Winfried Hartmeier Immobilized Biocatalysts An Introduction Translated by Jo y Wieser With 115 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Prof. Dr. WINFRIED HARTMEIER Department of Technical Biochemistry University of Hohenheim 7000 Stuttgart 70, FRG Translator: JOY WIESER Madleinweg 19 6064 Rum/Innsbruck, Austria Original German edition: W. Hartmeier , Immobilisierte Biokatalysatoren © Springer-Verlag Berlin Heidelberg 1986 Legend of cover motif: Propeller-Loop reactor with immobilized biocatalysts ISBN-13:978-3-540-18808-7 e-ISBN-13:978-3-642-73364-2 DOI: 10.1007/978-3-642-73364-2 Library of Congress Cataloging-in-Publication Data. Hartmeier, Winfried. Immobilized biocata lysts. Translation of Immobilisierte Biokatalysatoren. Includes index. 1. Immobilized enzymes Industrial applications 2. Immobilized enzymes-Synthesis. I. Title. TP248.E5H3813 1988 661'.8 88-489 Tbis work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9,1965, in its version of June 24,1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law. © by Springer-Verlag Berlin Heidelberg 1988 Tbe use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. 2132/3130-543210 Author's Preface to the English Edition The appearance of an English translation one year after the publication of the first German edition is due to the conviction of publisher and author that, despite the wealth of Anglo-American literature concerning immobi lized catalysts, there is a lack of inexpensive introductory books on the subject. Some slight extensions have been made to the original German text. In the practical section, two additional experiments introduce the student to recent techniques for using membrane reactors and for biocatalyst encap sulation using liquid membranes. Some of the most important publications that have appeared in the meantime have been taken into consideration and added to the list of recommended literature. I am greatly indebted to the translator, Mrs. Joy Wieser, and to Springer-Verlag, especially to Dr. Dieter Czeschlik, who encouraged and supported me in many ways. W. Hartmeier Contents THEORETICAL SECTION ............................................ . General Principles ........................................... 3 1.1 Principles of Biocatalysis .................................... 3 1.2 Structure of Enzymes .......................................... 5 1.3 Classification and Nomenclature of Enzymes .................... 10 1.4 Definition and Classification of Immobilized Biocatalysts ..... 14 1.5 Reasons for Immobilization .................................... 16 1.6 The History of Immobilization ................................. 18 1 .7 Economic Importance ........................................... 19 2 Methods of Immobilization .................................... 22 2.1 Adsorption .................................................... 22 2.2 Ionic Binding ................................................. 24 2.3 Covalent Binding .............................................. 26 2.4 Cross-linking........................... . . . . . . . . . . . . . . . . . . . . .. 33 2.5 Matrix Entrapment ............................................. 36 2.6 Membrane Confinement .......................................... 40 2.7 Combined Methods .............................................. 45 2.8 Immobilization of Coenzymes ................................... 48 3 Characteristics of Immobilized Biocatalysts .................. 51 3.1 Activity as a Function of Temperature ......................... 51 3.2 Stability as a Function of Temperature ........................ 54 3.3 Temperature Optimum in Long-term Processes .................... 57 3.4 The Influence of pH Value ..................................... 59 3.5 The Influence of Substrate Concentration ...................... 62 3.6 The Influence of Diffusion .................................... 65 3.7 Other Physical Properties ..................................... 68 4 Reactors for Immobilized Biocatalysts ........................ 72 4.1 Stirred Reactors .................... ,......................... 73 4.2 Loop Reactors ................................................. 75 4.3 Bed Reactors .................................................. 76 4.4 Membrane Reactors ............................................. 78 4.5 Special Forms of Reactors ..................................... 80 5 Industrial Applications ...................................... 82 5.1 Classical Fields of Application ............................... 83 5.2 Production of L-Amino Acids Using L-Aminoacylase .............. 86 5.3 L-Amino Acid Production in Membrane Reactors .................. 88 5.4 The Production of Fructose-containing Syrups .................. 90 5.5 Derivatives of Penicillins .................................... 93 5.6 Production of Aspartic Acid ................................... 95 5.7 Application of ß-Galactosidase ................................ 96 5.8 Further Possible Industrial Uses .............................. 98 VIII 6 Application in Analytical Procedures ........................ 103 6.1 Affinity Chromatography ...................................... 103 6.2 Automatie Analyzers .......................................... 104 6.3 Biochemical Electrodes ....................................... 106 6.4 Enzyme Thermistors ........................................... 108 6.5 Immuno Methods ............................................... 109 7 Uses in Medicine ............................................ 112 7.1 Intracorporeal Enzyme Therapy ................................ 112 7.2 Extracorporeal Enzyme Therapy ................................ 114 7.3 Artificial Organs ............................................ 115 8 Uses in Basic Research ...................................... 117 8.1 Structural Studies ........................................... 117 8.2 Properties of Enzyme Subuni ts ................................ 118 8.3 Degeneration and Regeneration ................................ 120 8.4 Simulation of Natural Systems ................................ 122 9 Special Developments and Trends ............ , .......... , ..... 124 9.1 Immobilized Plant Cells ...................................... 124 9.2 Immobilized Mamma li an Cells .................................. 126 9.3 Immobilized Organelles ....................................... 127 9.4 Co-immobilization of Enzymes and Cells ....................... 128 9.5 Other Co-immobilized Systems ................................. 132 9.6 Combination of Immobilization with Other Techniques .......... 134 PRACTICAL SECTION .............................................. 137 Exercise 1 Adsorptive Coupling of Invertase to Active Charcoal 139 E 1.1 Introduction ................................................ 139 E 1.2 Experimental Procedure ...................................... 140 E 1.3 Results and Evaluation ...................................... 142 Exercise 2 Ionic Binding of Catalase to CM-Cellulose .......... 145 E 2.1 Introduction ................................................ 145 E 2.2 Experimental Procedure ...................................... 146 E 2.3 Results and Evaluation ...................................... 147 Exercise 3 Covalent Binding of Glucoamylase to a Carrier Bearing Oxirane Groups ............................. 150 E 3.1 Introduction ................................................ 150 E 3.2 Experimental Procedure ...................................... 151 E 3.3 Resul ts and Evaluation ...................................... 152 Exercise 4 Immobilization of ß-Galactosidase by Cross-linking . 154 E 4.1 Introduction ................................................ 154 E 4.2 Experimental Procedure ...................................... 155 E 4.3 Results and Evaluation ...................................... 157 Exercise 5 Alginate Entrapment of Yeast Cells and Their Co-entrapment with Immobilized ß-Galactosidase 158 E 5.1 Introduction ................................................ 158 E 5.2 Experimental Procedure ...................................... 159 E 5.3 Results and Evaluation ...................................... 161 IX Exercise 6 Construction and Use of a Biochemical Electrode for Essaying Glucose ............................... 165 E 6.1 Introduction ................................................ 165 E 6.2 Experimental Procedure ...................................... 166 E 6.3 Results and Evaluation ...................................... 167 Exercise 7 Spinning of ß-Galactosidase into Cellulose Acetate Fibers ............................................. 169 E 7.1 Introduction ............................................... 169 E 7.2 Experimental Procedure ..................................... 170 E 7.3 Resul ts and Evaluation ..................................... 172 Exercise 8 Immobilization of L-Asparaginase in Nylon Microcapsules ...................................... 173 E 8.1 Introduction ............................................... 173 E 8.2 Experimental Procedure ..................................... 174 E 8.3 Resul ts and Evaluation ..................................... 175 Exercise 9 Degradation of Cellulose in a Membrane Reactor ..... 177 E 9.1 Introduction ............................................... 177 E 9.2 Experimental Procedure ..................................... 178 E 9.3 Results and Evaluation ..................................... 180 Exercise 10 Conversion of Fumaric Acid to Malic Acid in a Liquid-membrane Emulsion .......................... 182 E 10.1 Introduction .............................................. 182 E 10.2 Experimental Procedure .................................... 184 E 10.3 Results and Evaluation .................................... 184 APPENDIX ....................................................... 187 Abbreviations and Symbols ............................................ 189 Literature ........................................................... 192 Index ................................................................ 206 THEORETICAL SECTION 1 General Principles 1.1 Principles of Biocatalysis Enzymes are the biocatalytically active entities upon wh ich the metabolism of all living organisms is based. They speed up (bio)chemical reactions by lowering the energy of activation, without themselves appearing in the reaction products. In this, and in the fact that the catalyst itself is not used up, the action of enzymes resembles that of inorganic catalysts. For the continued existence of organic compounds, and hence for the presence of life on Earth, the existence of an activation barrier is indispensable for preventing continual breakdown. At moderate temperatures many substances are metastable; in other words, they do not break down even though their energy content is considerably higher than that of their breakdown products. Not until an adequate stimulus is provided by the addition of energy, or the energy of activation is sufficiently lowered by a catalyst, are such substances transformed at a greater speed. Figure 1 and Table 1 illustrate the effect of enzymes and inorganic catalysts on the energy of activation and the speed of reaction. The breakdown of hydrogen peroxide into water and oxygen has been taken as an example. -1- -,t; #" - .. ~, Wi thout catalyst ,t •, Ea I , o , , t I 'Wi th catalyst E a t _ t-_S_u_b_s_tr_a_t_e_{s _)_,_e_.g _._2_H.::2_02:......--+~ ___ AG 1 l Products 2 H20 + 02 ----------------------------~~ Direction of the reaction -+ fig. 1. Energy in areaction with and without catalyst 4 Table 1. Breakdown of hydrogen peroxide with and without catalyst I Catalyst I Energy of activation Relative reaction rate kJ/mol Without catalyst 75.4 Platinum (inorganic) 50.2 Catalase (enzyme) 8.4 The catalytic action of enzymes and inorganic catalysts involves their ability to alter the distribution of charges on the compound to be conver ted, thus bringing about a lowering of the energy of activation, Ea (cf. Fig. 1). As a rule, enzymes are more efficient in this respect, i.e., they lead to a much greater decrease in the energy of activation than inorganic catalysts (cf. Table 1). This is why enzyme-catalyzed reactions usually proceed under mild conditions, Le. at lower temperatures, atmospheric pressure and physiological pH values. Furthermore, in contrast to inor ganic catalysts, they are highly specific; in other words, a particular enzyme usually catalyzes only one reaction. This means that, to a large extent, side reactions can be avoided by employing enzymatic breakdown. The thermodynamic equilibrium of areaction is in no way affected by the use of catalysts, whether inorganic or enzymic. In the presence of a catalyst, the state of equilibrium is simply reached sooner. In nature, however, for instance in the living cell, it is quite possible for reac tions to take place even if the reaction products have considerably higher free energy (G) than the initial substrates. This is achieved by the coupling of such an energy-consuming (endergonie) reaction, whose6G value is positive, with an energy-liberating Cexergonic) reaction, where the second reaction must be sufficiently exergonic (negative 6G) for the sum of the changes in free energy of the two reactions, 6G, to be zero or negative. From what has been said so far it is clear that enzymes are specific biocatalysts. The term biocatalyst applies not only to single enzymes, but also includes chains of enzymes linked to form larger units. Even acelI, with its vast number of different enzymes, can be regarded as a complex biocatalyst capable, for example, of transforming sugar into ethanol and carbon dioxide, or of even more complicated biosynthetic feats.