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      Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs)

      review-article
      Applied Microbiology and Biotechnology
      Springer-Verlag
      Immobilization, Hydrolases, Oxidoreductases, Lyases, Magnetic, Sustainable

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          Abstract

          Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes.

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          Most cited references61

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          Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.

          Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
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            Glucose oxidase--an overview.

            Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of beta-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide. Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications of glucose oxidase in biosensors have increased the demand in recent years. Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucose oxidase by fermentation is detailed, along with recombinant methods. Various purification techniques for higher recovery of glucose oxidase are described here. Issues of enzyme kinetics, stability studies and characterization are addressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.
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              Directed evolution drives the next generation of biocatalysts.

              N. Turner (2009)
              Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.
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                Author and article information

                Contributors
                +31-15-2782675 , +31-15-2781415 , r.a.sheldon@tudelft.nl
                Journal
                Appl Microbiol Biotechnol
                Applied Microbiology and Biotechnology
                Springer-Verlag (Berlin/Heidelberg )
                0175-7598
                1432-0614
                2 September 2011
                2 September 2011
                November 2011
                : 92
                : 3
                : 467-477
                Affiliations
                Department of Biotechnology, Delft University of Technology, Julianalaan 136, NL-2628 BL Delft, The Netherlands
                Article
                3554
                10.1007/s00253-011-3554-2
                3189406
                21887507
                f49d869f-0c13-4e95-b5f5-3ae07cf27336
                © The Author(s) 2011
                History
                : 11 July 2011
                : 8 August 2011
                : 13 August 2011
                Categories
                Mini-Review
                Custom metadata
                © Springer-Verlag 2011

                Biotechnology
                oxidoreductases,hydrolases,lyases,sustainable,magnetic,immobilization
                Biotechnology
                oxidoreductases, hydrolases, lyases, sustainable, magnetic, immobilization

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