PAGE and zymogram analysis showed that the purified protein has a molecular weight of aproximatly 60 kDa. The optimum temperature for the purified -amylase is ranging from 70-90°C with a maximum o relative activity at 80 C without the addition of CaCl2. The relative activity was 92.3% and 56% at 90oC and 100oC respectively and it has revealed the enzyme is thermophilic in nature. The optimum pH was ranged from pH 6.0 to 9.0 (retained >80% activity) with a maximum activity at pH 7.0. The amylase activity was strongly stimulated by Fe3+ and Mg2+ (5 mM), Mn2+ showed slight enhancement in enzyme activity, while Ba2+, Ca2+ ions inhibited α-amylase activity. The chemical inhibitors and denaturants like dithiothreitol (DTT), β-mercaptoethanol (BME), ethylenediaminetetraacetic acid 1 (EDTA) and Urea, the activity of was slightly stimulated enzymatic activity, while phenylmethane sulfonyl fluoride (PMSF) and Sodium dodecyl sulfate (SDS) inhibited the activity. The analysis of kinetic showed that the enzyme has of 1.6 mg/mL and max of 28 mg/mL/min at pH 7. The Peptide mass fingerprinting using LC-MS analysis and subsequent Uniprot database search confirmed the enzyme as α-amylase. Our result showed that the enzyme was both, highly thermostable, calcium independent, with broad pH range and may be suitable in liquefaction of starch in high temperature, in starch based industrial applications. 2 Introduction Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and and greater catalytic activity (Gurung et al., 2013). The microbial enzymes are also more active and stable than the plant and animal enzymes and considered as a potential replacement, in absence of human enzymes (Anbu et al., 2013). The microbial world is considered as the largest unexplored reservoir of biodiversity on the earth (Vibha and Neelam, 2012). The number of microbial cells in aquatic environment has been estimated to be approximately 1.2 × 1029, while that in terrestrial environment is 4–5×1030 (Singh et al., 2009). Owing to their unpredictable ability to adapt to varying environmental conditions, microorganisms can occur in all biological niches on earth, from terrestrial environments to the oceans. Microorganisms numerically dominate terrestrial biodiversity which represents at least two to three orders of magnitude more than all the plant and animal cells combined. Thus, microorganisms are highly diverse group of organisms which constitute about 60% of the earth’s biomass (Singh et al., 2009). The variation in microbial diversity in response to environmental variability shows that microbial diversity changes with environmental factor (Zeglin, 2015). chemical reactions in living cells. The action of enzymes depends on their ability to bind the substrate at a domain on the enzyme molecule called the active site (Duza and Mustan, 2013). The industries and household catalysis are becoming more and more dependent on enzymes. The enzymes are able to catalyze all kinds of chemical reactions within few minutes or seconds (Sarrouh et al., 2012). Usually enzymes with the desired activity under industrial conditions could be obtained by optimizing process conditions and by protein engineering using directed evolution (De Carvalho, 2011). With better knowledge and purification of enzymes, the number of applications has 3 increased manifold, and with the availability of enzymes with robust characters (thermostability, alkaline, cold active etc) a number of new possibilities for industrial processes have emerged (Tiwari et al., 2015). The opportunity for discovery of new industrial applications from microorganisms is as large as the variety of environments they confront. The majority of existing biotechnological applications are of microbial origin and enzymes are the most important among them. Microbial enzymes are often more useful than enzymes derived from plants or animals because of the great variety of catalytic activities available, the high yields possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media. Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safe (Duza and Mastan, 2013). The exploration of important compounds produced by novel species from uncommon environments has considerable importance in drug discovery (Thornburg et al., 2010; Rateb et al., 2011; Pettit, 2011). Starch Starch and cellulose are the most abundant carbohydrate polymers on earth. Starch represents one of the most ubiquitous and an important renewable biological resource and also considered as a potential substrate for the production of fuels and chemicals by certain enzymatic processes (Vengadaramana, 2013). Starch-containing crops form an important constituent for the human diet and a large proportion of the world’s population consume it as the major food (Myat and Ryn, 2013). Native starch is a semi- crystalline material synthesized as roughly spherical granules in plant tissues. Pure starch is a heterogeneous polysaccharide consisting predominantly of α-glucan in the form of amylose and amylopectin (Fig 2A and B). These two polymers have different structure and physical properties (Mojsov, 2012). Starch is a polymer of glucose linked to one another through the C1 oxygen, known as the glycosidic bond. This glycosidic 4 bond is stable at high pH but hydrolyzes at low pH.
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