PROTEASES FROM AN ENVIRONMENTAL ISOLATE OF PSEUDOMONAS AERUGINOSA MCCB 123 AND THEIR APPLICATIONS Thesis submitted to Cochin University of Science and technology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Enviironmentall Biiotechnollogy Under the Facullty of Enviironmentall Studiies Schooll of Enviironmentall Studiies Cochiin Uniiversiity of Sciience and Technollogy by DIVYA JOSE Reg. No. 3065 NATIONAL CENTRE FOR AQUATIC ANIMAL HEALTH COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI 682016, KERALA November 2011 This is to certify that the research work presented in the thesis entitled “PROTEASES FROM AN ENVIRONMENTAL ISOLATE OF PSEUDOMONAS AERUGINOSA MCCB 123 AND THEIR APPLICATIONS” is based on the original work done by Ms. Divya Jose (Reg. No. 3065) under the guidance of Dr. A Mohandas, Professor Emeritus, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi -682016 and co- guidance of Dr. I.S Bright Singh, Coordinator, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi- 682016, in partial fulfilment of the requirements for the degree of Doctor of Philosophy and that no part of this work has previously formed the basis for the award of any degree, diploma, associateship, fellowship or any other similar title or recognition. Supervising Guide Co-Guide Dr. A Mohandas Dr. I.S Bright Singh Professor Emeritus, Coordinator, National Centre for Aquatic Animal Health, National Centre for Aquatic Animal Health, CUSAT CUSAT Kochi -682016 Kochi -682016 Kochi-682016 November, 2011 Decllaratiion I hereby do declare that the thesis entitled “PROTEASES FROM AN ENVIRONMENTAL ISOLATE OF PSEUDOMONAS AERUGINOSA MCCB 123 AND THEIR APPLICATIONS” is a genuine record of research work done by me under the guidance of Dr. A Mohandas, Professor Emeritus, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi-682016 and co-guidance of Dr. I.S Bright Singh, Coordinator, National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi – 682016, and that no part of this work has been presented for the award of any degree, diploma, associateship, fellowship or any other similar title or recognition. Divya Jose Kochi-682016 November, 2011 I give glory and honour to Almighty God for giving blessings, wisdom and guidance for enabling me to complete my thesis. The path towards this thesis spans several years of work and it is a great pleasure for me to thank the many people who made this thesis possible. My first debt of gratitude must go to my supervising guide Dr. I.S. Bright Singh, Professor, School of Environmental studies for his encouragement and advice necessary for me to proceed through the doctoral program and complete my thesis. I am extremely thankful to him for his continuous support to my research, for his patience, motivation, enthusiasm and immense knowledgement.His guidance greatly helped me in all the time of research. He patiently provided the vision, and without his help and support this thesis would not have been possible. The enthusiasm he has for his research was contagious and motivational for me. I wish to express my gratitude to my guide Dr. A Mohandas, Professor Emeritus, School of Environmental studies for his detailed and constructive comments and for his important support for this work. His understanding, encouragement and personal guidance have provided a good basis for this thesis. I wish to express my warm and sincere thanks to Dr. Rosamma Philip, Professor, School of Marine sciences for her warm encouragement and thoughtful guidance. I gratefully acknowledge Dr. Suguna Yesodharan, Director, School of Environmental studies for her support, guidance, helpful suggestions and all the help extended to me.I would also like to express my sincere gratitude to Dr. Ammini Joseph, Dr. S. Rajathy, Dr. V.Harindranathan Nair, Dr. V.Sivanandan Achari, Dr. E.P Yesodharan and Mr. M. Anand, Faculty members, School of Environmental studies for all their valuable suggestions. My sincere thanks are due to Dr. B. Sathyanathan and Dr. P. Rajalekshmi Subramanian, Technical Assistants, School of Environmental studies for their helpful suggestions. I am grateful to the librarian and office staffs at School of Environmental Studies for their valuable help. It has been a great privilege to spend several years in National Centre for Aquatic Animal Health for my research work and its members will always remain dear to me. I warmly thank Dr. Valsamma Joseph and Dr. Sajeevan T.P, faculty members at National Centre for Aquatic Animal Health for their valuable advice and friendly help. Individual acknowledgments are also owed to Ms. Sunitha Paulose for the timely help she has rendered to me in fermentation studies and statistical analysis. I specially thank Mr. Jayesh P, Mr. Prem Gopinath and Dr. Seena Jose for their help in tissue culture studies. Many thanks to Ms. Vrinda S and Ms. Priyaja P for their help in molecular biology works. I am deeply indebted to Ms. Sareen Sarah John for her unselfish support. I am deeply indebted to my Colleagues at National Centre for Aquatic Animal Health for providing a stimulating and fun environment in which to learn and grow. I am especially grateful to Ms. Surekhamol I.S, Ms. B. Sreelakshmi, Ms. Preena P.G, Ms. Deepa G.D, Dr. K. Sreedharan, Mr. Haseeb M, Ms. Rose Mary Jose, Mr. Renjith Kanjur, Ms. Ammu Thomas, Ms. Riya George, Ms. Gigi Paulose, Dr. Sudheer N.S, Dr. Sabu A.S, Mr. Suneesh,K, Ms. Blessy Jose, Ms. Surya Sugunan, Mr. Anish mon P.K, Mr. Devassy Christo, Ms.Asha P, Ms. Sanyo Sabu, Ms. Jisha Kumar and Ms. Ramya R. Nair for the support and caring they provided. I would like to thank Ms. Manjusha K, for being a co-worker and for all her helpful discussions. I am also grateful to my senior colleagues Deepesh V, Dr. Manju N.J, Dr. Somanath Pai, Dr. Preetha P, Dr. Rejish Kumar V.J and Dr. Anas Abdul Aziz. Dr. Somnath Pai for their support. Many thanks to Soman chettan ,Kusumam chechi and Parisa chichi for their valuable help they have offered throughout my research period. I owe a huge debt of gratitude to Dr. Sally N. Thomas, Scientist, Central Institute of Fisheries Technology for her help and encouragement throughout the study. The financial assistance provided by Cochin University of Science and Technology is gratefully acknowledged. Lastly and most importantly, I wish to thank my family. Words are not enough to express my sincere gratitude to my beloved husband, Mr. Anand Thekkekara, whose love, encouragement and great support allowed me to finish my journey. I wish to thank my entire extended family for providing a loving environment for me. I have no words to express my gratitude to my beloved father Mr. Jose J. Kallumkal and Mrs. Liza Jose. They raised me, supported me, taught me and loved me. To them I dedicate this thesis. I am extremely grateful to my father-in-law Mr. Antony Thekkekara and mother-in-law Aleece Anotny for their valuable support and prayers they have given me throughout the period, which made this thesis possible. I am extremely thankful to my grandparents Late John J. Kallumkal and Mrs. Annakutty John for their prayers and blessings. I am thankful to my brother Mr. Johnes Jose for all his great support and affection. I also thank all my family members for their great support, love and encouragement. Chapter 1 General Introduction .................................................. 01 - 50 1.1 Introduction ---------------------------------------------------------- 01 1.2 Microbial proteases ------------------------------------------------- 01 1.2.1 Classification of proteases --------------------------------------- 02 1.2.1.1 Exopeptidases ----------------------------------------------- 02 1.2.1.1.1 Aminopeptidases -------------------------------------- 03 1.2.1.1.2 Carboxypeptidases ------------------------------------ 06 1.2.1.2 Endopeptidases ---------------------------------------------- 08 1.2.1.2.1 Serine proteases -------------------------------------- 08 1.2.1.2.2 Metallo protease ------------------------------------- 17 1.2.1.2.3 Aspartic protease -------------------------------------- 23 1.2.1.2.4 Cysteine protease ------------------------------------- 23 1.2.2 Specificity of proteases ------------------------------------------ 24 1.2.3 Protease production ----------------------------------------------- 26 1.2.3.1 Solid state fermentation ------------------------------------ 26 1.2.3.2 Submerged fermentation ----------------------------------- 28 1.2.4 Optimization of protease production---------------------------- 28 1.2.5 Downstream processing of protease ---------------------------- 30 1.2.5.1 Precipitation ------------------------------------------------- 30 1.2.5.2 Ultrafiltration ------------------------------------------------ 31 1.2.5.3 Ion-exchange chromatography ---------------------------- 32 1.2.5.4 Hydrophobic interaction chromatography --------------- 34 1.2.5.5 Affinity chromatography ----------------------------------- 35 1.2.5.6 Gel filtration chromatography ----------------------------- 35 1.2.5.7 Aqueous two phase extraction ----------------------------- 35 1.2.6 Properties of protease -------------------------------------------- 37 1.2.6.1 pH and Temperature kinetics ------------------------------ 37 1.2.6.2 Effect of metal ions and inhibitors ------------------------ 37 1.2.7 Application of