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Characterization of Alginate Lyase from Microbulbifer Mangrovi Sp. Nov

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Characterization of Alginate Lyase from Microbulbifer Mangrovi Sp. Nov Characterization of alginate lyase from Microbulbifer mangrovi sp. nov. DD-13T Thesis Submitted to Goa University For the degree of Doctor of Philosophy in Biotechnology by Ms. Poonam Vashist Department Of Biotechnology Goa University Taleigao- Goa 2014 Characterization of alginate lyase from Microbulbifer mangrovi sp. nov. DD-13T Thesis Submitted to Goa University For the degree of Doctor of Philosophy in Biotechnology by Ms. Poonam Vashist Under the supervision of: Dr. S. C. Ghadi Department Of Biotechnology Goa University Taleigao- Goa 2014 CERTIFICATE This is to certify that the thesis entitled “Characterization of alginate lyase from Microbulbifer mangrovi sp.nov. DD-13T” submitted by Ms. Poonam Vashist, for the award of the Degree of Doctor of Philosophy in Biotechnology is based on original studies carried out by him under my supervision. The thesis or any part thereof has not been submitted for any other degree or diploma in any university or institution. Place : Goa University Date : 25/06/2014 Dr. S.C. Ghadi (Research Guide) Professor, Department of Biotechnology Goa University, Goa -403 206, India STATEMENT As required by the Goa university ordinance OB-09.9(ii), I state that the present thesis entitled “Characterization of alginate lyase from Microbulbifer mangrovi sp.nov. DD-13T” is my original contribution and that the same has been submitted on any previous occasions for any degree. To best of my knowledge, the present study is the first comprehensive work of its kind from the area mentioned. The literature related to the problem investigated has been cited. Due acknowledgments have been made wherever facilities and suggestions have been availed of. Place: Goa, India Date: 25/06/2014 Poonam Vashist ACKNOWLEDGEMENT It gives me immense pleasure and satisfaction to convey my sincere thanks and gratitude to Dr. S.C. Ghadi, my guide and patron for enlightening me into the world of research, for teaching me to see things from different perspective, for sharing his vast knowledge in microbial biotechnology and for his patience, persistence and tolerance. Each and every day was new door-step of adventure and significance. Dr. S.C. Ghadi has been inspiring me to face courageously and intelligently the usual ups and downs of voyage of research. It’s my fortune to gratefully acknowledge the support and encouragement of Dr. Usha Muraleedharan, Dr. Urmila Barros, Dr. Savita Kerkar, Prof. U.M.X. Sangodkar, Dr. S K Dubey (Dept.of Microbiology) and Dr. Shanta Nair, Scientist (NIO, Goa). I will be privileged to thank Dr. Yuichi Nogi (JAMSTEC, Japan) for their kind cooperation and collaboration during this study. I also thank Dr. Y. Shouche and Mr. Pankaj Verma, National Centre for Cell Sciences, Pune, Mr. Sanjay Singh, NIO, Goa. I would also like to acknowledge the Vice-Chancellor and the Dean of life sciences, Goa University for providing necessary infrastructure to carry out my research. I would also like to to thank Dr. Rahul Mohan Sharma from NCAOR , Goa; Mr. Khedekar and Mr. Arif from NIO in helping me with the SEM pictures. I wish to express my gratitude to Shayna and Mr. Suhas NCAOR. Words are short to express my deep sense of gratitude towards my friends and colleague Lillian and Shahin for being with me as confidence in my good and bad time. I also sincerely convey my thanks to Late Shri Ravi Chand for their encouragement and being there as my friendly guide. I am privileged to thank Dr. Kanchana, Sudheer, Asha, Kuldeep, Nirmal Prasad, Tonima, Surya, Imran, Preethi, Judith, Priyanka, Samantha, Parantho, Alisha, Amruta, Michelle, Shuvankar, Kirtidas, Delicia, Dr. Tomchou Singh, Hanumanth, Deepa and M.Sc (Biotechnology) students for sharing delightful moments, help and co-operation during the study. I convey my sincere thanks to Mr. Martin, Serrao, Ulhas, Amonkar, Ruby, Vandana, Sadanand, Anna, Concessa, Neelima, Tulsidas, Bharath, Sumati and Samir for their assistance and great help in day to day laboratory work; Administrative staff and library staff for their help and providing necessary facilities; Goa University for their financial assistance for this study. I would like to pay high regards to my parents S.S. Vashisth and Shakuntala Vashisth for their sincere encouragement and inspiration throughout my research work and lifting me uphill this phase of life. I owe everything to them. I wish to express my gratitude to my husband Amir Kumbhar, in-laws Somanath D Kumbhar & Jyoti Kumbhar and my little daughter Shaivi and son Neehan, whose constant patience helped me to excel through my degree. Without their enduring support and sacrifices, this journey would not have been possible. I would also like to convey my thanks to Asha Gavali who has proved to be a great help during this phase of my life. I also express gratitude to those who have contributed to my research directly or indirectly even though they remain anonymous. Above all I thank the ALMIGHTY for giving me this opportunity and helping me to be patient & optimistic throughout my struggles and failures. Poonam Table of contents Chapter 1 General introduction 1- 5 Research goal and significance 6 – 8 Chapter 2 Literature review 9- 35 Chapter 3 Screening for multiple polysaccharide degrading bacteria 36- 59 Chapter 4 Identification of Multiple polysaccharide degrading bacterial 60- 121 strain DD-13 Chapter 5.Optimization of the growth and culture conditions for 122- 148 enhancing production of alginate lyase from Microbulbifer mangrovi sp.nov. DD-13T Chapter 6. Purification and characterization of the alginate lyase from 149- 189 Microbulbifer mangrovi sp.nov. DD13T Chapter 7 Applications of alginate lyase. 190- 216 Summary and Conclusions 217- 221 Future prospects 222 References 223- 275 Appendix Paper published LIST OF ABBREVIATIONS A Absorbance at the given wavelength APS Ammonium persulphate BSA Bovine serum albumin O.D. Optical Density EDTA Ethylenediaminetetraacetic acid PAGE Polyacrylamidegel electrophoresis rpm Revolutions per minute SDS Sodium dodecyl sulphate TEMED NNN'N'- tetramethyl ethylene diamine TLC Thin layer chromatography U Unit SCD Single cell detritus G Guluronic acid M Mannuronic acid DP Degree of polymerization SCD Single cell detritus CPC Cetylpyridinium chloride LPS Lipoopolysaccharides Dedicated to my family & Almighty…… CHAPTER 1: INTRODUCTION 1 Polysaccharides also known as glycans are branched or linear carbohydrate polymeric structure composed of repeating monosaccharide units bond together by glycosidic linkages. Based on the structure the glycans are either homoglycans (single type monosaccharides units) or heteroglycans (different monosaccharide units). Polysaccharides are known to be an important class of biological polymer which usually functional as structural and/ or energy reservoir in living organisms. Based on the charge on the polysaccharides, these are divided into three categories i.e. neutral such as guar gum, amylopectin, amylase, cellulose etc.; anionic polysaccharides such as alginates, carrageenan, xanthan, gellan etc.; and cationic polysaccharides such as chitin. Due to solubility and various substituent functional groups, these polysaccharides are recalcitrant and also referred as insoluble complex polysaccharides (ICPs). ICPs are the most abundant renewable resources on the earth. Marine ecosystem comprises a major part of the biosphere and annually produces more than 2 billion tons of ICPs. These ICPs are associated with biofilms, algal blooms, planktonic organisms and shells of marine invertebrates (Kloareg and Quatrano, 1988; Pakuski and Benner, 1994; Kurita, 2006). Degradation of ICPs is an important component within global nutrient recycling and act as major sink of carbon in nature (Arrigo, 2005). Microorganisms produce various extra and intracellular polysaccharide hydrolyzing enzymes. These enzymes depolymerize long chains of polysaccharides by hydrolyzing the glycosidic linkages. Various mechanisms involved in ICPs degradation by bacterial systems has been extensively reviewed (Salyers, et al., 1996). The ability of these microorganisms to ferment ICPs to simple sugars using polysaccharase and other enzymes has made the 1 utilization of these recalcitrant compounds easier. Hydrolysis of ICPs by enzymes produced intermediates that can be used as the valuable feedstock in aquaculture, antioxidants, nutraceuticals, antitumor, antidiabetic, skin whitening agents, thickeners, gelling agent or stabilizers of emulsions and dispersions. With rising understanding of biological functions of these marine ICPs, the utilization of these sources in pharmaceuticals, aquaculture and other biotechnology related industries has been improved significantly. Alginate is an uronic acid co-polymer comprising β- D- mannuronic acid (M) joined by (1- 4) linkage to α-L- guluronic acid (G) (Haug and Larsen, 1962). These moities can be arranged in homopolymeric (Poly G / Poly M) or heteropolymeric (Poly MG) blocks (Haug, et al., 1966). Alginate isolated from various sources differs in properties and molecular structures. These variations are either because of the arrangement of moities or different substitutions such as galactose substituted alginate, propylene glycol alginate, sulphated alginate or methylated alginate etc. Alginate is found in cell walls and intracellular spaces in brown seaweeds. Commercially used alginate is generally obtained from Laminaria, Ascophyllum and Macrocystis (Skjak-Barek, et al., 1991). Heterotrophic bacteria belonging to two families Azotobacteriaceae and Pseudomonadaceae also produce alginate.
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