Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

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1 Methods in Molecular Biology Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to

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3 Enzyme Stabilization and Immobilization Methods and Protocols Edited by Shelley D. Minteer Department of Chemistry, Saint Louis University, St. Louis, MO, USA

4 Editor Shelley D. Minteer, Ph.D. Department of Chemistry Saint Louis University St. Louis, MO USA ISSN e-issn ISBN e-isbn DOI / Springer New York Dordrecht Heidelberg London Library of Congress Control Number: Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (

5 Preface Enzyme stabilization has been an area of research interest since the 1950s, but in the last decade, researchers have made tremendous progress in the field. This has opened up new opportunities for enzymes in molecular biology as well as industrial applications, such as bioprocessing. The first chapter introduces the reader to the field of enzyme stabilization and the different theories of enzyme stabilization, including the use of immobilization as a stabilization technique. The first half of the book will focus on protocols for enzyme stabilization in solutions including liposome formation, micelle introduction, crosslinking, and additives, while the second half of the book will focus on protocols for enzyme stabilization during enzyme immobilization including common techniques like sol gel encapsulation, polymer encapsulation, and single enzyme nanoparticle formation. Protocols for a variety of enzymes are shown, but the enzymes are chosen as examples to show that these protocols can be used for both enzymes of biological importance as well as enzymes of industrial importance. The final chapter will detail spectroscopic protocols, methods, and assays for studying the effectiveness of the enzyme stabilization and immobilization strategies. The chapters of this volume should provide molecular biologists, biochemists, and biomedical and biochemical engineers with the state-of-the art technical information required to effectively stabilize their enzyme of interest in a variety of environments (i.e., harsh temperature, ph, or solvent conditions). St. Louis, MO, USA Shelley D. Minteer v

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7 Contents Preface Contributors v ix 1 Introduction to the Field of Enzyme Immobilization and Stabilization Michael J. Moehlenbrock and Shelley D. Minteer 2 Stabilization of Enzymes Through Encapsulation in Liposomes Makoto Yoshimoto 3 Micellar Enzymology for Thermal, ph, and Solvent Stability Shelley D. Minteer 4 Lipase Activation and Stabilization in Room-Temperature Ionic Liquids Joel L. Kaar 5 Nanoporous Silica Glass for the Immobilization of Interactive Enzyme Systems Andreas Buthe, Songtao Wu, and Ping Wang 6 Enzyme Stabilization and Immobilization by Sol-Gel Entrapment Allan E. David, Arthur J. Yang, and Nam Sun Wang 7 Nanoporous Gold for Enzyme Immobilization Keith J. Stine, Kenise Jefferson, and Olga V. Shulga 8 Enzyme Stabilization via Bio-templated Silicification Reactions Glenn R. Johnson and Heather R. Luckarift 9 The Immobilization of Enzyme on Eupergit Supports by Covalent Attachment Zorica D. Knežević-Jugović, Dejan I. Bezbradica, Dušan Ž. Mijin, and Mirjana G. Antov 10 Micellar Polymer Encapsulation of Enzymes Sabina Besic and Shelley D. Minteer 11 Enzyme Stabilization via Cross-Linked Enzyme Aggregates Munishwar N. Gupta and Smita Raghava 12 Enzyme Immobilization in Polyelectrolyte Microcapsules Michael J. McShane 13 Macroporous Poly(GMA-co -EGDMA) for Enzyme Stabilization Nenad B. Milosavić and Radivoje M. Prodanović 14 Enzyme Nanoparticle Conjugates for Biomedical Applications Alexey A. Vertegel, Vladimir Reukov, and Victor Maximov 15 Enzyme Nanoparticle Fabrication: Magnetic Nanoparticle Synthesis and Enzyme Immobilization Patrick A. Johnson, Hee Joon Park, and Ashley J. Driscoll 1 vii

8 viii Contents 16 Cytochrome c Stabilization and Immobilization in Aerogels Amanda S. Harper-Leatherman, Jean Marie Wallace, and Debra R. Rolison 17 Kinetic Measurements for Enzyme Immobilization Michael J. Cooney Index

9 Contributors Mirjana G. Antov University of Novi Sad, Faculty of Technology, Novi Sad, Serbia Sabina Besic Department of Chemistry, Saint Louis University, St. Louis, MO, USA Dejan I. Bezbradica Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia Andreas Buthe Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN, USA Michael J. Cooney School of Ocean and Earth Science and Technology, Hawaii Natural Energy Institute, University of Hawaii-Manoa, Honolulu, HI, USA Allan E. David Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA; Industrial Science & Technology Network (ISTN) Inc., York, PA, USA Ashley J. Driscoll Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY, USA Munishwar N. Gupta Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India Amanda S. Harper-Leatherman Chemistry Department, Fairfield University, Fairfield, CT, USA Kenise Jefferson Department of Chemistry and Biochemistry, Center for Nanoscience, One University Boulevard, University of Missouri Saint Louis, Saint Louis, MO, USA Glenn R. Johnson Microbiology and Applied Biochemistry, Air Force Research Laboratory, Tyndall Air Force Base, FL, USA Patrick A. Johnson Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY, USA Joel L. Kaar Department of Chemical Engineering, McGowan Institute for Regenerative Medicine, University of Pittsburg, Pittsburg, PA, USA Zorica D. Kneževic -Jugovic Department of Biochemical Engineering and Biotechnology, University of Belgrade, Belgrade, Serbia Heather R. Luckarift Microbiology and Applied Biochemistry, Air Force Research Laboratory, Tyndall Air Force Base, FL, USA Victor Maximov Department of Bioengineering, Clemson University, Clemson, SC, USA Michael J. McShane Biomedical Engineering Department, Texas A&M University, College Station, TX, USA Dušan Ž. Mijin University of Belgrade, Belgrade, Serbia Nenad B. Milosavic Department of Chemistry, Belgrade University, Belgrade, Serbia Shelley D. Minteer Department of Chemistry, Saint Louis University, St. Louis, MO, USA ix

10 x Contributors Michael J. Moehlenbrock Department of Chemistry, Saint Louis University, St. Louis, MO, USA Hee Joon Park Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY, USA Radivoje M. Prodanovic University of Belgrade, Belgrade, Serbia Smita Raghava Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India Vladimir Rekov Department of Bioengineering, Clemson University, Clemson, SC, USA Debra R. Rolison Naval Research Laboratory, Surface Chemistry Branch, Washington, DC, USA Olga V. Shulga Cross-Link Technologies, Jordan Valley Innovation Center, Springfield, MO, USA Keith J. Stine Department of Chemistry and Biochemistry, University of Missouri Saint Louis, Saint Louis, MO, USA Alexey A. Vertegel Department of Bioengineering, Clemson University, Clemson, SC, USA Jean Marie Wallace Nova Research, Inc., Alexandria, VA, USA Nam Sun Wang Department of Chemical and Biochemical Engineering, University of Maryland, College Park, MD, USA Ping Wang Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN, USA Songtao Wu Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN, USA Arthur J. Yang Industrial Science & Technology Network (ISTN) Inc., York, PA, USA Makato Yoshimoto Department of Applied Molecular Bioscience, Yamaguchi University, Ube, Japan