Contributors

José M. Abad • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Olga Abian • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Patrick Adlercreutz • Department of Biotechnology, Lund University, Lund, Sweden

Noelia Alonso-Morales • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Silvana Andreescu • Department of Chemistry, Clarkson University, Potsdam, New York

Francisco Batista-Viera • Cátedra de Bioquímica, Facultad de Química,

Universidad de la República, Montevideo, Uruguay Ezio Battistel • Istituto Guido Donegani, Polimeri Europa SpA, Novara, Italy Lorena Betancor • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Ana Blandino • Department of Chemical Engineering, Food Technology, and Environmental Technologies, Facultad de Ciencias, University of Cadiz, Cadiz, Spain

Beatriz M. Brena • Cátedra de Bioquímica, Facultad de Química, Universidad de la República, Montevideo, Uruguay Bogdan Bucur • BIOMEM, Centre de Phytopharmacie, Université de Perpignan,

Perpignan Cedex, France Gema Cabrera • Department of Chemical Engineering, Food Technology and Environmental Technologies, Facultad de Ciencias, University of Cadiz, Cadiz, Spain

Monica Campàs • BIOMEM, Centre de Phytopharmacie, Université

de Perpignan, Perpignan Cedex, France Laurent Coquet • Laboratory of Polymers, Biopolymers, and Membranes (PBM), UMR 6522 CNRS and European Institute for Peptide Research (IFRMP 23), University of Rouen Cedex, France Pascal Cosette • Laboratory of Polymers, Biopolymers, and Membranes (PBM), UMR 6522 CNRS and European Institute for Peptide Research (IFRMP 23), University of Rouen Cedex, France Gisella Dellamora • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Teresa de Diego • Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus del Espinardo, Spain

Amaia Esquisabel • Laboratorio de Farmacia y Tecnologia Farmacéutica, Facultad de Farmacia, Universidad del País Vasco (EHU-UPV), Vitoria-Gasteiz, Spain

Pedro Fernandes • Centro de Engenharia Biologica e Química, Instituto Superior

Técnico, Lisboa, Portugal Víctor M. Fernández • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Roberto Fernandez-Lafuente • Institute of Catalysis, CSIC, Campus UAM-

Cantoblanco, Madrid, Spain Gloria Fernández-Lorente • Institute of Catalysis, CSIC, Campus UAM-

Cantoblanco, Madrid, Spain Lutz Fischer • Institute of Food Technology, Department of Biotechnology,

University of Hohenheim, Stuttgart, Germany Manuel Fuentes • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Alicia Rodríguez Gascón • Laboratorio de Farmacia y Tecnologia Farmacéutica, Facultad de Farmacia, Universidad del País Vasco (EHU-UPV), Vitoria-Gasteiz, Spain

Peter Gemeiner • Department of Glycobiotechnology, Institute of Chemistry, Slovak

Academy of Sciences, Bratislava, Slovakia Raquel de Lima Camargo Giordano • Departamento de Engenharia Química,

Universidade Federal de Sao Carlos, Sao Carlos, Brasil Roberto Campos Giordano • Departamento de Engenharia Química, Universidade

Federal de Sao Carlos, Sao Carlos, Brasil Valeria Grazu • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Serge R. Guiot • Groupe de bioingenierie de l'environnement, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada

Jose M. Guisan • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Munishwar N. Gupta • Department of Chemistry, Indian Institute of Technology,

Delhi, N. Delhi, India Rosa María Hernández • Laboratorio de Farmacia y Tecnologia Farmacéutica, Facultad de Farmacia, Universidad del País Vasco (EHU-UPV), Vitoria-Gasteiz, Spain

Aurelio Hidalgo • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

José L. Iborra • Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus del Espinardo, Spain

Thierry Jouenne • Laboratory of Polymers, Biopolymers and Membranes (PBM), UMR 6522 CNRS and European Institute for Peptide Research (IFRMP 23), University of Rouen Cedex, France Guy-Alain Junter • Laboratory of Polymers, Biopolymers and Membranes (PBM), UMR 6522 CNRS and European Institute for Peptide Research (IFRMP 23), University of Rouen Cedex, France

Contributors xiii

Martina Koneracká • Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia Peter KopCansky • Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia

Fernando López-Gallego • Institute of Catalysis, CSIC, Campus UAM-

Cantoblanco, Madrid, Spain Pedro Lozano • Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Campus del Espinardo, Spain Nirupama Mallick • Agricultural and Food Engineering Department, Indian

Institute of Technology, Kharagpur, West Bengal, India Carmen Manta • Cátedra de Bioquímica, Facultad de Química, Universidad de la

República, Montevideo, Uruguay Jean-Louis Marty • BIOMEM, Centre de Phytopharmacie, Université de

Perpignan, Perpignan Cedex, France Cesar Mateo • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Tamara Montes • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Peyman Moslemy • Sr. Scientist, Spherics, Inc., Lincoln, RI

Marián Navrátil • Department of Biochemical Technology, Faculty of Chemical and

Food Technology Slovak University of Technology, Bratislava, Slovak Republic Ronald J. Neufeld • Department of Chemical Engineering, Queen's University,

Kingston, Ontario, Canada Gilvanda Silva Nunes • Technological Chemistry Department, CCET, Federal

University of Maranhao, Sao Luís, Maranhao, Brazil Gorka Orive • Laboratorio de Farmacia y Tecnologia Farmacéutica, Facultad de

Farmacia, Universidad del País Vasco (EHU-UPV), Vitoria-Gasteiz, Spain Claudia Ortiz • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Ignacio de Ory • Department of Chemical Engineering, Food Technology, and Environmental Technologies, Facultad de Ciencias, University of Cadiz, Cadiz, Spain

Karen Ovsejevi • Cátedra de Bioquímica, Facultad de Química, Universidad de la

República, Montevideo, Uruguay Jose M. Palomo • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

José Luis Pedraz • Laboratorio de Farmacia y Tecnologia Farmacéutica, Facultad de Farmacia, Universidad del País Vasco (EHU-UPV), Vitoria-Gasteiz, Spain Benevides C. C. Pessela • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Marcos Pita • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain Chenyl Nynitapal Ramchand • Kemin Nutricional Technologies Pvt Ltd., Tamil Nadu, India

Martin Ramirez • Department of Chemical Engineering, Food Technology, and Environmental Technologies, Facultad de Ciencias, University of Cadiz, Cadiz, Spain

Manfred T. Reetz • Max-Planck-Institut für Kohlenforschung, Mülheim/Ruhr, Germany

Giovanni Rialdi • ISMAC-Istituto per lo Studio delle Macromolecole, CNR,

Sezione di Genova, Genova, Italy Ipsita Roy • Department of Chemistry, Indian Institute of Technology, Delhi, N. Delhi, India

Zainul M. Saiyed • School of Mechanical and Systems Engineering, University of Newcastle upon Tyne, Newcastle, United Kingdom Marc Schlieker • geniaLab GmbH, Braunschweig, Germany Rob Schoevaart • CLEA Technologies, Delft, The Netherlands Rosa L. Segura • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Anil de Sequeira • School of Science and Environment, Bath Spa University

College, Newton Park Campus, Bath, United Kingdom Roger A. Sheldon • Delft University of Technology, Biocatalysis and Organic

Chemistry, Delft, The Netherlands Juraj v VITEL • Pure and Applied Biochemistry, Center for Chemistry and Chemical

Engineering, Lund University, Lund, Sweden Milan Timko • Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia

Rodrigo Torres • Institute of Catalysis, CSIC, Campus UAM-Cantoblanco, Madrid, Spain

Michael Trevan • University of Westminster, London, United Kingdom Alankar Vaidya • Institute of Food Technology, Department of Biotechnology,

University of Hohenheim, Stuttgart, Germany Luuk M. van Langen • CLEA Technologies, Delft, The Netherlands Sébastien Vilain • Laboratory of Polymers, Biopolymers, and Membranes (PBM), UMR 6522 CNRS and European Institute for Peptide Research (IFRMP 23), University of Rouen Cedex, France Klaus-Dieter Vorlop • geniaLab GmbH, Braunschweig, Germany

Immobilization of Enzymes as the 21st Century Begins

An Already Solved Problem or Still an Exciting Challenge? Jose M. Guisan

Summary

The main goal of enzyme immobilization is the industrial re-use of enzymes for many reaction cycles. In this way, simplicity and improvement of enzyme properties have to be strongly associated with the design of protocols for enzyme immobilization. In spite of their excellent catalytic properties, enzymes have many other characteristics that are not very suitable for their use in chemical industries: low stability, inhibition by high concentrations of substrates and products, low activity and selectivity toward nonnatural substrates under nonconventional conditions, and so on. The possibility of improving these unsuitable characteristics via the design of simple immobilization protocols is a very exciting goal. There are many protocols for immobilization of enzymes but very few are also very simple and/or very capable of improving enzyme properties. Novel immobilization protocols are still needed in order to achieve a massive implementation of enzymes as catalysts of the most complex chemical processes under the most benign experimental and environmental conditions. A critical review of enzyme immobilization under this point of view is still necessary.

Key Words: Enzymes and sustainable chemical industries; enzyme immobilization protocols; enzyme immobilization techniques; enzyme properties.

1. Advantages and Limitations of Enzymes as Industrial Catalysts

Because of their excellent functional properties (activity, selectivity, and specificity), enzymes are able to catalyze the most complex chemical processes under the most benign experimental and environmental conditions (1). Enzymes are able to catalyze, under very mild conditions, very fast modifications of a unique functional group (between several similar groups) existing in only one substrate in the presence of other very similar molecules. Therefore, enzymes may be excellent

From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ

Enzyme Immobilization Biotechnology
Fig. 1. Biological and chemical tools to improve enzyme properties.

industrial catalysts in a number of areas of chemical industry such as fine chemistry, food chemistry, and analysis.

However, enzymes have been modified during biological evolution in order to optimize their behavior in the framework of complex catalytic chains, inside living things under stress and needing regulation. Obviously, enzymes have not been optimized to work inside industrial reactors. In this way, enzymes, in addition to their excellent catalytic properties, also have some characteristics that are not very suitable for industrial applications: they are soluble catalysts, they are usually very unstable, they may be strongly inhibited by substrates and products, and they only work well on natural substrates and under physiological conditions. In most cases, enzymes have to be greatly improved before their use in industrial processes. The engineering of enzymes, from biological to chemical industries, is one of the most exciting, complex, and interdisciplinary goals of biotechnology. The large-scale implementation of enzymes as industrial catalysts requires a multidisciplinary utilization of very different techniques (see Fig. 1): (1) the screening of enzymes with suitable properties (2), (2) the improvement of enzyme properties via techniques of molecular biology (3), (3) the improvement of enzyme properties via immobilization and postimmobilization techniques (4,5), and (4) the improvement of enzyme properties via reaction and reactor engineering (6,9). Such a successful improvement of enzyme properties should be one of the key solutions for the development of a much more sustainable chemical industry that is able to synthesize very complex and useful compounds under very mild and cost-effective conditions.

2. Immobilized Enzymes as Catalysts of Industrial Chemical Processes

For both technical and economic reasons most chemical processes catalyzed by enzymes require the re-use or the continuous use of the biocatalyst for an extended period of time (10,11). Under this perspective, immobilization of enzymes may be

Silvana Andreescu
Fig. 2. Factors to be considered in design of a biocatalyst.

defined as any technique that is able to allow the re-use or continuous use of the biocatalysts. From this industrial point of view, simplicity and cost-effectiveness are the key properties of immobilization techniques. On the other hand, a long-term industrial re-use of immobilized enzymes also requires the preparation of very stable derivatives that also have suitable functional properties for a given reaction (e.g., activity and selectivity). At first glance, the practical development of protocols for immobilization of enzymes is intimately related to simplicity, cost-effectiveness and stability, and stabilization of enzymes (see Fig. 2).

With the above-mentioned parameters in mind, the thousands of protocols for enzyme immobilization that are already reported in scientific literature should be reassessed according to a number of criteria:

1. No need to use toxic or hazardous reagents during and after the immobilization process.

2. The use (in biotechnology companies) of very stable activated pre-existing supports that could have been prepared by other companies with high levels of expertise in synthesis, as well as activation of supports and security protocols.

3. The possibility of associating immobilization of enzymes and improvement of functional properties (activity, stability, and selectivity). In general, we can assume that the preparation of very active and very stable immobilized derivatives is a key requirement for a successful industrial application of such derivatives. Poorly activated and unstable derivatives could be useful for laboratory trials but they are unlikely to be useful for industrial applications. From a practical point of view, very stable enzymes need to be immobilized or enzyme stability needs to be greatly improved as a consequence of their immobilization.

4. The preparation of immobilized derivatives useful in different reactions (e.g., those involving soluble or insoluble substrates, a need for cofactor regeneration, or presence of oxygen as enzyme substrate), in different reaction media (e.g., water, organic solvents, supercritical fluids), and in different reactors (e.g., stirred tanks, fluidized beds) 5. The preparation of immobilized derivatives for use in different applications such as fine chemistry, biosensors, and therapeutic applications.

3. The Simplicity of Immobilization of Enzymes

Enzymes can be immobilized in different places for different purposes. In general, it can be assumed that simplicity and the cost-effectiveness of immobilization are not very important when working at the laboratory scale but would be critical for industrial applications.

3.1. Immobilization of Enzymes at the Laboratory Scale

This may be performed for preliminary trials of interesting biotransformations or for more basic structural and functional tests. In these cases, use of toxic or harmful reagents before, during, and after immobilization is not necessary. In addition, unstable activated supports can be used for very rapid immobilization of a low amount of enzyme. Moreover, cost-effectiveness is hardly relevant.

3.2. Immobilization of Enzymes at an Industrial Scale

Many companies (e.g., those in fine chemistry and food chemistry) are able to produce industrial enzymes but are not able—and do not want to be able—to synthesize and activate supports. In these cases, the development of very simple and cost-effective protocols for enzyme immobilization is critical. In addition, the use of very stable and ready-to-use activated supports would also be very convenient.

4. The Improvement of Enzyme Properties Via Immobilization and Postimmobilization Techniques

Functional properties of industrial enzymes can be greatly improved by using suitable protocols for controlled and directed immobilization (see Fig. 3) (12). In this way, immobilization of enzymes is a technique necessary for the re-use of enzymes and can also become a very powerful tool for the improvement of enzyme properties. Moreover, enzyme properties could also be improved through physical and chemical modification of immobilized derivatives. Both techniques of enzyme engineering are fairly compatible with additional engineering via preliminary biological techniques (e.g., microbiology, molecular biology) (13-15).

The following are some protocols for enzyme improvement via immobilization that have already been reported in literature.

4.1. Stabilization of Enzymes by Random Immobilization

Covalent immobilization or strong physical adsorption of enzymes, fully dispersed on the internal surface of porous supports, may promote very interesting stabilizing effects such as (16) (1) the immobilized enzyme is not able to undergo any intramolecular process (e.g., autolysis, proteolysis, aggregation), and (2) the immobilized enzyme (inside a porous structure) is not able to undergo undesirable interactions with large hydrophobic interfaces (e.g., air/oxygen bubbles, immiscible organic solvents). In this way, under certain experimental conditions, these random immobilization protocols may promote very important stabilizations of

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