Post on 06-Mar-2018
Indian Journal of Biotechnology Vol 1, October 2002, pp 321-338
Trends in Immobilized Enzyme and Cell Technology
S FD'Souza*
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 improvement 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 perturbations 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, immobilized enzymes, immobilization techniques
Introduction
Bioprocessing is currently gaining importance as a useful eco-friendly alternative to conventional process technology. This is mainly because unlike the chemical catalysts, the biological systems have the advantages of accomplishing the complex chemical conversions under mild environmental conditions, with high specificity and efficiency resulting in better product yields with less energy consumptior .. The current demand for better utilization of renewable resources and pressure on industry to operate within environmentally 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
*Fax: +91-22-5505151 E-mail : sfdsouza@apsara.bare.erne!.in
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 significantly 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 enzymes are soluble in aqueous media they are not amenable for their economical reuse. The cost of bioprocessing can in turn be brought down using crude enzyme preparations like fermentation broths for extracellular 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 association 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
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with the biological system that is used, thus paving the use of crude enzyme preparations like the whole cells in bioprocessing. The importance of immobilization 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 toluene, 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 lysozyme or papain (Patil & D ' Souza, 1997). The permeabilisation process, however, renders the cell nonviable but can serve as an economical source of intracellular enzymes. They can be used for simple bioconversions 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 enzymes 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 without 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 explored 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 systems like anchoring proteins/enzyme onto the surface of lactic acid bacteria to obtain recombinant E. coli cells with surface expressed oragnophosphorus hydrolase (OPH), an enzyme useful in the detection of organophosphate compounds (and also for the expression 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 biospecific 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 unwanted side reactions. These can be avoided by inactivating 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 sucrose 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 biotechnologists in the future is in improving the fermentation techniques. The classical fermentation suffers from various constraints such as low cell density, nutritional 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 process 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 freedom to determine the cell density prior to fermentation. It also facilitates operation of fermentation on a continuous mode without cell wash out even at high dilution rates. The immobilized cell technology process also decouples microbial growth from cellular synthesis of favoured compounds. In addition to microbial cells the fermentation technology using bioreactors is also gaining importance for the production of high value compounds using plant and animal cell cultures. Immobilization of such cells have been shown to offer them stability against shear force when used in continuous stirred tank bioreactors (Doernenburg & Knorr, 1995; Liang et ai, 2000; Shoji et ai, 2000).
Techniques for the Immobilization of Biocatalysts Biocatalysts can be immobilized either through ad
sorption, entrapment, covalent binding, cross-linking or a combination of all these techniques (Bickerstaff, 1997; D'Souza, 1989 a; 1998; 1999 a) Covalent binding is a commonly used technique for the immobilization of enzymes and antibodies. A variety of techniques are now available for covalent binding of enzymes to natural and synthetic polymers and inorganic supports. A number of reviews and books deal with such techniques (D'Souza, 1999 a; Hartmier, 1988; Mosbach, 1987 a, b, c). The common approach is to introduce highly active electrophilic or nucleophilic groups through activation of the support matrix followed by the covalent bond formation with biological systems under mild reaction conditions. When covalent binding or cross-linking is used precaution needs to be taken so as to bind the enzyme without significantly affecting its conformational flexibility and activity. Use of substrate or substrate analogues (Godbole et ai, 1984; Marolia & D' Souza, 1999; Melo et ai, 1986; Melo & D'Souza, 2000) during immobilization has been often used to protect the active site from inactivation. Conformational flexibility can be retained by covalent binding of the enzymes using spacer arms (Bonnington et ai, 1995; Sarfo et ai, 1995). Glycoprotein enzy mes like glucose oxidase, peroxidase and invertase can also be covalently bound via their carbohydrate moiety (Husain & Jafri, 1995; Melo & D'Souza, 1992). Such an approach often
results in better retention of enzyme activity as it avoids the chemical modification of functional groups in the protein moiety of the enzyme. Covalent binding however has not been very useful for the immobilization of cells. One of the general problems with covalent binding is that the cells are exposed to potent reactive groups and other harsh reaction conditions thus affecting their viability. There may also be a loss in the structural integrity of the cell during continuous use, leading to loss of intracellular enzymes. Among others is the very low cell loading that is achieved as compared to entrapment and other techniques. A few recent reports are, however, available on the covalent binding of cells for specific applications. Jirku (1999) has covalently bound Saccharomyces cervisiae cells to an epoxide derivative of hydroxyalkylmethacrylate gel via glutaraldehyde-diamine spacers. Direct covalent binding on glutaraldehyde activated proteinc supports like wool has also been reported (Krastanov, 1997). Covalent binding of cells by introduction of active aldehyde groups on cell surface using periodate oxidation has also shown promise (Abelyan, 2000). Useful techniques have been developed in our laboratory for the covalent binding of cells on Se ph arose for use as affinity ligand for the purification of enzymes (D'Souza & Marolia, 1999). These include the direct covalent linkage using Epoxy-activated Sepharose or to amino-Sepharose using glutaraldehyde.
Entrapment is a useful technique for the immobilization of cells. However it is not a good technique for the immobilization of cell free enzymes in view of their possible leakage from the entrapment matrix. Enzymes have been encapsulated in liposomes for their controlled release (Laloy et ai, 1998) and also inside the reversed micelle (Das et aI, 1997). Cells have been immobilized in a variety of synthetic and natural polymers like polyacrylamide, polyvinyl alcohol, polyurethane foams, carrageenan, agarose, alginate, pectin and chitosan (Bickerstaff, 1997; D' Souza, 1999 a, b; Ramakrishna & Prakashan, 1999). Acrylic polymers have shown promise (Hsiau et aI, 1997). Entrapment of cells in polyacrylamide (blocks or beads) using gamma-ray polymerisation has been extensively investigated in our laboratory (Deshpande et ai, 1987; D'Souza 1998; 1999 a; D'Souza & Nadkarni, 1980 a, b, c; Ghosh & D'Souza, 1989; Godbole et ai, 1983 a, b). Basic advantage of polymerization using gamma rays is that unlike the routinely used chemical polymerization technique, radiation polymerization can be carried out even at -75°C. This not
324 INDIAN J BIOTECHNOL, OCTOBER 2002
only prevents heat inactivation (Deshpande et ai, 1987; Gupte & D'Souza, 1999) but also the samples can be frozen in any required geometry helping in obtaining entrapment system in bead, tube or membrane forms. Radiation polymerization technique has also been extended to entrap cells in gelatine. This technique may be useful for immobilization of enzymes which are otherwise sensitive to glutaraldehyde (Deshpande et ai, 1986).
Entrapment in alginate by ionotropic gelation using a variety of divalent and trivalent cations has found extensive use in immobilized viable cell technology (Kierstan & Bucke, 2000; Smidsord & Skjak-Braek, 1990). The major limitations of Ca-alginate gels is their destabilization and subsequent solubi lisation by the Ca-chelators present in the processing solution or waste and their low mechanical strength and density. The common approaches used for stabilizing alginate gels include direct covalent cross-linking of the carboxyl groups and" covalent grafting of alginate with synthetic polymers. Some of these include crosslinking with polyvinyl alcohol and treatment with polyethylenimine followed by cross-linking (Hertzber et ai, 1995; Kokofuta et ai, 1987; Smidsord & Skjak-Braek, 1990). A novel technique has been developed at BARC, Mumbai for stabilizing the alginate beads towards Ca-'Chelators by reinforcing them with gamma ray polymerised polyacrylamide (Gupte & D'Souza, 1999). Unlike the Ca salt; Ba-alginate gels have been shown to have better mechanical compression and have been shown to have low oxygen permeability for use under anoxic conditions like denitrification (Yamagiwa et ai, 1997). Density and strength of alginate gels can be enhanced by incorporation of inorganic materials like silica, sand and alumina (Ramakrishna & Prakasham, 1999). Effect of sterilization of alginate prior to its use as support has been discussed (Leo et ai, 1990).
Other promising synthetic polymers include polyurethane based hydrogels, photo- cross-linkable resins and polyvinyl alcohol (Koenig et ai, 1997; Fukui et ai, 1987; Yang et ai, 1997; Tag et ai, 2000). Polyvinyl alcohol is one of the most widely studied polymers, as it can form beads, membranes, fibres , etc. Enzymes and cells have been immobilized in them either by entrapment, covalent binding, cross-linking, freezing and thawing, y-irradiation, photo-cross linking or entrapment followed by cro~s-linking (Uhlich et aL, 1996). A technique has also been reported using polyvinyl alcohol crosslinked with sodium nitrate.
This new technique can simultaneously eliminate the agglomeration of PV A beads and the toxicity of boric acid caused by the PV A-boric acid and PV Aorthophosphate methods (Chang & Tseng, 1998). Photo-cross linkable polyvinyl alcohol bearing styrylpyridinium groups has been shown to entrap cellular organelles and cells under very mild conditions retaining their biological activity (Rouillon et ai, 1995; 1999). Polyacrylonitrile membranes (Ulbricht & Papra, 1997) and albumin-poly (ethylene glycol) hydrogel (D' Urso & Fortier, 1996) have aiso shown promise in the immobilization of enzymes. Albumin-poly (ethylene glycol) hydrogels, in view of their biocompatibility, may be useful in medical applications (D'Urso & Fortier, 1996). Others include Poly carbamoylsulphonate, a hydrogel matrix of low toxicity retaining survival rates of microorganisms greater than 99% (Wilke et ai, 1994; Lehmann et ai, 1999). Highly stable immobilized lipase preparations have been obtained by entrapment in poly (N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate) hydrogel, with divinylbenzene as the crosslinking agent (Basri et ai, 1999). The use of a novel immobilization technique utilizing an oil-in-water macroemulsion, termed as colloidal liquid aphron has been developed for the entrapment of enzymes for use in non-aqueous media (Lamb & Stuckey, 1999). Microbial cells especially fungal cells have been immobilized by passive entrapment in polyurethane or vegetable sponges (Federici et ai, 1996; Manohar et aL, 200 I; Pinheiro & Cabral, 1992; Slokoska & Angelova, 1998). In addition to microbial cells entrapment techniques have also been used for the immobilization of viable animal cells and cellular organelles (Bugarski et ai, 1993; Lee & Palsson, 1990; Shen et aI, 1993). Major limitation of entrapment technique is the additional diffusional barrier offered by the entrapment materials, which can be minimized by increasing the porosity of the matrix using open pore entrapment techniques (Miranda & D'Souza, 1988; SivaRaman et ai, 1982). Others include entrapment in hollow fibre modules (Ju et ai, 2000; Lloyd et ai, 1999; Piret & Cooney, 1991; Rucka & Sroka, 1989) A highly porous sponge type proteinic matrix has been developed in our laboratory which allows for the diffusion of even bacterial cells into the vicinity of the bound enzyme (Marolia & D'Souza, 1993; 1999).
Immobilization of enzymes and cetl s through ad · sorption perhaps is the simplest of all the techniques. Enzymes have been immobilized through adsorption
D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 325
on a variety of commercial ion exchange resins and gels. The basic advantage is the reversibility of binding which also helps in economic recovery of the support. This has been successfully adapted in industry for the resolution of racemic mixtures of amino acids using amino acid acylase. In addition to ion exchange resins, enzymes have also been immobilized through adsorption on hydrophobic supports as well as affinity supports (0' Souza, 1999 a).
Adsorption or more appropriately termed as adhesion is perhaps the oldest method of immobilizing cells. Many cells have natural tendency to adhere to solid surfaces. Naturally adhered cells have played an important role in many biotechnological applications such as wastewater treatment and fermentations like vinegar (Marshall, 1984). Techniques for the adhesion of whole cells on polymeric surfaces has gained considerable importance (0' Souza, 1990). A variety of approaches are being applied. The most common is the passive adsorption of cells on surfaces such that a natural biofilm is obtained (Ho et al. 1997; Lewis & Yang, 1992; Yang & Huang, 1995). The major limitation of this approach is the long time ranging from days to number of weeks required for the formation of the biofilm. Under natural pH conditions most of the cells have a net negative charge and can hence be adsorbed on ion exchangers (Bar et al, 1986). However ion exchangers often possess poor binding capacity. These limitations have been reduced by adhesion of cells using metallic ions like Al+3 or by coating the support or the cells with colloidal particles which act as binding agents between the cell surface and the support most commonly used being glass surface (Van Haecht et al, 1985). Activated or oxidised carbon filaments were found to efficiently adsorb a variety of bacterial cells (Kalenyuk et al, 1999). Adhesion of cells has also been facilitated by nutrient starvation (Bringi & Dale, 1985). Novel techniques have been developed in our laboratory for immobilizing viable or non-viable cells through adhesion on a variety of polymeric surfaces including glass, cotton fabric and synthetic polymeric membranes using polyethylenimine (PEI) (D'Souza et al, 1986, D'Souza, 1990; D'Souza & Kamath, 1988; D'Souza & Melo, 2001; Kamath & D'Souza, 1992; Melo & D'Souza, 1999). The adhesion is found to be rapid and the cells adhere as a monolayer. Adhesion being very strong, the high ionic concentrations and extreme pH conditions, which normally disrupt the ionic interactions, fail to desorb the cells. Cells can be adhered by
coating either the cells, the support or both, with PEI (D'Souza et al, 1986; D'Souza & Kamath, 1988). Viability of the cell was not affected by this treatment. The technique has also been used for the simultaneous filtration and immobilization of cells from a flowing suspension, thus integrating downstream processing with bioprocessing (Melo & 0' Souza, 1999). These studies were recently extended for immobilization of invertase containing yeast cells through adhesion on jute fabric for use in an annular column reactor for the inversion of concentrated sucrose syrups (D'Souza & Melo, 2001). The PEI technique developed in our laboratory has been applied by others for the adhesion of cells and proteins (Nandkumar & Mattiasson, 1999; Senthuran et al. 1997; Tampion & Tampion, 1987; Guilbault, 1989).
Cross-linking using bifunctional reagents like glutaraldehyde has been successfully used for the immobilization of enzymes and cells in various supports. Of these, proteinic supports such as gelatine, collagen (0' Souza, 1989 a; Deshpande et al, 1986; Srivastava et al, 2001; Svitel et al, 1998), albumin (Loranger & Carpentier, 1994) and hen egg white (D'Souza et al, 1985; D'Souza & Nadkarni, 1981; Marolia & 0' Souza, 1994; 1999) have been extensively used. Novel techniques have been developed at BARC, Mumbai for immobilizing enzyme and cells in hen egg white either in a powder (D'Souza et al, 1982; D'Souza & Nadkarni , 1981 ; Kaul et al, 1984); or bead form (D'Souza et al. 1985; Kubal et al, 1986). A highly porous sponge type cross-linked proteinic matrix has also been developed (Marolia & D'Souza, 1993; 1994; 1999). The unique feature of this support is the large concentration of lysozyme naturally present in hen egg white which gets co-immobilized thus imparting the bacteriolytic property to the support (Kaul et al, 1983; Marolia & D'Souza, 1993; 1999). The technique of cross-linking in the presence of an inert protein can be applied to either enzymes or cells. The chemical cross-linking reagents used, often affect the cell viability. Thus cross-linking technique will be useful in obtaining immobilized non-viable cells. The technique can also be used for the immobilization of enzymes by crosslinking the cell homogenates (D'Souza 1989 a; 1999 a). Enzymes from halophilic organisms have been immobilized by cross-linking the crude homogenate in the presence of an inert protein (0' Souza et al, 1997). Osmotic stabilization of cellular organeJles (0' Souza, 1983) or halophilic cells (D'Souza et al,
326 INDIAN J BIOTECHNOL, OCTOBER 2002
1992) prior to immobilization using cross-linkers has also shown promise.
Adsorption followed by cross-linking has been extensively used in the immobilization of enzymes (D' Souza 1999 a; Hartmier, 1988; Mosbach, 1987 a, b, c) One of the techniques which has gained importance is the use polyethylenimine for imparting polycationic characteristics to many of the neutral supports based on cellulose or inorganic materials (Bahulekar et ai, 1991). Enzymes with low pI like invertase (Yamazaki et ai, 1984; Godbole et ai, 1990), glucose oxidase (Sankaran et ai, 1989), catalase (Sankaran et ai, 1989) and urease (Kamath et ai, 1988; Kamath et ai, 1991) have been bound through adsorption followed by cross-linking on polyethylenimine coated supports.
A variety of other techniques have also been developed. One of the current interest in further economizing on the cost of the purified enzyme is through development of simultaneous purification and immobilzation approaches. Recent studies from our laboratory have shown the possibility of simultaneous purification and reversible imrnobilization of D-amino acid oxidase from Trigonopsis variabilis on hydrophobic support using the crude cell extracts (D'Souza & Deshpande, 2001). Reversibility of immobilization process helps in the reuse of the expensive support material and should find applications for the economic utilization of otherwise labile enzymes like Damino acid oxidase. Extraction and immobilization in one step of ~-galactosidase released from a strain Debaromyces hansenii using hydroxyapatite (Riccio et ai, 1999) and maltose phosphorylase and trehalose phosphorylase from the crude extract of a strain of Plesiomonas on an anion-exchange resin has been reported (Y oshida et al, 1998). A simple approach for the simultaneous isolation and immobilization of invertase using crude extracts of yeast and jack bean meal has been reported from BARC (Melo & D'Souza, 2000). rDNA technology is gaining importance in tailoring enzymes (fusion proteins) by introducing recognition sites (e.g. streptavidinlbiotin) into a specific protein. This approach is currently gaining importance in one-step purification and simultaneous immobilization of enzymes from crude cell lysate (Clare et al, 200 I; Huang et al, 1996). Utilisation of molecular recognition ability of biomolecules like avidin-biotin or streptavidin-biotin in conjunction with a lithographic technique is being investigated for the micro immobilization of enzymes on silicone wa-
fers for biosensor applications (Koyano et ai, 1996). Immobilization of enzymes on silicone supports has attracted attention in biosensor chip technology and a variety of classical techniques have been proposed (Subramanian et ai, 1998). Other approaches in this direction include immunoaffinity technique using specific anti-enzyme antibodies immobilized on polymeric supports (Farooqui et al, 1999) and techniques for oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function (Turkova, 1999). Enzymes have also been immobilized on reversibly watersoluble polymers like Eudragit S-100 (Sardar et ai, 1997). In addition to enzymes submitochondrial particles prepared from beef liver mitochondria have been immobilized on Fractosil, a porous form of silica, through adsorption in order to stabilize their enzymatic activity (Habibi & Nemat, 1998). Human cells have been immobilized in macroporous microcarriers for the onsite evaluation of environmental waters (Soji et al, 2000).
Stability Characteristics, Protective Effects and Physiological Alterations
Immobilization, in general, has been shown to stabilize the enzymes as well as the cells. Most of the stabilisation efforts have involved either limited intramolecular chemical cross-linking or protein engineering. In this respect useful strategies have been developed for immobilization-stabilization of enzymes by multi point covalent attachment to gels (Blanco et al, 1988; Femandez et al, 1995; Mateo et ai, 2000). Such approaches have been proposed to show more resistance to conformational changes induced by heat, drastic change in pH, organic solvents, etc. On the other hand a very intensive enzymesupport multi point attachment may also promote unwanted conformational changes in the enzyme structure leading to loss of catalytic activity. So, a very careful control of these enzyme-support multiinteraction processes is necessary in order to get derivatives with promising activity and stability characteristics. Amorphous enzyme aggregates prepared by chemical cross-linking with glutaraldehyde have shown enhanced stability under stress conditions like temperature, and exposure to organic solvent (Tyagi et al, 1999). Formation of such aggregates is generally attributed to both intramolecular and intermolecular cross-links introduced in the protein molecule. Horseradish peroxidase which has applications in a variety
D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 327
of analytical systems has been extensively investigated in this respect to enhance the stability either through chemical modification or immobilization (Miland et al, 1996; Ryan et al, 1994). Immobilization has also been shown to enhance the stability of enzymes by preventing the change in the ionization state of active site Fe in metalloenzyme like lipoxygenase by modulation of the ligand environment in the active site (Chikere et al, 200 I). Such stabilization due to changed microenvironment in the vicinity of the active site of the enzyme has also been demonstrated for carboxypeptidase A (Vertesi et ai, 1999). Thermal stability of glycosidases was found to increase considerably through immobilization (Hernaiz & Crout, 2000).
A large number of bioprocess in the future will be carried out in organic solvents. Immobilization in general has been shown to enhance their stability in organic solvents (Barros et ai, 1999; Bouwer et ai, 1997; Cabral et ai, 1997). In this direction crosslinked enzyme crystals, microcrystals grown from aqueous solution and cross-linked with a bifunctional reagent such as glutaraldehyde have shown promise. Cross-linked enzyme crystals help in obtaining highly concentrated immobilized enzyme particles exhibiting better stability at elevated temperatures, in nearanhydrous organic solvents and a variety of other conditions including attack by proteases (St Clair, 1992). Methods have been developed to introduce highly hydrophilic nano-environment surrounding immobilized enzymes like penicillin acylase leading to its dramatic stabili.zation in organic solvents (Fernandez et ai, 1998 a) .
In addition to protein stabilisation there is also an interest in the stabilisation of cells and cellular organells. The animal and plant cellular organells can exhibit highly specific biological activity. However the major drawback is their osmotic instability thus limiting their applications for use only in isotonic solutions. Osmotic stabilisation of cellular organells like the animal mitochondria using glutaraldehyde has shown promise (D'Souza, 1983). Halophilic cells are gaining importance in biochemical conversions under high salt conditions where the normal microbial cells fail to grow or function. The major limitations of these cells which have potentials in processing under saline conditions is their lysis with slight changes in the external salt concentration from the optimum. Cross-linking techniques developed in our laboratory can obviate these problems (D'Souza et ai, 1992).
Cross-linking technique can also be used for the stabilization of halophilic enzymes towards denaturation under low salt concentration (D'Souza et ai, 1997) and for the stabilization of microbial cells towards lysis by lytic enzymes (D'Souza & Marolia, 1999). Immobilization has been shown to improve the stability of hybridoma antibody productivity in serum free media (Lee & Palsson, 1990).
One of the other important advantages of immobilization is the protection of the enzyme/cells from external environmental perturbations like abiotic stresses such as freeze thawing, wet dry cycles, toxic chemicals and organic solvents (Cassidy et al, 1996; Joshi & D'Souza, 1999) and other biotic stresses like phage attack and lytic enzymes (Steenson et al, 1987). These can be controlled by the proper selection of the immobilization matrix. Protective effects have been suggested due to adsorption of toxic compounds by the matrix; restricted diffusion of macromolecules like phages or lytic enzymes or alteration in the membrane composition of the immobilized cells (Cassidy et al, 1996; Doran & Bailey, 1986). Immobilization of cells in alginate has been shown to provide protection to cells against a variety of organic solvents, viz. esters, phthalates, alkanes, alcohols, phenols and perfiuorochemicals (Buitelaar et al, 1990; Cassidy et al, 1996; Joshi & D'Souza, 1999). These attributes have special significance in the effective use of microbial cells in fermentation and especially in developing newer strategies for introduction of the organisms into soil for agricultural and other environmental applications as discussed in the later part of this review.
Microbial metabolism of immobilized viable cells has been shown to be different than cells in their free state of fermentation. Metabolic rates especially with respect to final product and rate of respiration has been show to be enhanced (Doran & Bailey, 1986). No detailed studies are available, however, there is an indication that this may be due to the result of what is termed as immobilization stress (Rao et al, 1994). The enhancement phenomenon as a uniform and stable effect of the whole cell immobilization is often discussed in relation to the effect of multi point cell-sohd surface contact, potentially bringing positive modulation of complex cellular functions. Recently covalent attachment of Candida utilis cells, possibly simulating natural microbial immobilizations, stimulated stable and significant enhancement of extracellular production of alkaline protease as compared to other
328 INDIAN J BIOTECHNOL, OCTOBER 2002
proteolytic enzymes (Jirku, 1997). Covalent immobilization of Saccharomyces cerevisiae cells has been shown to improve their tolerance to ethanol which has been attributed to membrane compositional changes accompanying immobilization (Jirku, 1999). Studies from our laboratory have shown the enhancement of B-galactosidase activity in cells on immobilization and use in the desugaring of milk (Rao et ai, 1994). DeAlteriis et al (1995) reported subtle differences in the electrophoretic mobility of external invertase from free and gel-immobilized yeast cells which has been attributed to different levels of glycosylation of the protein moiety . Immobilization has also been shown to alter the biosynthesis spectrum of pectin-degrading enzymes. The free cell cultures of Aspergillus niger produced four pectinolytic enzyme actIvIties, viz.polymethygalcturonase (PMG), polygalacturonase (PG), pectinesterase, and pectinlyase, while entrapped mycelium synthesized only PMG and PG (Pashova et al, 1999). Other attribute is the possibility of controlled growth of the immobilized viable cells through nutrient starvation. This interest has stemmed from the increased use of genetically manipulated biological cells. One of the difficulties which arises from the insertion of foreign DNA into microbes is the reversion which can be overcome by placing the cells in an environment in which cellular replication can be minimized while cellular activity is maintained at high levels. Immobilization of cells has greatly helped in achieving this objective. (Kumar & Schugerl, 1990).
Immobilized Enzymes and Cells in Bio-processing and Fermentation
Immobilized enzymes and nonviable cells have been investigated for a variety of bioconversions both in aqueous and organic solvents (Balcao, 1996; D'Souza, 1999 a). Some immobilized preparations based on such systems have been commercialized (D'Souza, 1999 a). Some of these include production of high fructose syrups using glucose isomerase, invert sugar using invertase, aspartic acid using aspartase, lactose hydrolysed milk and whey using lactase, 6-aminopenicillanic acid using penicillin acylase and resolution of recemic amino acids using amino acid acylase. Studies from our laboratory have shown their possible potentials in the hydrolysis of sucrose (invertase) (D'Souza & Melo, 2001; D'Souza & Nadkarni, 1980 b; Ghosh & 0' Souza, 1989; Godbole et al, 1990; Melo et al, 1992), preparation of keto acids
(D-amino acid oxidase) (Deshpande et al , 1987; D'Souza & Deshpande, 2001), removal of hydrogen peroxide using catalase (D'Souza & Nadkarni, 1980 a; 0' Souza et al, 1987), hydrolysis of lactose in milk (B-galactosidase) (Kaul et ai, 1984), and removal of glucose using glucose oxidase (Sankaran et al, 1989; Marolia & D'Souza, 1994). Enzyme cellcoimmobilizates have been investigated for the conversion of sucrose to fructose and gluconic acid (invertase, glucose oxidase and catalase) (0' Souza, 1989 b; D'Souza & Melo, 1991; D'Souza & Nadkarni , 1980 c) and in the initiation of lactoperoxidase antimicrobial system (B-galactosidase and glucose oxidase) (Kaul et ai, 1986). Immobilized viable cells have also been investigated in a variety of fermentation processes including antibiotics, organic acids, enzymes and alcohols (0' Souza 1999 b; Ramakrishna & Prakasham, 1999). The most extensively studied system has been the use of immobilized viable yeast cell s in the preparation of fuel ethanol and alcoholic beverages. The extensive studies on immobilized cell carriers, viability, vitality, mass transfer characteristics and bioreactor design indicate that an industrial scale immobilized cell system for primary beer fermentation may become a reality in the modern breweries (Linko et al, 1998; Pilington et al, 1998). Studies from our laboratory have shown the usefulness of immobilized viable yeast cells for the rapid fermentative removal of lactose from milk (Rao Pt al, 1988) and glucose from eggs (0' Souza & Godbole, 1989). In general, entrapment technique is being increasingly investigated for the preservation of cultures as well as a source of continuous inoculum for a variety of fermentation applications (Broadent & Kondo, 1993; Cassidy et al, 1996; Morin et al, 1992). Immobilized viable cells have also gained importance in the continuous production of enzymes (Bagai & Madamwar, 1997; Slokoska & Angelova, 1998). A few typical examples of immobilized enzymes and cells have been summarized in Table 1.
Agricultural and Environmental Applications Immobilized cells are being investigated as an al
ternative technology for a variety of environmental applications in agriculture, biocontrol, pesticide application and pollutant (e.g. pesticide/xenobiotics) degradation in contaminated soils. Microbial inoculants have been investigated for soil applications such as enhancement of symbiotic or associative nitrogen fixation, biological control of soil-borne plant
D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 329
Table I-Applications of Immobilized Biocatalysts
Biosystem Immobilization Application Reference
G I ucoamy I ase Cross-linking on cotton cloth Hydrolysis of starch D'Souza & Kubal, 2002 Glucose oxidase Cross- linking in raw hen egg Removal of glucose from egg Marolia & D'Souza, 1994
white Trypsin-streptavidin fusion Biotinylated porous glass Limited proteolysis of milk proteins Clare et ai, 2001 protein Aspergi llus niger Alginate Polymethylgalacturonase produc- Pashova et ai, 1999
tion Hydantoinase and L-N- Eupergit-C Optically pure L-amino acids. Ragnitz et ai, 200 1 carbamoy I ase Proteases Agarose-glutaraldehyde Hydrolysis of whey proteins Lamas et ai, 200 I Yeast Alginate Asymmetric ketone reduction Griffin et ai, 200 I Lipase Algi nate Hydrolysis of blackcurrant oi l Vacek et ai, 2000 Lipase Hydrophobic zeolite Palm oi l hydrolysis Knezevic et ai, 1998 Lipase Poly(methylmethacrylate Synthesis of fatty esters Basri et ai, 1996 Tannase Chitosan Hydrolysis of tannins Abdel et ai, 1999 Psuedomonas dacullhae Carrageenan L-Aalanine Calik et ai, 1998 a-L-arabino- furanosidase Ch itosan Increase the aroma of wine Spagna et ai, 1998 and P-D-glucopyronisidase p -glucosidase Hydroxyapatite Release of specific-bound aroma in Riccio et ai, 1999
wine and fruit juices Maltose phosphorylase and Anion-exchange resin Production of trehalose from mal- Y oshida et ai, 1998 trehalose phosphorylase to se Ca 2+-independent micro- Chitosan Deamidation of food proteins Nonaka et ai, 1996 bial transglutaminase Pectinlyase Acrylic resin Fruit juice treatment Spagna et ai, 1995 Pectinlyase Nylon Fruit juice clarification Alkorta et ai, 1996 Protease Liposome Cheese ripening Laloy et ai, 1998 Invertase Affinity precipitation using Sucrose hydrolysis Melo & D'Souza, 2000
lectins Yeast cells (invertase) Adhesion to cotton cloth or Sucrose syrup hydrolysis Melo & D'Souza, 1999;
jute; entrapment in alginate D'Souza & Melo, 2001; stabilized with polyacrylamide Gupte & D'Souza, 1999
Inulinases Porous glass beads Sucrose hydrolysis Ettalibi & Baratti, 2001 D-amino acid oxidase Adsorption on hydrophobic Preparation of a-keto acids D'Souza & Deshpande,
support 2001 Basidomycetes cells Alginate or carrageenan Decolurisation of molasses Tamaki et ai, 1989 Escherichia coli Copolymer of methacrylamide 6-Aminopenicillanic acid Hsiau et ai, 1997 (penicillinG acylase) and
N ,N' methylenebisacrylamide D-amino acid oxidase from Duolite A35-polystyrene resin Deamination of cephalosporin c.. Golini et ai, 1995 different yeasts Penicillin V acylase Alginate 6-Ami nopenicillanic acid and 7- Shewale & Sudhakaran,
aminodesaacetoxy-tephalosporanic 1997 acid
Psuedofl/onas sp. Alginate Salicylic acid production Manohar et ai, 1999 D amino acid oxidase and Entrapment in porous support D-Phenylalanine to phenylpyruvic Femadez et ai, 1998 b catalase acid Micrococcus lysodeikticus Covalent binding to Sepharose Purification of lysozyme D'Souza & Marolia, 1999 cells Lysozyme Cross-linking of raw hen egg Bacterial cell lysis Marolia & D'Souza, 1999
white Coimmobi lized lactate de- Adsorption on Amberlite NAD+INADH recycling Le & Means, 1998 hydrogenase and glutamate XAD-7 (non ionie polyacrylate dehydrogenase beads) Carboxypeptidase A Polyacry lamide C-Terminal amino acid analysis Vertesi et ai, 1999
(Comd)
330 INDIAN J BIOTECHNOL, OCTOBER 2002
Table I- Applications of Immobilized Biocatalysts-Contd
Biosystem
Activated sludge Mercury resi stant AzotobaCTer chroococcIIIII microbes Escherichia coli and DeslI/f() viiJrio deslI/filricalls De.IIII[oviiJrio de.l'lIljuriclIlIs
Ureolytic cell s
PSlledolllOllas sp. Bacilllls sp. Catalase
Atoxigellic Aspergilllls/la-1'11.\'
Ch/orella vulgaris + Azo.l'pirillulII brasilellse Glucose oxidase Urease
Glucose oxidase. urease, lipase Islets
Immobilization
Entrapment in alginate Alginate
Hollow tiber reactor
Biofilm on Pd-Ag membrane surface Flocculation and adhesion on cotton Polyurethane foam Polyurethane foam or alginate Crosslinked on alumina carrier
Alginate
Coimmobilized in alginate
Cotton cloth Chitosan beads
Elctropolymerised polyaniline conducting polymer Alginate microcapsules
pathogens, reduction of aflatoxins, and biodegradation of xenobiotic compounds (Cassidy et al, 1996; Daigle & Cotty, 1997). One of the major limitations for the applications of microbial consortium into soil is their survival under biotic and abiotic stresses. Soil moisture content, heavy metal toxicity, temperature, pH, texture, oxygen availability, rate of oxygen diffusion and nutrient availability have been suggested as abiotic factors controlling survival of introduced bacteria in soils. Biological factors include predation by protozoans, phages, a lower level of starvation resistance of the introduced bacteria and lack of suitable soil niches for extended cell survival (Cassidy et al, 1996). Immobilization of the microbes through encapsulation or entrapment in certain defined polymers has been shown to afford protection to cells under such adverse conditions (Cassidy et aI, 1996; Joshi & D' Souza, 1999; Leung et ai, 1995 ; Morin el aI, 1992; Smit et ai, 1996; Steenson et aI, 1987; Trevors et aI, 1993). The physical soil environment is heterogeneous and changing environmental conditions can result in various alterations of the soil s. Encapsulation provides not only protection, but a more stable microenvtronment for the entrapped microbial cells. For example, microbial cells entrapped in alginatL' remained stable after a few drying/wetting cycles in soil , whereas free cells under similar conditi()n~ were reduced by about
Application
Degradation of phenol Volatilization of mercury
Reduction of technetium
Palladium recovery
Treatment of urea effluent
Degradation of naphthalene Degradation of dimethylphthalate Removal of H20 2 from textile bleaching effluent Introduction into soil s
Effective means of increasing microalgal population Glucose biosensor Estimation of urea
Glucose, urea, lipids biosensor
Artificial pancreas
Reference
Joshi & D'Souza. 1999 Ghosh el ai, 1996
Lloyd et al. 1999
Yong et al. 2002
Kamath & D'Souza, 1992
Manohar et ai, 200 I Niazi & Karegoudar, 200 I Costa el al. 2002
Daigle & Cotty, 1995
Gonzalez & Bashan. 2000
Kumar el al. 1994 Kayastha & Srivastava, 2001 Sukeerthi & Contractor, 1999 Mullen el al. 2000
log 2 CFUlg (Cassidy et ai, 1996; Trevors et aI , 1993).
The entrapment techniques discussed earlier have been modified to include in addition to the microbial consortia, nutritional amendments like milk proteins, oil seed meal, etc. and fillers like clay (Cassidy et ai, 1996). Such immobilization techniques can help in devising newer strategies in the future in the introduction of microbes into the soils. Using this approach it is now possible to provide the microbial consortium with a more defined microenvironment , quite different from that encountered when directly in contact with soil environments. In this context encapsulation process adds a modicum of control, potentially becoming a miniature reactor in the environment. Immobilized cells can also act as synthetic inoculation carriers for the slow release of plant growth related organisms like Rhizobium into soils. Microenvironment in the bead may initially protect cells from the soil microenvironmenl. Microorganisms are released after adaptation to prevailing environmental conditions. This may enable cells to overcome the numerous changing conditions in soil and increase microbial survival. Entrapment in gel-matrix offers a stable, defined, consistent. protective environment. without the immediate release of large number of cells, where cells can survive and metabolic activity
D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 331
can be maintained for extended periods of time (Trevors et aI, 1993). Immobilization can provide additional benefits for commercial purposes especially in terms of ease of storage and transportation and also in terms of biosafety features that limit contamination and bioaerosol formation. Coimmbilization of the fresh water micro-alga, Chlorella vulgaris and the plant-growth-promoting bacterium, Azospirillum brasilense in alginate beads resulted in a significant increased growth of the micro-alga. Such an approach has been suggested as an effective means of increasing micro-algal population within confined environment for agricultural and allied applications (Gonzalez & Bashan, 2000). Entrapped cells have also been extensively investigated for pollutant degradation with an emphasis on in situ bioremediation of chemically contaminated soils (Cassidy et al, 1996; D'Souza, 1999c; Romantschuck et al, 2000). The approach has also been used for the restoration/rejuvenation of degraded ecosystems. More research is required in the future, to establish the potential effectiveness of immobilization technology for use in the environment in varied soil systems.
Bioremediation is currently gaining considerable importance as an economically alternative technology for the treatment of heavy metal and radionuclide waste. Bacteria, yeast, fungi, algae and agro biomass can remove heavy metals and radionuclides from aqueous solutions in substantial quantities either through the process of biosorption or bioaccumulation (Bhainsa & D'Souza, 1999; D'Souza 1999c; D'Souza et al, 2001; Gadd, 2000; L10yd & Macaskie, 2000; Sar & D'Souza, 2001; 2002; Veglio & BeoIchini, 1997). One major requirement of any such process is a biosorbent of mechanical stability and integrity in addition to its biosorption capacity, particularly for multiple adsorption-desorption cycles (Veglio & BeoIchini, 1997). Other important criteria for the use of microbial biomass for bioremediation of heavy metal /radionuclides is their ability to be retained in a bioreactor. This is mainly because unlike the organic compounds which can be biodegraded heavy metals are immutable at elemental level thus methods to retain them in the bioreactor, forms one of the important step in their utilization. Unlike the fungal pellets, which can be used directly, for the effective use in the bioremediation processes, microbial biomass including yeast and bacteria consists of very small particle with low density, poor mechanical strength and little rigidity . Hence these have to be pelletized. This can
be achieved by immobilization of the biomass. Immobilization imparts more operational flexibility and solves the problems associated with solid-liquid separation in settling tanks. Immobilization also helps in the recovery of metal through desorption and its subsequent reuse over a number of cycles thus economizing on the process. Some of the techniques discussed above for the immobilization of cells are being investigated for this purpose (D'Souza, 1999c; D'Souza et al, 200 I; Leenen et al, 1996; Macaskie et al, 1995; Michael & Reeves, 1997; Rus et aI, 1995, Siedel & Jeffers, 1991; Tucker et al, 1998). Studies are under progress in our laboratory for the preparation of bioresins containing a variety of microbes for use in the treatment of radionuclide and allied wastes.
Analytical (Biosensor) and Medical Applications
Immobilized proteins, enzymes, cells and cellular organelles have been widely used in the field of analysis and medicine. The use of immobilized biomaterials in these directions can be divided into two major categories: biosensors and artificial organs. Biosensors are gaining applications in a variety of analytical fields. Biosensor consists of a transducer in conjunction with a biologically active material thus converting a biochemical signal into a quantifiable electric response to yield a measurable signal. The specificity of the biosensor will depend on the selection of the biomaterial. As biological sensing elements enzymes, antibodies, DNA, receptors, organelles and microorganisms as well as animal and plant cells or tissues have been used (D'Souza, 1999b; 2001a, b; MuIchandani & Rogers, 1998; Rogers & Mulchandani, 1998; Shanmugam et al, 2001 ; Turner et al, 1987). The basic requirements of a biosensor are that the biological material should bring the physicochemical changes in close proximity of a transducer. In this direction immobilization technology has played a major role. Immobilization not only brings about the intimate contact of the biological catalysts with the transducer, but also helps in the stabilization of the biological system thus enhancing its operational and storage stability. The biological material has been immobilized directly on the transducer or in most cases, in membranes, which can subsequently be mounted on the transducer. Most of the techniques described above have been used for the immobilization of biocatalyst for biosensor applications. Selection of a technique and/or support would depend on the nature of biomaterial, nature of substrate and
332 INDIAN J BIOTECHNOL, OCTOBER 2002
configuration of the transducer used. The choice of support and technique for the preparation of membranes has often been dictated by the low diffusional resistance of the membrane (Kumar et al, 1992, 1994). Gentle techniques need to be applied when viable cell preparations are to be used. The development of molecular devices incorporating a sophisticated and highly organized biological information processing function is a long-term goal of bioelectronics . For this purpose, it is necessary in the future to develop suitable methods for micro immobilizing the proteins /enzymes into an organized array/pattern, (Farooqi et al, 1999; Koyano et al, 1996; Subramanian et ai, 1998; Yang et ai, 1997). There is also a current interest in developing techniques for immobilization of biomolecule on electrode surfaces by entrapment or attachment to electrochemically polymerized conducting or non-conducting films. (Cosnier, 1999; Sukeerthi & Contractor, 1999). Some of the current developments and future potentials in biosensor field has been recently reviewed by the authour (D'Souza 2001a, b).
Ever since the feasibility of preparing artificial cells was first demonstrated in 1957 their importance in medicine especially in the fabrication of artificial organs has gained interest. An increasing number of approaches to their preparation and use have become available (Chang, 1984; Liang et aI, 2000). Such entrapment systems can be obtained as beads, micro capsules or membranes and can be formed using a variety of synthetic or natural polymeric materials with desired variation in their permeability, surface properties and biocompatibility. A variety of materials including enzymes, cells, cellular organelles, vaccines, antibodies, adsorbents, etc. have been trapped especially for use in medicine (Chang, 1984; Liang et ai, 2000). One of the important current interest in such an approach is to form immunobarriers . Basically low-molecular weight substances such as nutrients, electrolytes, oxygen, bioactive secretory products and cellu lar waste products can diffuse freely across the arti fIc ial membranes while immunoglobulins and other immune effector mechanisms are excluded . This is gaining considerable interest in the creation of bioartifical organs like bioartificial pancreas using entrapped islets (Jwata et aI, 1994; Lanza et ai, 1995 a, b; Lim & Sun, 1980; Mikos et aI, 1994; Mullen et al, 2000). Transplantation of tissues and cells across discordinate barriers remains a challenge both in experimental and clinical settings in view of
the immunolgical problems. Immobilization especially the entrapment technique has been proposed to play an important role in this direction (Iwata et al, 1994; Lanza et aI, 1995 a, b; Mikos et ai, 1994) .
Conclusion There are interesting possibilities within the field
of immobilized enzymes and it is imminent that in the future many applications will be replaced by immobilized systems and many more new systems will become technically as well as commercially feasible. Biotechnology in general is a highly interdisciplinary area of research. A fruitful fusion in the future of various scientific, engineering and medical di sciplines will allow biotechnology to realise its full industrial , environmental, analytical and medical potential
Acknowledgement The author is thankful to all colleagues who have
contributed to the work reviewed in this paper. Thanks are also due to Dr (Mrs) A M Samuel, Director Biomedical Group, BARC for her encouragement.
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