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

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 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.

References Abdel N M A et al, 1999. Immobilization of A.lpergillus OIyzae

tannase and properties of the immobilized enzyme. Appl Mi­crobiol, 87, 108- 114.

Abe lyan V A, 2000. A new method for immobilization of micro­bial cells by cross- linking. Prikl Biokhim Mikrobiol, 36, 359-364.

Alkorta I et ai, 1996. Immobilization of pectin lyase from Peni­cilliulll italiclIIll by covalent binding to nylon. Enzyme Mi­crob Technol, 18, 141 - 146.

Bagai R & Madamwar D, 1997. Continuous production of halo­philic alpha-amylase through whole cell immobili zation of Halobacterium salillariull1. Appl Biochem Biotechllol, 62, 213-218.

Bahulekar R et ai, 199 1. Polyelhylenimine in immobilization of biocatal ys ts. Enzyme Microb Technol, 13. 858-868.

Balcao V M et al, 1996. Bioreactors with immob ili zed lipase: State o f the art. Enzyme Microb Teclmol, 18. 392-416.

Bar R et al, 1986. Immobili zation of Acetobacler aceti on cellu­lose ion exchangers: Adsorption isotherms. Biotee/1Ilo1 Bio­eng, 28, 1166-1171.

Barros R J et al, 1999. Enhancement of immobili zed protease catalyzed dipeptide synthes is by the presence of insoluble protonated nucleophile. Enzyme Microb Techllol, 24, 480-488 .

Basri M et ai, 1999. Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction. Appl Biochem Biotecli ­/lol, 81,205-217.

Basri M et ai, 1996. Immobilization of lipase from Calldida rt/­

f:osa on synthetic po lymer beads for use in the synthesis of fatty esters. J Chem Tee/mol Biotechllol, 66. 169-173 .

Bhainsa K C & D'Souza S F, 1999. Biosorption of uranium (VI) by Aspergillusjumigatus. Biotechllol Tech, 13,695-699.

D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 333

Bickerstaff G F. (Ed), 1997. Immobilization of Enzymes and Cells. Humana Press, Totowa, New Jersey.

Blanco R M et ai, 1988. Immobilization-stabilization of enzymes; variables that control the intensity of the trypsin (amine)­agarose (aldehyde) multipoint attchment. Enzyme Microb Teclmol, 11 ,353-359.

Bonnington L S et ai, 1995. p-(CH20H)rpolyetheramine-derived polymeric films for enzyme immobilization. Enzyme Microb Technol, 17,746-750.

Bouwer STet ai, 1997. The performance of enzyme-membrane reactors with immobilized lipase. Enzyme Microb Tec1l1lol, 21, 291-296.

Bringi V & Dale B E, 1985. Enhanced yeast immobilization by nutrient starvation. Biotechnul Lelt , 7,905-908.

Broadent J R & Kondo J K, 1993. Biotechnology of dairy starter cultures. in The Dairy Science and Technology Handbook 3 Application Science, Technology, and Engineering, edited by Y.H. Hui, VCH Publishers, Inc, New York, Pp 77-104.

Bugarski B et ai, 1993. Methods for animal cell immobilization using lec trostatic droplet generation. Biotechnol Tech, 7, 677-682.

Buitelaar R M et ai, 1990. The influence of various organic sol­vents on the respiration of free and immobilized cells of Tageei.\· minuta. Biotechnol Tech, 4,415-418.

Cabral J M S et al, 1997. Biotransformation in organic media by enzymes and whole cells. J Biotechnol, 59, 133-143.

Calik G et ai, 1998. Growth and kappa-carrageenan immobiliza­tion of Pseudomonas dacunhae cells for L-alanine produc­tion . Enzyme Microb Technol, 24,67-74.

Cassidy M B et ai, 1996. Environmental applications of immobi­lized microbial cells: a review. J Ind Microbiol, 16, 79-10 I.

Chang C C & T seng S K, 1998. Immobilization of Alcaligenes eutrophus using PV A crosslinked with sodium nitrate. Bio­tecl1llo1 Tech, 12, 865-868.

Chang T M S, 1984. Artificial cells in medicine and biotechnol­ogy. Appl Biochelll Biotechnol, 10, 5-24.

Chikere A C et ai, 200 I. Stability of immobilized soybean lipoxy­genases: Influence of coupling conditions on the ionization state of the active site Fe. Enzyme Microb Technol, 28, 168-175.

Clare D A et ai, 2001. Molecular design, expression, and affinity immobilization of a trypsin-streptavidin fusion protein. En­zyme Microb Technol, 28, 483-491.

Cosnier S, 1999. Biomolecule immobilization on electrode sur­faces by entrapment or attachment to electrochemically po­lymerized films . A review. Biosens Bioelectron, 14,443-456.

Costa S A et ai, 2002. Recycling of textile bleaching effluents for dyeing using immobilized catalase. Biotechnol Letl, 24, 173-176.

Cruz N et ai, 2000. Engineering the Escherichia coli outer mem­brane protein OmpC for metal bioadsorption. Bioteclmol Lelt, 22, 623-629.

D'Souza S E et ai, 1992. A novel technique for the preparation of osmotically stabilised and permeabili sed cells of extremely halophilic bacteria. J Biochem Biophys Methods, 24, 239-247.

D' Souza S E et ai, 1997. Immobilization of Haloferax mediterra­nei aldolase by cross-linking in a proteinic matrix : Stability and halophilic characteristics. World J Micrbiol Biotechnol, 13,561-564.

D'Souza SF & Nadkarni G B, 1980a. Immobilized catalase con­taining yeast cells: Preparation and enzymatic properties. Biotechnol Bioellg, 22, 2191-2205.

D'Souza S F & Nadkarni G B, 1980b. Continuous inversion of sucrose by gel-entrapped yeast cells. Enzyme Microb Tech-1101, 2,217-222.

D'Souza S F & Nadkarni G B, 1980c. Continuous conversion of sucrose to fructose and gluconic acid by immobilized yeast cell multienzyme complex. Biotechnol Bioeng, 22, 2 179-2189.

D' Souza S F & Nadkarni G B, 1981. Hen egg white a novel sup­port for the immobilizati on of enzymes. Biotecl1llol Bioellg, 23, 431-436.

D ' Souza SF et al, 1982. Immobilization of microbial cells in hen egg white. Biotechllol Bioeng, 24, 1701-1704.

D'Souza S F, 1983. Osmotic stabilisation of mitochondria using chemical cross-linkers. Biotechnol Bioeng, 25, 1661-1 664.

D' Souza S F et ai, 1985. A method for the preparation of hen egg white beads containing immobilized biocatalysts. Biotechllol Lelt, 7,589-592.

D 'Souza S F et ai, 1986. Immobilization o f yeast cells by adhe­sion to glass surface using polyethylenimine. Biotechnol Lelt, 8,643-648.

D 'Souza S F et ai, 1987. Effect of permeabilization on thermo­stability o f catalase in immobilized yeast cells. Biotecl1llo1 Lelt, 9, 625-628.

D'Souza S F & Kamath, N, 1988. Cloth bioreactor containing yeast cells immobilized on cotton cloth using polyethyle n­imine. Appl Microbiol Biotechnol, 29, 136-140.

D ' Souza SF, 1989a. Immobilized cells: Techniques and applica­tions. Indian J Microbiol, 29, 83-117.

D'Souza S F, 1989b. Potentials of co-immobilizates in biochem i­cal processing: The current state of the art. J Microbial Bio­technol, 4, 63-73 .

D'Souza S F & Godbole SS, 1989. Removal of glucose from egg prior to spray drying by fermentation with immobilized yeast cells. Biotechnol Lelt, 11, 211-212.

D 'Souza S F, 1990. Surface immobilization of food relevant mi ­crobial cells through adhesion . Food Biotecllllol, 4, 373-382.

D' Souza S F, 1991. Immobilized yeast in food industry. in The Yeast Molecular Biology and Biotechnology, edited by R Prasad, Omega Science Publications, New Delhi. Pp 223-236.

D'Souza S F & Melo J S, 1991. A method for the preparation of co-immobilizates by adhesion using polyethylenimine. EIl­zyme Microb Technol, 13,508-511.

D' Souza S F, 1998. Current developments in immobilized bio­catalysts for industrial and biosensor applications. in The Applications of Biotechnology in Molasses Based and Allied Industries: Trends and prospects, edited by V.KJain, Wi s­dom Publishing House, New Delhi . Pp 232-241.

D'Souza S F, 1999a. Immobilized enzymes in bioprocess. Curl' Sci, 77,69-79.

D 'Souza S F, 1999b. Immobilized cells in biochemical process development and monitoring. in The Advances in Bioproc­essing and r-DNA Technology, edited by V Bihari & S C Agarwal, Modem Printers, Lucknow, India. Pp 107-125.

D'Souza S.F, 1999c. Bioremediation of heavy metal and radionu­c1ide waste, IANCAS Bull, 15, 46-54.

D' Souza S F & Marolia K Z, 1999. Stabilization of MicrocOCCIIS

334 INDIAN J BIOTECHNOL, OCTOBER 2002

Iysodeikticus cells towards lysis by lysozyme using glutaral­dehyde: Application as a novel biospecific ligand for the pu­rification of lysozyme. Biotechllol Tech. 13,375-378.

D' Souza S F, 2001a. Immobilization and stabilization of biomate­rials for biosensor applications. Appl Biochem Biotechnol. 96, 225-238.

0' Souza S F, 200 I b. Microbial Biosensors. Biosells Bioelectroll. 16,337-353.

D'Souza S F & Deshpande A, 2001. Simultaneous purification and reversible immobilization of 0 amino acid oxidase from Trigollopsis variabilis. Appl Biochem Bioteclmol. 95, 83-92.

D'Souza S F & Melo J S, 2001. Immobilization of bakers yeast on jute fabric through adhesion using polyethylenimine: Appli­cation in an annular column reactor for the inversion of su­crose. Process Biochem. 36, 677-681 .

D' Souza S F et ai, 2001. Bioremediation of heavy metal and ra­dionuclide waste: An overview. Proc fill COllf flldustr Polhl­tioll COllfrol Technol (fCIPACT-200f). Jawaharlal Nehru Technological University, Hyderabad, India. 7-10 Decem­ber.Pp 102-111.

D' Souza S F & Kubal B S, 2002 . Cloth-strip bioreactor containing immobilized glucoamylase. J Biochem Biophys Methods (in press).

D'Urso E M & Fortier G, 1996. Albumin-poly(ethylene glycol) hydrogel as matrix for enzyme immobilization: Biochemical characterization of crosslinked acid phosphatase. Enzyme Microb Teclll/f)l. 18, 482-488.

Daiglc 0 J & Cotty P J, 1995. Formulating atoxigenic Aspergillus flavus for field release. Biocolltrol Sci Techllol. 5, 175-184.

Daigle 0 J & Cotty P J, 1997. The effect of sterilization, pH, filler and spore inoculum concentration on the preparation of algi­nate pellets. Biocolltrol Sci Teclmol. 7, 3-10.

Das N et ai, 1997. Enzyme entrapped inside the reversed micelle in the fabrication of a new urea sensor. Biotechnol Bioeng. 54, 329-332.

De-Alteriis E et ai, 1995. Electrophoretic mobility of external invertase from free and gel immobilized yeast cells. Res Mi­crobiol, 146,217-225.

Deshpande A et ai, 1986. Immobilization of microbial cells in gelatine using gamma-irradiation. flldiall J Biochem Biophys. 23. 353-354.

Deshpande G et ai, 1987. Co-immobilization of D-amino acid oxidase and catalase by entrapment of Trigonopsis variabilis in radiation polymerised polyacrylamide beads. J Biosci. 11, 137-144.

Doernenburg H & Knorr 0 , 1995. Strategies for the improvement of secondary metabolite production in plant cell cultures. El/­zyme Microb Technol, 17,674-684.

Doran P M & Bailey J E, 1986. Effects of immobilization on growth, fermentation properties, and macro molecular com­position of Saccharomyces cerevisiae attached to gel. Bio­techllol Bioeng 28, 73-87 .

Ettalibi M & Baratti J C, 200 I. Sucrose hydrolysis by thermosta­ble immobilized inulinases from Aspergillus ficuum. En zyme Microb Techl/ol. 28,596-601.

Farooqi M et ai, 1999. Immunoaffinity layering of enzymes: Sta­bilization and use in flow injection analysis of glucose and hydrogen peroxide. Appl Microbiol Biotechllol, 52, 373-379.

Fcderici F et ai, 1996. Glucose oxidase and catalase activities of Penicillium variable P 16 immobilized in polyurathane sponge. J fnd Microbiol. 17, 15-19.

Felix H R, 1982. Permeabilized cells. Anal Biochem. 120. 710-715.

Fernandez L R et ai, 1995. Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents. En­zyme Microb Technol. 17.517-523.

Fernandez L R et al. 1998a. Facile synthesi s of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives: Dramatic stabilization of penicillin acylase ver­sus organic solvents. Enzyme Microb Techllol. 24. 96-103 .

Fernandez L R et ai, 1998b. The coimmobilization of D-amino acid oxidase and catalase enables the quantitative transfor­mation of D-amino acids (D-phenylalan ine) into alpha-keto acids (phenylpyruvic acid). Enzyme Microb Technol. 23. 28-33.

Fukui S et ai, 1987. Entrapment of biocatalysts with photo­crosslinkable resin prepolymers and urethane resin prepo­Iymers. Methods Enzymol. 135. 230-252.

Gadd G M. 2000. Bioremediation potential of microbial mecha­nisms of metal mobilization and immobilization. Curr Opill Bioteclmol, 11.271-279.

Ghosh S & D' Souza S F. 1989. Crushing strength as a tool for reactor height determination for invertase containing yeast cells immobilized in polyacrylamide. Enzyme Microb Tech­nol. 11.376-378.

Ghosh S et al. 1996. Volatilization of mercury by immobilized mercury-resistant bacterial cells. J Appl Bacteriol. 81 , 104-108.

Godbole S S et al,1980. Immobilization of alcohol dehydrogenase by gel entrapment of cells of Saccharomyces cerevisiae. EIl­zyrne Microb Technol. 2. 223-226.

Godbole S S et al. 1983 a. Immobilization of fumarase by en­trapment of rat liver mitochondria in polyacrylamide gel us­ing gamma-rays. Biotechnol Bioeng. 25, 217 -224.

Godbole S S et al. 1983 b. Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells. Enzyme Microb Teclmol. 5, 125-128.

Godbole S S et ai, 1984. Effect of modifying agents on immobi­lized alcohol dehydrogenase. Biotechnol Bioeng. 26, 544-545.

Godbole S S et ai, 1990. Hydrolysis of concentrated sugar syrup by invel1ase immobilized on anion exchanger waste cotton threads. Enzyme Microb Techllol. 12,214-217.

Golini P et al. 1995. Immobilization of D-amino acid oxidase from different yeasts: Characterization and application in the deamination of cephalosporin C. Enzyme Microb Techllol. 17,324-329.

Gonzalez L E & Bashan Y, 2000. Increased growth of microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirilllllll brasilel1se. Appl Ellvirol1 Microbiol. 66. 1527-1531 .

Griffin 0 R et ai, 2001. Asymmetric ketone reduction with immo­bilized yeast in hexane: Biocatalyst deactivation and regen­eration . Biotechnol Prog. 17, 304-310.

Guilbault G G, 1989. Immobilization methods for piezoelectric biosensor. Biotechnology. 7. 349-351.

GlIpte A & D'Sollza S F, 1999. Stabilization of alginate beads using radiation polymerized polyacrylamide. J Biochem Bio­phys Methods, 40, 39-44.

D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 335

Habibi R M & Nemat GM, 1998. Adsorptive immobilization of submitochondrial particles on Fractosil. Int J Biochem Cell Bioi, 30, 417-22.

Hartmier W, 1988. Immobilized Biocatalyst : An Introduction. Spinger Verlag, Berlin, Heidelberg,Germany.

HernaizM 1 & Crout D H G, 2000. Immobilization/stabilization on Eupergit C of the beta-galactosidase from B. circulans and an alpha-galactosidase from Aspergillus oryzae. Enzyme Microb Techllol27, 26-32.

Hertzber S et ai, 1995 Mixed photo-cross-linked polyvinyl alco­hol and calcium-alginate gels for cell entrapment. Appl Mi­crobiol Biotechnol, 43, 10-17.

Ho K L G et ai, 1997 Ingredient selection for plastic composite supports for L-(+}-Iactic acid biofilm fermentation by Lacto­bacillus casei subsp. rhamnosus. Appl Enviroll Microbiol, 63,2516-2523.

Hsiau LT et ai, 1997. Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads. Appl Biochem Biotechnol, 62, 303-315.

Huang X L et ai, 1996. Simultaneous isolation and immobiliza­tion of streptavidin-beta-galactosidase: Some kinetic charac­teristics of the immobilized enzyme and regeneration of bio­reactors. En zyme Microb Technol, 19,378-383.

Husain S & lafri F, 1995. Covalent immobilization of invertase and horseradish peroxidase on concanavalin A-Seralose via carbohydrate moieties. Biochem Mol Bioi 1111, 36, 669-677.

Iwata H et ai, 1994. Strategy for developing microbeads applica­ble to islet transplantation into a spontaneous diabetic NOD mouse. J Biomed Mat Res, 28, 1201-1207.

lirku V, 1997. Changes in the starvation response through cova­lent cell attachment. AnIonic Leeuwenhoek Microbiol, 71, 369-373.

lirku V, 1999. Whole cell immobilization as a means of enhanc­ing ethanol tolerance. J Ind Microbiol Biotechnol,22, 147-151.

10shi M S et ai, 1989. Permeabilization of yeast cells (Kluyvero­mycesfragilis) to lactose by digitonin. Enzyme Microb Tech-1101, 11, 439-443.

Joshi N T & D'Souza S F, 1999. Immobilization of activated sludge for the degradation of phenol. J Ellviron Sci Health-A, 34, 1689-1700.

Ju Y H et ai, 1995. Starch slurry hydrolysis using alpha-amylase immobilized on a hollow-fiber reactor. Enzyme Microb Technol, 17,685-688.

Kalenyuk K K et ai, 1999. Immobilization of the bacterial cells on modified carbon filaments for use in biosensors. Bio­teknologiya, 5, 71-78.

Kamath N & D'Souza SF, 1992. Immobilization of ureolytic cells through flocculation and adhesion on cotton cloth using polyethylenimine. Enzyme Microb Technol, 13,935-938.

Kamath N et ai, 1988. Urease immobilized on polyethylenimine coated cotton cloth. Appl Biochem Biotechnol, 19, 251-258.

Kamath N et ai, 1991. Covalent binding of urease on polymeric surfaces. Trellds Biomaterials Artificial Organs, 5, 67-71.

Kaul R et ai, 1983. The bactericidal effect of hen egg white sup­port: A step towards the use of self-sterilising enzyme sup­ports. Biotechnol Bioeng, 25,887-889.

Kaul R et ai, 1984. Hydrolysis of milk lactose by immobilized p-galactosidase-hen egg white powder. Biotechnol Bioeng, 26,901-904.

Kaul R et ai, 1986. Co-immobilized glucose oxidase-E.coli cell conjugate system for the hydrolysis of lactose in milk and its preservation. J Microbiol Biotechnol, 1, 12-19.

Kayastha A M & Srivastava P K, 2001. Pigeonpea (Cajanus cajall L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications. Appl Biochem Biotech­nol, 96, 41-53.

Kierstan M & Bucke C, 2000. The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Biotechnol Bioellg, 67,726-736.

Knezevic Z el ai, 1998. Palm oil hydrolysis by lipase from Call­dida cylindracea immobilized on zeolite type Y. Enzyme Mi­crob Tee/mol, 22,275-280.

Koenig A et ai, 1997. Microbial sensors for PAH in aqueous so­lution using solubilizers. in Field Screening Europe, edited by 1 Gottlieb, H Hotz, K Huck and R Niessener; Kluwer Academic Publishers, Netherlands. Pp 203-206.

Kokofuta E et ai, 1987. Coimmobilizaion of Nitrosomollas ell­ropaea and Paracoccus denitrijicalls cells using polyelec­trolyte complex stabilized calcium alginate gel. J Fermellt Techllol, 65, 659-664.

Koyano T et ai, 1996. Development of a technique for microim­mobilization of proteins on silicone wafers by streptavidin­biotin reactions. Biotechllol Prog, 12, 141-144.

Krastanov A, 1997. Continuous sucrose hydrolysis by yeast cells immobilized to wool. Appl Microbiol Biotechnol, 47. 476-481.

Kubal B S et ai, 1986. Preparation and characterization of mag­netic hen egg white beads containing co-immobilized glu­cose oxidase, magnetite and Mn02. Indian J Biochelll Bio­phys, 23, 240-241.

Kubal B S el al, 1990. Immobilization of yeast invertase by ad­sorption on magnetite. J Microbiol Biotechnol, 4, 98-102.

Kumar P K R & Schugerl K, 1990. Immobilization of genetically engineered cells: A new strategy for higher stability. J Bio­techllol, 14, 255-272.

Kumar S D et ai, 1992. Cyclic voltametric studies at the glucose oxidase enzyme electrode. Bioelectrochem Bioenerg, 27, 153-160.

Kumar S D et al. 1994. Potentiometric studies at the glucose oxi­dase enzyme electrode. Bioelectrochem Bioenerg, 34, 195-198.

Laloy E et al, 1998. Release of enzymes from liposomes during cheese ripening. J COllfrolled Release, 54,213-222.

Lamas E M el al, 2001. Hydrolysis of whey proteins by proteases extracted from CYllara cardunculus and immobilized onto highly activated supports. Enzyme Microb Technol, 28, 642-652.

Lamb S B & Stuckey 0 C, 1999. Enzyme immobilisation on col­loidal liquid aphrons (CLAs): The influence of protein prop­erties. Enzyme Microb Technol, 24,541-548.

Lanza R P et al, 1995a. A simple and inexpensive method for transplanting xenobiotic cells and tissues into rats using algi­nate gell spheres. Transplant Proc, 27,3322.

Lanz R P et ai, 1995b. Xenotransplantation of porcine and bovine islets without immunosuppression using uncoated alginate microspheres. Transplallfation, 69,1377-1384.

Le M & Means G E, 1998. NAD+INADH recycling by coimmo­bilized lactate dehydrogenase and glutamate dehydrogenase. Enzyme Microb Technol, 23,49-57.

336 INDIAN J BIOTECHNOL, OCTOBER 2002

Lee G M & Palsson B 0, 1990. Immobilization can improve sta­bility of hybridoma antibody productivity in serum-free me­dia. Biotecllol Bioellg, 36, 1049-1055.

Leenen E J T M et aI, 1996. Characteristics of and selection crite­ria for support materials for cell immobilization in wastewa­ter treatment. Wat Res. 30. 2985-2996.

Lecnhouts K et aI, 1999. Anchoring of proteins to lactic acid bacteria. Antonie Leeuwellhoek Microbiol. 76,367-376.

Lehmann M et aI, 1999. Measurement of biodegradable sub­stances using thc salt tolerant yeast Arxula adellillivorans for a microbial sensor immobi lized with poly(carbamoyl) sul­fonate (PCS) Part 11. Application of the novel biosensor to real samples of coastal and island regions. Biosens Bioelec­troll , 14, 295-302.

Leo W J et al. 1990. Effects of sterilization treatments on some properties of alginate solutions and gels. Bio/echllol Prog. 6, 51-53.

Leung K T et aI, 1995. Survival of k-carageeenan-encapsulated and unencapsulated Psuedo/llollas aerugillosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating. FEMS Microbiol Ecol, 16, 71- 82.

Lewis V P & Yang S T, 1992. Continuous propionic acid fer­mentation by immobilized Propiollibacleriulll achlij)ropi­ollici in a novel packed-bed bioreactor. Biotechllol Bioellg. 40.465-474.

Liang J F el al. 2000. Biomedical application of immobilized enzymes. J Pharlll Sci, 89, 979-90.

Lim F & Sun A M, 1980. Microencapsulated islets as bioartificial endocrine pancreas. Sciellce. 210,908-910.

Linko M et al. 1998. Recent advances in the malting and brewing industry. J BiolecilllOl, 65. 85-98.

L10yd J R & Macaskie L E. 2000. Bioremediation of radionu­cl ides-containing waste water. ill Environmental Microbe­Metal Interactions, edited by D R Lovley . American Society of Microbiology Press, Washington DC. Pp 277-327.

L10yd ] R el al. 1999. Microbial reduction of technetium by Es­cherichia coli and Desulfovibrio desulfuricalls: Enhancement via the use of high-acti vity strains and effect of process pa­rameters. Biotecimol Bioeng. 66, 122-130.

Loranger C & Carpentier R. 1994. A fast assay for phytotoxicity measurements using immobilized photosynthetic mem­branes. Bio/echllol Bioeng. 44, 178- I 83.

Macaskie L E el aI, 1992. Uranium bioaccumulation by a Citro­bacter sp. as a result of enzymatically mediated growth of polycrystalline HU02P04. Sciellce, 257, 782-785.

Macaskie L E et aI, 1995. Enzymatically-mediated uranium ac­cumulation and uranium recovery using a Citrobacter sp. immobili zed as a biofilm within a plug-flow reactor. J Chelll Technol Bio/echllol, 63,1-16.

Manohar C el aI, 1999. Production of salicylic acid from naph­thalene by immobilized Psuedomonas sp. Strain NGKI. J Microbiol Biolechllol. 9,482-487.

Manohar C el aI, 2001. Enhanced degradation of naphthalene by immobilization of Psuedomonas sp. Strain NGKI in polyu­rethane foam. Appl Microbiol Biotechllol, 55,311-316.

Marolia K Z & D' Souza S F, 1993. A simple technique for the immobilization of lysozyme by cross-linking in hen egg white foam. J BiociJem Biophys Methods, 76, 143-147.

Marolia K Z & D' Souza S F, 1994. Removal of glucose from eggs using glucose oxidase and catalase co-immobilized in hen egg white foam matrix. J Food Sci Technol, 31, 153-155.

Marolia K Z & D'Souza S F, 1999. Enhancement of the lysozyme activity of the hen egg white foam matrix by cross-linking in the presence of N-acetyl glucosamine. J Biochelll Biophys Me/hods. 39,115-117.

Marshall R C (Ed), 1984. Microbial Adhesion and Aggregation . Springer Verlag, New York.

Mateo C et ai, 2000. Increase in conformational stability of en­zymes immobilized on epoxy-activated supports by favoring additional mUltipoint covalent attachment. Enzyme Microb Teclmol. 26,509-515.

Mattiasson, B (Ed), 1983. Immobilized Cell s and Organelles, Vol.l-2, CRC Press, Boca Raton, FL, USA.

Melo J S et ai, 1986. Ocilllum baciliculII seeds as a pellicular sup­port for immobilizing enzymes. Biotechnol Le((, 8,885-888.

Melo J S & D'Souza S F, 1992. Immobilization of invertase through its carbohydrate moiety on Ocimum basiliclIm seeds. Appl Biochem Biotechnol, 32, 159-170.

Melo J S el aI, 1992. Production of inverted sucrose syrup using yeast cells adhered to polyethylenimine treated cotton threads. Food Biotechnol, 6, 175-186.

Melo J S & D'Souza S F, 1999. Simultaneous filtration and im­mobilization of cells from a flowing suspension using a bio­reactor containing polyethylenimine coated cotton threads: Application in the continuous inversion of sucrose syrups. World J Microbiol Biotechnol, 15,25-27.

Melo J S & D'Souza S F, 2000. A simple approach for the simul­taneous isolation and immobilization of invertase using crude extracts of yeast and jack bean meal. J Biochem Biophys Methods, 42, 133-135.

Michael Z C H & Reeves M, 1997. Biosorption of uranium by Pseudomonas aeruginosa strain CSU immobilized in a novel matrix. Biotechnol Prog. 13, 60-70.

Mikos A G et aI, 1994. Islet transplantation to create a bioartifi­cial pancreas. Biotechnol Bioeng. 43,673-677.

Miland E e/ ai, 1996. Increased. thermal and solvent tolerance of acetylated horseradish peroxidas. En zyme Microb Tec'/1I1ol, 18,63-67.

Miranda C & D'Souza S F, 1988. Clarification of pectin using pectinolytic fungi immobilized in open pore gelatin block . J Microbiol Bio/echnol. 3,60-65.

Morin N e/ ai, 1992. Production of concentrated Lac/ococcus lac/is sub sp cremoris suspensions in calcium alginate beads. Appl Ellviron Microbiol, 58, 545-550.

Mosbach K, (Ed), 1987a. Immobilized Enzymes and Cells. Part B. Methods in Enzymology. Vol 135. Academic Press, London, UK.

Mosbach K, (Ed), 1987b. Immobilized Enzymes and Cells. Part C. Methods in Enzymology. Vol 136. Academic Press, Lon­don, UK.

Mosbach K, (Ed), 1987c. Immobilized Enzymes and Cells. Part D. Methods in Enzymology. Vol 137. Academic Press, Lon­don, UK.

Mulchandani A & Rogers K R (Eds), 1998. Enzyme and Micro­bial Biosensors: Techniques and Protocols . Humana Press. Totowa, New Jersey.

Mulchandani A et aI, 1998. Biosensor for direct determination of organophosphate nerve agents using recombinant Escher­ir.hia coli with surface-expres~ed organophophorous hydro­lase. I . Potentiometric microbial electrode. Anal Chem, 70, 4140-4145.

D'SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 337

Mullen Y et ai, 2000. Current progress and perspectives in immu­noi solated islet transplantation. J Hepatobiliary Pancreat Surg, 7,347-357.

Murai T et ai, 1997. Genetc immobilization of cellulase on the cell surface of Saccharomyces cerevisiae. Appl Microbiol Bioteel1l101, 48, 499-503 .

Nandakumar R & Mattiasson B, 1999. A microbi al biosensor using Psuedomonas putida cells immobilized in an expanded bed reactors for the on-line monitoring of phenolic com­pounds. Anal Lelt, 32,2379-2393 .

Neelakantan S et ai, 1999. Production and use of microbial en­zymes for dairy processing. CII,.,. Sci,77, 143-148.

Navratil M & Sturdik E, 1999. Bioactive components in produc­tions using immobilized biosystems. Bioi Bratislava, 54, 635-648.

Niazi J H & Karegoudar T B, 2001. Degradation of dimethyl­phthalate by cells of Bacillus sp. Immobilized in calcium alginate and polyurethane foam. J Enviroll Sci Health PartA, Environ Sci Eng. 36, 1135-1144.

Nonaka M et ai, 1996. Deamidation of several food proteins usi ng free and immobilized Ca-2+-independent microbial trans­g lutaminase. Biosci Bioteel, Biochem, 60,532-533.

Pashova S et ai, 1999. Induction of po lymethylgalacturonase bio­synthesis by immobilized cells of Aspergillus niger. Ellzyme Microb Teel1l101, 24, 535-540.

Patil A & D'Souza SF, 1997. Measurement of in situ halophilic glyceralddehyde-3-phosphate dehydrogenase ac tivity from the permeabilised cells of archaebacterium Haloarcula val­/ismortis. J Cen Appl Microbiol, 43, 163-167.

Pilkington P H eT ai, 1998. Fundamenta ls of immobilized yeast cells for continuous beer fermentation : A review. J Inst Brew, 104, 19-3 1.

Pinhe iro H M & Cabral J M S, 1992. Screening of whole -cell immobili zation procedures for the DEL T A-l­dehydrogenation of steroids in organic medium. Enzyme Mi­crob Technol, 14,6 19-624.

Piret J M & Cooney C L, 1991. Model of oxygen transport limita­tions in hollow fiber bioreactors. Bioteel1l101 Bioellg, 37, 80-92.

Ragnitz K et ai, 2001. Optimization of the immobilization pa­rameters and operational stability of immobilized hydanto­inase and L-N-carbamoylase from Arthrobacter aurescells for the production of optically pure L-amino acids. Enzyme Microb Teclmol, 28, 713-720.

Ramakri shna S V & Prakasham R S, 1999. Microbial fermenta­tion with immobilized cells. Currellf Sci, 77, 87-100.

Rao B Y K et ai, 1988. Preparation o f lactose free milk by fer­mentation using immobilized Saccharomyces fragilis . Bio­technol Lell, 10, 427-430.

Rao B Y K et ai, 1994. Alteration of metabo lic aspects of cells under stress due to immobilization . DAE Symp Stress Adap­Tive Responses Bioi System. M S University, Baroda, India, March 23-25, 1994. Pp 258-263.

Rao D S & PanJa T , 1994. Comparative analysi s of different whole cell immobi lized Aspergilus niger catalysts for glu­conic ac id fermentation usin)! pretreated cane molasses. Bio­process Ellg. 11, 209-2 12.

Riccio P et ai, 1999. Extraction and immobilization in one step of two beta-glucosidases re leased from a yeast strain of De­baryomyces hallsenii. Enzyme Microb Teel1l1ol, 24, 123-129.

Rogers K R & Mulchandani A, 1998. Affinity iosensors: Tech­niques and protocols. Humana Press, Totowa, New Jersey.

Romantschuk M et ai, 2000. Means to improve the effect of insitu biore mediation of contaminated soil : An overview of novel approaches. Environ Pollut, 107, 179-185.

Rouillon R et ai, 1995. Immobilization of thylakoids in polyvinyl alcohol for the detection of herbic ides. Sells Actuators, 27 , 477-479.

Rouillon R et ai, 999. A photochemical cell for detecting pollut­ant-induced effects on the activity of immobilized cyano­bacterium Synechococclls sp. PCC 7942 . Enzyme Microb Technol, 25. 230-235 .

Rucka M & Sroka Z, 1989. Hollow liber modules as a tool for enzy me and cell immobilization. Acta Biotechnol. 9, 467-472.

Rus E et ai, 1995. A spiral bioreactor for removal and recovery of metals from aqueous wastes. Bioprocess Eng, 13, 13- 17.

Ryan 0 et ai, 1994. Thermostabilized chemical derivati ves o f horseradish peroxidase. Ellzyme Microb Technol, 16, 501-505 .

Sankaran K et ai, 1989. Preparation of spray dried, sugar- free egg powder using g lucose oxidase and catalase coimmobilized on cotton cloth. Enzyme Microb Technol, 11, 617-619.

Sar P & D'Souza S F, 2001. Biosorptive uranium uptake by a Pselldomonas strain: characterization and equilibrium stud­ies. J Chem Tech Biotechnol, 76, 1286-1 294.

Sar P & D' Souza S F, 2002 . Biosorption of thorium (IV) by a Pseudomonas strain. Biotechnol Lell, 24. 239-243.

Sardar M et ai, 1997. Noncovalent immobilization of enzymes on an enteric polymer Eudragit S-100. Enzyme Microb Technol, 20,361-367.

Sarfo K et ai, 1995. Polymeric phosphine oxide polyether-derived copolymer as a support for urease immobilization . Enzyme Microb Technol, 17, 804-808.

Seidel D C & Jeffers T H, 1991. Polymer bead containing immo­bilized metal extractant. US Pat(429 236).

Senthuran A et ai, 1997. Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei. Bio­tecl1ll01 Bioeng 55, 841-853.

Shanmugam K et ai, 2001. 2,4-toluene diamines-Their carcinoge­nicity, biodegradation, analytical techniques and an approach towards development of biosensors. Analytical Sciences 17, 1369- 1374.

Shen B Q et ai, 1993. Examination of polysaccharides for their uses in hybridoma cell culture. Biotechnol Tech, 7, 193-198.

Shewale J G & Sudhakaran V K, 1997. Penicillin V acylase: Its potential in the production of 6-aminopenicillanic acid. En­zyme Microb Technol, 20,402-4 10.

Shoji R et ai, 2000. Development of a rapid and sensitive bioassay device using human cells immobilized in macroporolls mi­crocarriers for the on-site evaluation of environmental wa­ters . Appl Microbiol Biotechnol, 54,432-438.

SivaRaman H et ai, 1982. Continuous ethanol production by yeast cells immobilized in open pore gelatine matrix. Bioteclll101 Lett, 4, 359-364.

Slokoska L S & Angelo\' ;) M B, 1998. Immobilization of po lymethylgalacturonase producing Aspergillus niger on Luffa sponge material. Z Natureforsch Sec C Biosci, 53, 968-972.

338 INDIAN J B10TECHNOL, OCTOBER 2002

Smidsord 0 & Skjak-Braek G, 1990. Alginate as immobilization matrix for cells. Trends Biotechllol, 8, 71-78.

Smit E et aI, 1996. Interactions between a genetically engineered PSI/ec/omolla.\' fluorescence and bacteriophage jR2f in soil: effec t of nutrients, alginate encapsulation and wheat rhizos­phere. MicrobEcol31, 125-140.

Spagna G et ai, 1995. Immobilizatio n of a pectinlyase from As­pergillu.\' niger for application in food technology. Enzyme Microb Techllol, 17, 729-738.

Spagna G et ai, 1998. Immobilization of the glycosidases: alpha­L-arabinofuranosidase and beta-D-glucopyranosidase from Aspergillus niger on a chitosan derivative to increase the aroma of wine: Part 11. Enzyme Microb Technol, 23, 413-421.

Srivastava P K et ai, 2001. Characterization of gelatin­immobilized pigeonpea urease and preparation of a new urea biosensor. Biotec/mol Appl Biochem, 34, 55-62

SI. Clair N L & Navia M A 1992. Cross-linked enzymes as robust biocatalysts. JAm Chem Sac, 114, 7314-7316.

Steenson L R et ai, 1987. Calcium alginate-immobilized cultures of acid Streptococci are protected from bacteriophages. J Dairy Sci, 70,1121-1127.

Subramanian A et al. 1998. Comparison of techniques for enzyme immobilization on silicon supports. Ellzyme Microb Tee/mol, 24, 26-34.

Sukeerthi S & Contractor A Q, 1999. Molecular sensors and sen­sor arrays based on polyaniline microtubules. Anal Chem, 71,2231-2236.

Svitel J et ai, 1998. Microbial cell-based biosensor for sensing g lucose, sucrose or lactose. Biotechnol Appl Biochem, 27, 153-158.

Tag K et ai, 2000. Measurement of biodegradable substances with a mycelia-sensor based on the salt tolerant yeast Arxula adellinivoralls LS3. Sells Actuators B, 67, 142-148.

Tampion J & Tampion M D, 1987. Immobilized Cells: Principles and Applications. Cambridge University Press, Cambridge, UK

Tamaki H et ai, 1989. Decolorization of molasses by using immo­bilized growing basidomycete cells: Decolorization of cane sugar molasses by action of basidomycete : Part IV. J JPN Sac Food Sci Technol, 3, 827-831.

Trevors J T et ai, 1993.Survival of alginate encapsulated Psuedo­monas fluoresence cells in soil. Appl Microbial Biotechnol, 39,637-643.

Tucker M D et ai, 1998. Removal of U and Mo from water by immobilized Desulfovibrio desulfuricans in column reactors.

Biotec!Jnol Bioeng, 60, 88-96. Turkova J, 1999. Oriented immobilization of biologically active

proteins as a tool for revealing protein interactions and func­tion. J Chromatogr B, 722, 11-31

Turner A P F, Karube I. & Wilson, G S (Eds), 1987. Biosensors Fundamentals and Applications. Oxford University Press, Oxford.

Tyagi R et ai, 1999. Amorphous enzyme aggregates: Stability towards heat and aqueous organic cosolvent mixtures. En­zyme Microb Tee/mol, 24, 348-354.

Uhlich T et ai, 1996. Immobilization of enzymes in photochemi­cally cross- linked polyvinyl alcohol. Enzyme Microb Tech­nol,19, 124-131.

Ulbricht M & Papra A, 1997. Polyacrylonitrile enzyme ultrafiltra­tion membranes prepared by adsorption, cross-linking, and covalent binding. Enzyme Microb Technol, 20,61-68.

Vacek M et aI, 2000. Lipase-mediated hydrolysi s of blackcurrant oil. En zyme Micro l} Technol, 27,53 1-536.

Van Haecht J L et ai, 1985. Immobilization of Saccharomyces cerevisiae by adhesion:Treatment of cells by Al ions. Bio­tec/mol Bioeng, 27, 217-224.

Veglio F & Beolchini , F, 1997. Removal of metal by biosorption: a review. Hydrometallurgy, 44,301-316.

Vertesi A et aI, 1999. Preparation, characterization and applica­tion of immobilized carboxypeptidase A. Enzyme Microb Tec1mol, 25,73-79.

Willke B et ai, 1994. Poly(carbamoylsulphonate) as a matrix for whole cell immobilization: biological characterization. Bio­tee/mol Tech, 8, 623-626.

Yamagiwa K et ai, 1997. Immobilization of denitrifier within a matrix with low oxygen permibiability. Biotech Tech. 11 , 95-98.

Yamazaki H et ai, 1984. Immobilization of invertase on polyeth­ylenimine coated cotton cloth. Biotechllol Lell, 6, 165-170.

Yang S T & Huang Y, 1995. A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose. Biotechnol Bioeng, 45, 379-386.

Yang Z et aI, 1997. Fabrication of oxygen electrode arrays and their incorporation into sensors for measuring biochemical oxygen demand. Anal Chim Acta, 357,41-49.

Yong P et ai, 2002. Palladium recovery by immobilized cells of Desulfovibrio desulfuricans using hydrogen as the donor in a novel electrbioreactor. Biotechllol Lell, 24, 205-212.

Yoshida M et ai, 1998. Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehal ose phosphorylase. Ellzynle Microb Technol, 22, 71-75.