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Catalysis by Enzymes Immobilizedon CATALYSIS BY ENZYMES IMMOBILIZED ON TUNED MESOPOROUS SILICA Thesis submitted to Cochiin Uniiversiity of Sciience and Technollogy in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry Under the Faculty of Science by RENI GEORGE Under the Supervision of Dr. S. SUGUNAN Department of Applied Chemistry Cochin University of Science and Technology Kochi - 682 022 May 2013 Catalysis by Enzymes immobilized on Tuned Mesoporous Silica Ph. D. Thesis under the Faculty of Science Author: RENI GEORGE Research Fellow, Department of Applied Chemistry Cochin University of Science and Technology Kochi -682 022 E mail: [email protected] Research Guide: Dr. S. SUGUNAN Emeritus Professor Department of Applied Chemistry Cochin University of Science and Technology Kochi - 682 022 Email: [email protected] Department of Applied Chemistry Cochin University of Science and Technology Kochi - 682 022 India May 2013 DEPARTMENT OF APPLIED CHEMISTRY COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI - 682 022, INDIA Dr. S. Sugunan Emeritus Professor 20-05-2013 Certified that the thesis work entitled "Catalysis by enzymes immobilized on tuned mesoporous silica " submitted by Ms. Reni George is an authentic record of research work carried out by her under my supervision at the Department of Applied Chemistry in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry of Cochin University of Science and Technology and has not been included in any other thesis previously for the award of any other degree. Dr. S.Sugunan (Supervising Guide) I hereby declare that the work presented in the thesis entitled “Catalysis by enzymes immobilized on tuned mesoporous silica” is my own unaided work under the supervision of Dr. S. Sugunan, Emeritus Professor in Department of Applied Chemistry, Cochin University of Science and Technology, Kochi‐22, and not included in any other thesis submitted previously for the award of any other degree. Kochi-22 Reni George 20 -05-2013 I would like to express my sincere and heartfelt gratitude to my supervising guide Dr. S. Sugunan, for his invaluable advice and inspiration rendered throughout my research work. I could share academic as well as personal snags with him. On many occasions I felt his caring nature and support. I would like to thank Dr. K. Sreekumar, who was the Head of the department during my research period. I remember with such high regard of his constant support and guidance throughout the period of my work. I wish to express my sincere thanks to Former head Dr. K. Girishkumar for his motivational approach and encouragement. I am grateful to Dr. S. Prathapan my doctoral committee member, for providing me academic and creative thoughts that helped me a lot on several occasions. I would never forget nor miss to mention all my teachers thankfully, for their blessings and all non-teaching staff members for their generous hand holding. Dr. Sanjay Gopinath who led me to the field of enzyme catalysis is one person whom I would render a special word of thanks. Dr. Reshmi. R who was my senior and friend as well helped by a long haul for many of my technical clarifications whenever I was in need of them. I could share a lot of enjoyable moments with her. Dr. Rani Abraham is like an elder sister for me and I would like to thank her for giving me a pleasant time. I would like to equally raise my heartened gratitude to my seniors S. Ajitha, Joyce Jacob, Bolie Therattil, .Ambili V. K and Rajesh K.M for their timely interventions and support. A gentle and helpful team around me has always been a gift to get rid of many difficult and unmaneuverable situations that I have been through, especially my labmates Cimi, Nissam, Rosemiss, Dhanya, Soumini, Mothi, Sandhya, Soumya and Honey. Special thanks to Rose miss for her friendship from M.phil class days onwards. Cimi, my friend helped me in every situation with matured advice, positive energy and confidence. Mothi, like my younger brother, always bestowed his caring nature and support. Nissam and Soumini helped me a lot through their creative criticisms and suggestions. Sandhya with her sound advice has helped me in dealing with many day to day junctures and matters. I would as well wish to express my untoward appreciations to my dear friends Jomon, Eason, Mridula, Anoop and Mahesh for their sensible help and support. Jiby my vibrant friend, from the days of DAT exam onwards with her funs, made the evening times very enjoyable and memorable. Jesny and my dear polymer lab mates for their talks and friendship made dry leisure time lively. I would like to express my sincere obligations to all my pals in the DAC. All the people and well-wishers around me, who have been instrumental directly or indirectly, to every small and big accomplishment found in my renderings, are remembered with such a great level of cheers from the bottom of my heart. I also wish to express my gratitude to Dr. P. R Rajmohan, NCL, for NMR analysis, Dr. K. Sreekumar, NCL, Mr. Nilesh Kulkarni for SXRD, Mr. Biji Bal, University Korea for SEM analysis, Dr.Salim Al-Harthi, SQ University, Muscat, Dr. M. K. Jayaraj, Dr. M. R Anatharaman, DOP, CUSAT and SAIF CUSAT & Mumbai for various analysis. Anees, Jithesh, Mithra, Raman, Shoy and Senoy helped me a lot for various analysis. I am grateful to DST- purse and CUSAT for financial assistance. Travel through University bus for past 4 years….one of the most enjoyable moments in my life. I found those silent hours giving me an opportunity for imagination, planning for my work and dreaming. I never would like to forget those peaceful memories… My personal elevated thoughts reminds me of the M.tech lab and work done in that lab….thanks to God for giving that wonderful, peaceful experience. That journey fetched me many friends and they are always with me to share the bad and good times in life, and to appreciate and motivate me with their suggestions and advice. My beloved teachers Dr. P. Ananthapadmanabhan and Jerly sir for their blessings and because of them I loved chemistry. Lastly it is the place where and when I feel so tired and stressed out, with a bunch of love, care of my Appa and Mummy that makes me fresh and calm. I can’t express my acknowledgment ….it can be beyond the words. They travelled through many painful occasions during my studies. I can’t express with words, my everlasting memories to the world for their love and care. My brother has always been there for me with his silent prayers. The great force from heaven showered blessings upon me and gave me inner strength to move confidently towards my goals. He showed light to my path to travel .I realized that I can do all the things through GOD who can strengthen me as and when needed. I have felt the presence of God in all painful situations beholding in his hands and walked ahead of me leading to this victory. Reni George Enzyme has its own unique advantages and disadvantages, and has been successfully utilized in various applications. Enzymes were immobilized into supports to preserve their stability and catalytic activity under extreme conditions. Mesoporous silica nanoparticles provide a non-invasive and biocompatible delivery platform for a broad range of applications in therapeutics, pharmaceuticals and diagnosis. Additionally, mesoporous silica materials can be synthesized together with other nanomaterials to create new nanocomposites, opening up a wide variety of potential applications. The ready functionalization of silica materials makes them ideal candidates for bioapplications and catalysis. These properties of mesoporous silica like high surface areas, large pore volumes and ordered pore networks allow them for higher loading of drugs or biomolecules. Comparative studies have been made to evaluate the different procedures; much of the research to date has involved quick exploration of new methods and supports. Requirements for different enzymes may vary, and specific conditions may be needed for a particular application of an immobilized enzyme such as a highly rigid support. In this endeavor, mesoporous silica materials having different pore size were synthesized and easily modified with active functional groups and were evaluated for the immobilization of enzymes. In this work, Aspergillus niger glucoamylase, Bovine liver catalase, Candida rugosa lipase were immobilized onto support by adsorption and covalent binding. The structural properties of pure and immobilized supports are analyzed by various characterization techniques and are used for different reactions of industrial applications. Still there is no universal support and optimal technique that it is suitable for all enzymes and their applications. Search for a good support is a fascinating challenge in biotechnology. Chapter 1 INTRODUCTION AND LITERATURE REVIEW..................................................01 - 29 1.1 Introduction-----------------------------------------------------------02 1.2 Enzymes as biocatalyst----------------------------------------------02 1.3 Immobilization -------------------------------------------------------02 1.4 Criteria for an ideal enzyme supports ----------------------------03 1.4.1 Mesoporous silica materials as support for enzyme immobilization------------------------------------------------------------ 04 1.4.2 Mesocellular foam (MCF) -----------------------------------------06 1.4.3 Synthesis of mesostructured materials ---------------------------09 1.4.4 Mechanism of formation of mesosilica --------------------------10 1.5 Enzyme
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