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Indian Journal of Biotechnology Vol 1, October 2002, pp 321-338
Trends in Immobilized Enzyme and Cell Technology
Nuclear Agriculture and Biotechnology Divi sion , Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India
Enzyme and microbial technology has influenced the process industry significantly in the recent years by im-provement of existing processes as well as in the development of new eco-friendly industrial bioprocesses. One of the techniques, which have played a significant role, is the immobilization of enzymes and cells. Immobilization helps in the retention of the biomass in a reactor geometry thus enabling in their economic reuse and in the development of continuous processes. Immobilization also improves stability and prevents product contamination thus paving the use of crude enzyme preparations like whole cells in bioprocessing. Protection of cells from environmental perturba-tions on immobilization has helped to introduce them into soils for agricultural and environmental applications. In the fabrication of biosensors immobilization helps in establishing intimate contact of the biomaterial on transducer surface and in medicine for the formation of immunobarrier. The current review delineates some of these aspects.
Keywords: bioprocessing, bioremediation, biosensors, enzyme stabilization, fermentation, immobilized cells, immo-bilized enzymes, immobilization techniques
Bioprocessing is currently gaining importance as a useful eco-friendly alternative to conventional process technology. This is mainly because unlike the chemi-cal catalysts, the biological systems have the advan-tages of accomplishing the complex chemical conver-sions under mild environmental conditions, with high specificity and efficiency resulting in better product yields with less energy consumptior .. The current de-mand for better utilization of renewable resources and pressure on industry to operate within environmen-tally compatible limits, has also been a stimulus to the development of new eco-friendly enzyme catalyzed industrial processes. The increasing use of enzymes by the biotech industries to produce specific products with characteristic attributes can be emphasized by the world sale of industrial enzymes approximating to US $ 1.6 billion which is expected to reach US $ 3.0 billion by the year 2008. Over 45% of the enzymes produced are being used in the food industry and the remaining is shared by detergent (34.4%), textile (11 %), leather (2.8%), paper and pulp 0 .2%) and other industries (5.6%) excluding enzymes for use in diagnostics and therapeutics (Neelkantan et aI, 1999). The use of enzymes in Indian industries is also on the rise. Basic hesitation in the switching over from the
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classical chemical technology to bioprocessing has been the cost of the biological catalysts and also their labile nature and to some extent lack of awareness. Biotechnology has influenced enzyme industry sig-nificantly in the recent years especially in the more efficient production of enzymes, their stabilization and economic reuse with a view to economize on the overall process.
Major limitations to the use of purified enzymes in bioprocessing is their high cost. In addition since en-zymes are soluble in aqueous media they are not ame-nable for their economical reuse. The cost of bio-processing can in turn be brought down using crude enzyme preparations like fermentation broths for ex-tracellular enzymes and cell homogenates or whole cells for intracellular enzymes. However, the major limitation of this approach especially in food and pharmaceutical industries, is in view of their very low specific activities, large excess of the crude enzyme preparations need to be employed, which leads to product contamination. The important technique which has emerged in the past two decades to solve these problems of enzyme cost and product purity is the immobilization of either enzyme preparations or the cells. Immobilization which deals with the asso-ciation of a biological system with an insoluble matrix not only stabilizes the biological system but helps in their economic reuse in batch as well as continuous bioreactor systems. One of the greatest advantages is that it also prevents contamination of the final product
322 INDIAN J 1310TECHNOL, OCTOBER 2002
with the biological system that is used, thus paving the use of crude enzyme preparations like the whole cells in bioprocessing. The importance of immobili-zation technology has now been well established and number of books and reviews have appeared from time to time (Bickerstaff, 1997; 0' Souza, 1989 a; 1991; 1999 a, b; 200 I a; Hartmier, 1988; Mattiasson, 1983 ; Mosbach, 1987 a, b, c ; Tampion & Tampion, 1987).
Immobilized Cells CUITently whole cells are gaining importance as a
source of immobilized enzymes. Whole cells can be immobilized either in a vi able or non-viable form. Important limitation in the utilization of whole cells as an intracellular source of enzymes is the diffusion of substrate and products through the cell membrane. One of the ways to obviate this problem is to use permeabilised cells. The cell s can be permeabilised using physical (freezing and thawing) or chemical (organic solvents/detergents) techniques. The most common technique uses organic solvents such as tolu-ene, chloroform, ethanol and butanol or detergents like N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB), Na-deoxycholate and digitonin (D' Souza, 1989 a; 1999 a, b; Felix, 1982; Patil & D'Souza, 1997). Our recent studies have shown that certain cells of halophilic bacteria can also be effectively permeabilised without lysing using enzymes like ly-sozyme or papain (Patil & D ' Souza, 1997). The per-meabilisation process, however, renders the cell non-viable but can serve as an economical source of intra-cellular enzymes. They can be used for simple bio-conversions that do not require cofactor regeneration or metabolic respiration (0' Souza, 1999 a). Number of techniques have been developed in our laboratory for obtaining permeabilised cells containing catalase (D'Souza & Nadkarni, 1980 a, D'Souza et al, 1987), alcohol dehydrogenase (Godbole et al, 1980), amino acid oxidase (Deshpande et ai, 1986) and some en-zymes from halophilic organisms (0' Souza et al, 1992; Patil & D'Souza, 1997).
Alternatively in the case or periplasmic enzymes, such as invertase and catalase in yeast and urease, phosphatases and penicillin G acylase in bacteria (whole cells can be used as a source of enzymes with-out permeabilisation (0' Souza & Nadkarni, 1980 a, b; Svitel et ai, 1998; Hsiau et ai, 1997; Kamath & D ' Souza, 1992; Macaskie er oi 1992 . One of the recent advances is in 'using genetic engineering
techniques to transport the intracellular proteins and anchor it into the periplasmic space (Cruz et al, 2000) . Different types of anchoring domains have been ex-plored for their efficiency in attaching hybrid proteins to the cell wall or cell membrane. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall (Leenhouts et al, 1999). This approach, which holds promise has been demonstrated for a variety of sys-tems like anchoring proteins/enzyme onto the surface of lactic acid bacteria to obtain recombinant E. coli cells with surface expressed oragnophosphorus hy-drolase (OPH), an enzyme useful in the detection of organophosphate compounds (and also for the expres-sion of cellulase activity on the cell surface for the hydrolysis of cellulose from the media (Leenhouts et ai, 1999; Mulchandani et al, 1998; Murai et al, 1997) . These types of approaches may help in the utilization of whole cells as a source of enzymes without the need for their permeabilisation, and may have major significance in the future in immobilized enzyme technology. Studies from our laboratory and others have shown the possibility of introducing enzymes onto a cell wall surface through chemical or biospeci-fic affinity techniques (0' Souza 1989 b; 0' Souza & Melo, 1991; D ' Souza & Nadkarni , 1980 c; Kaul et al, 1986). The other major limitation in the use of whole cells as an enzyme source is the possibility of un-wanted side reactions. These can be avoided by inac-tivating such enzymes if any, using inhibitors, heat or chemicals (Godbole et al, 1983 a). Permebilisation of the cells often empties the cell of most of its cofactors thus minimizing side reactions. Thus unlike a whole viable cell of yeast which can ferment sugars like su-crose or lactose to ethanol, permeabJised cells results only in their hydrolysis to monosaccharides (Joshi et al, 1989; Rao et al, 1988).
Immobilized viable cells are gaining importance in fermentation (D'Souza, 1989 a; 1999 a, b; Navratil & Sturdik, 1999; Ramakrishna & Prakasham, 1999). One of the important challenges for the biotechnolo-gists in the future is in improving the fermentation techniques. The classical fermentation suffers from various constraints such as low cell density, nutri-tional limitations and batch mode of operation with high down times. It has been well recognized that microbial cell density is of prime importance to attain higher volumetric productivity. The major limitation in the development of continuous fermentation proc-ess has been the wash out of cells from the bioreactor.
D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 323
Use of f10cculating strains, cell recycle and membrane reactors are being investigated to solve some of these problems. The immobilized viable cell technology can eliminate most of the constraints faced with the free cell systems. The remarkable advantage is the free-dom to determine the cell density pri