Industrial enzymes enzyme immobilization

Choice of Method. Numerous enzyme immobilization techniques have been described in the Hterature comprehensive books on this and related subjects, including industrial appHcations, are available (33—36). The more general techniques and some selection criteria are included herein. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The hydrolysis of nitriles can be carried out with either isolated enzymes or immobilized cells. Eor example, resting cells of P. chlororaphis can accumulate up to 400 g/L of acrylamide in 8 h, provided acrylonitrile is added gradually to avoid nitrile hydratase inhibition (116). The degree of acrylonitrile conversion to acrylamide is 99% without any formation of acryUc acid. Because of its high efficiency the process has been commercialized and currentiy is used by Nitto Chemical Industry Co. on a multithousand ton scale. 

Immobilized enzymes and immobilized cells are being employed as specific heterogeneous catalysts by several of the chemical industries. An evaluation of the mode of action and efficiency of... 

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

For some recent reviews, see Pfenosil, J.E., Kut, O.M., Dunn, I.J. and Heinzle, E., Immobilized biocatalysts. In Ullman s Biotechnology and Biochemical Engineering, vol. 2. Wiley-VCH, Weinheim, 2007, pp. 683-734 Sheldon, R. A., Enzyme immobilization the quest for optimum performance. Adv. Synth. Catal., 2007, 349, 1289-1307 End, N. and Schoning, K.-U., Immobilized biocatalysts in industrial research and production. Topics Curr. Chem., 2004, 242, 273-317 Bornscheuer, U.T., Immobilizing enz3mies how to create more suitable biocatalysts. Angew. Chem. Int. Ed., 2003,42, 3336-3337 Cao, L. Immobilised enzymes science or art Curr. Opin. Chem. Biol., 2005, 9, 217-226. 

It is also important to note that molecular biology, while it is a very powerful tool, is probably most effective in industrial process development when used in conjunction with other techniques such as enzyme formulation, immobilization and appropriate process design engineering. ... 

In this paper we have immobilized an enzyme within a thermally reversible hydrogel. Immobilized enzymes have been used in a variety of applications, ranging from treatment of diseases to sensors, assays, and industrial processes (15-20). When an enzyme is immobilized within a gel which exhibits reversible shrinking and swelling as the ten rature is raised and lowered through the LCST of the gel matrix polymer, the enzyme may be switched off and on as the substrate diffusion rate is regulated by the gel pore size (5). In adcfition to enzymes, a variety... 

The method is widely used for immobilization of viable cells, where the open gel makes transport of nutrients/metabolites to and from the cell possible, but it has not gained any industrial importance in immobilizing enzymes. The reasons for this are the... 

Tosa, T. and Shibatani, T. (1995) Industrial Applications of Immobilized Biocatalysts in Japan. Enzyme Engineering XU, edited by M.-D.Legoy and D.N.Thomas. Aimals of the New York Academy of Science, Vol. 750, 364-375. 

W. II. Pitcher Design and Operation of Immobilized Enzyme Reactors. - S. A Barker Biotechnology of Immobilized Multienzyme Systems. - R. A Messing Carriers for Immobilized Biologically Active Systems. -P. Brodelius Industrial Applications of Immobilized Biocatalysts. - B. Solomon Starch Hydrolysis by Immobilized Enzymers. 

Dohan, L. A., Baret, J.-L., Pain, S. and Delalande, P. 1980. Lactose hydrolysis by immobilized lactase Semi-industrial experience. Enzyme Eng. 5, 279-293. 

A difficult problem in utilizing enzymes as catalysts for reactions in a non-cellular environment is their instability. Most enzymes readily denature and become inactive on heating, exposure to air, or in organic solvents. An expensive catalyst that can be used only for one batch is not likely to be economical in an industrial process. Ideally, a catalyst, be it an enzyme or other, should be easily separable from the reaction mixtures and indefinitely reusable. A promising approach to the separation problem is to use the technique of enzyme immobilization. This means that the enzyme is modified by making it insoluble in the reaction medium. If the enzyme is insoluble and still able to manifest its catalytic activity, it can be separated from the reaction medium with minimum loss and reused. Immobilization can be achieved by linking the enzyme covalently to a polymer matrix in the same general manner as is used in solid-phase peptide synthesis (Section 25-7D). 

The successful conversion of D-glucose into D-fructose on the industrial scale with immobilized D-glucose isomerase was a brilliant demonstration of the value of this kind of approach. Then followed a huge technical literature on enzyme immobilization, reviewed in Ref. 9 (page 353). We shall here restrict ourselves to the methods which have been utilized in the syntheses outlined in Tables II to X. We suggest to readers interested in theses techniques that they first use these methods. If they prove unsatisfactory, as there is a plethora of alternatives, other techniques, described in Refs. 8-10, may be tried a majority of readily available carbohydrate enzymes have been immobilized, often in several different ways. 

The use of industrial enzymes for the synthesis of bulk and fine chemicals represents a somewhat specialized application for biocatalysts relative to their broader uses, as outlined above. Industrial biocatalysis is, however, becoming increasingly relevant within the chemical industry for the production of a wide range of materials (see Table 31.3).1,2,4-8 Broadly defined, a biocatalytic process involves the acceleration of a chemical reaction by a biologically derived catalyst. In practice, the biocatalysts concerned are invariably enzymes and are used in a variety of forms. These include whole cell preparations, crude protein extracts, enzyme mixtures, and highly purified enzymes, both soluble and immobilized. 

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