Trenduri in imobilizarea celuleor si tehnologiei celulare.pdf

8/9/2019 Trenduri in imobilizarea celuleor si tehnologiei celulare.pdf

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Indian Journal of Biotechnolo gyVol 1, October 2002, pp 321-338

Trends in Immobilized Enzyme and Cell Technology

S FD'Souza *Nucle ar 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 thetechniques, which have played a significant role, is the immobilization of enzymes and cells. Immobilization helps inthe retention of the biomass in a reactor geometry thus enabling in their economic reuse and in the development ofcontinuous processes. Immobilization also improves stability and prevents product contamination thus paving theuse 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. nthe fabrication of biosensors immobilization helps in establishing intimate contact of the biomaterial on transducersurface 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

ntroduction

Bioprocessing is currently gaining importance as auseful eco-friendly alternative to conventional processtechnology. This is mainly because unlike the chemical catalysts , the biological systems have the advantage s of accomplishing the complex chemical conversions under mild environmental conditions, with highspecificity and efficiency resulting in better productyields with less energy consumptior . The current demand for better utilization of renewable resources andpressure on industry to operate within environmentally compatible limits, has also been a stimulus to thedevelopment of new eco-friendly enzyme catalyzedindustrial processes . The increasing use of enzymesby the biotech industries to produce specific productswith characteri stic attribute s can be emphasized bythe world sale of industrial enzymes approximating toUS $ 1.6 billion which is expected to reach US $ 3.0billion by the year 2008. Over 45 of the enzymes

produced are being used in the food industry and theremaining is shared by detergent (34.4 %), textile11 %), leather (2.8 ), paper and pulp 0 .2 ) and

other industries (5.6%) excluding enzymes for use indiagnostics and therapeutics (Neelkantan et aI, 1999 .The use of enzymes in Indian industries is also on therise. Basic hesitation in the switching over from the

*Fax : +91-22-5505151E-mail : sfdsouza@ apsara.bare.erne .in

classical chemical technology to bioprocessing hasbeen the cost of the biological catalysts and also theirlabile nature and to some extent lack of awareness .Biotechnology has influenced enzyme industry significantly in the recent years especially in the moreefficient production of enzymes, their stabilizationand economic reuse with a view to economize on theoverall process .

Major limitations to the use of purified enzymes inbioprocessing 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 crudeenzyme preparations like fermentation broths for extracellular enzymes and cell homogenates or wholecells for intracellular enzymes . However, the majorlimitation of this approach especially in food andpharmaceutical industries, is in view of their very lowspecific activities, large excess of the crude enzymepreparations need to be employed, which leads toproduct contamination. The important techniquewhich has emerged in the past two decades to solvethese problems of enzyme cost and product purity isthe immobilization of either enzyme preparations orthe cells. Immobilization which deals with the association of a biological system with an insoluble matrixnot only stabilizes the biological system but helps intheir economic reuse in batch as well as continuousbioreactor systems. One of the greatest advantages isthat it also prevents contamination of the final product


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322 INDIAN J 1310TECHNOL OCTOBER 2002

with the biological system that is used, thus pavingthe use of crude enzyme preparations like the wholecells in bioprocessing. The importance of immobilization technology has now been well established andnumber of books and reviews have appeared fromtime 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 ells

CUITently whole cells are gaining importance as asource of immobilized enzymes. Whole cells can beimmobilized either in a vi a ble or non-viable form.Important limitation in the utilization of whole cellsas an intracellular source of enzymes is the diffusionof substrate and products through the cell membrane.One of the ways to obviate this problem is to usepermeabilised cells. The cell s can be permeabilisedusing physical (freezing a nd thawing) or chemical(organic solvents/detergents) techniques. The most

common technique uses organic solvents such as tolu-ene, chloroform , ethanol and butanol or detergentslike 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 certaincells of halophilic bacteria can also be effectively

permeabilised without lysing using enzymes like ly-sozyme or papain (Patil D ' Souza , 1997). The per-meabilisation process, however, renders the cell nonviable but can serve as an economical source of intra-cellular enzymes. They can be used for simple bio-conversions that do not require cofactor regeneration

or metabolic respiration (0 Souza, 1999 a). Numberof techniques have been developed in our laboratoryfor obtaining permeabilised cells containing catalase(D Souza Nadkarni, 1980 a, D Souza et al, 1987 ,alcohol dehydrogenase (Godbole et al, 1980), aminoacid 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 Souz a Nadkarni, 1980 a, b;Svitel et ai, 1998; Hsiau e t ai , 1997; KamathD ' Souza, 1992; Macaskie o 1992» . One of th erecent 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 domain s have been explored for their efficiency in attaching hybrid protein sto the cell wall or cell membrane. The most exploitedanchoring regions are those with the LPXTG box thatbind the proteins in a covalent way to the cell wall(L e e nhouts et al, 1999) . This approa c h , which hold spromise has been demonstrated for a variety of sys-tem s like anchoring proteins/enzyme o nto the surfaceof lactic acid bacteria to obtain recombinant E colicells with surface expressed oragnophosphorus hydrolase (OPH), an enzyme useful in the detection oforganophosphate compounds (and also for the expression of cellulase activity on the cell surface for thehydrolysis of cellulose from the media (Leenhouts etai , 1999; Mulchandani et al, 1998; Murai et al 1997 .

These types of approaches may help in the utilizationof whole cells as a source of enzymes without th eneed for their permeabilisation, and may have major

significance in the future in immob i lized enzymetechnology. Studies from our laboratory and othershave shown the possibility of introducing enzymesonto a cell wall surface through chemical or biospecific affinity techniques (0 Souza 1989 b; 0 SouzaMelo, 1991; D ' Souza Nadkarni , 1980 c; Kaul et al ,1986). The other major limitation in the use of wholecells as an enzyme source is the po ssibility of un-wanted side reactions. These can be avoided by inac-tivating such enzyme s if an y , using inhibitors, heat orchemicals (Godbole et al , 1983 a). Permebilisation ofthe cells often empties the cell of most of its cofactor sthus minimizing side reactions. Thus unlike a wholeviable cell of yeast which can ferment sugars like sucrose or lactose to ethanol, permeabJised cells resultsonly in their hydrolysis to monosaccharides (Joshi e tal 1989; Rao t al, 1988 .

Immobilized viable cells are gaining importance infermentation (D Souza, 1989 a; 1999 a, b; NavratilSturdik, 1999; Ramakrishna Pr a k asham, 1999) .One of the important challenges for the biotechnolo -gists in the future is in improving the fermentationtechniques. The classical fermentation suffers from

various constraints such as low cell density, nutri-tional limitations and batch mode o f operation withhigh down times. It has been well recognized th a tmicrobial cell density is of prime impo r tance to att a in

hi g her volumetric productivity. Th e major limitat ionin the development of continuous fe rmentation pr o c -ess has been the wash out of cells from the bioreactor.


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Use of f10cculating strains, cell recycle and membranereactors are being investigated to solve some of theseproblems. The immobilized viable cell technology caneliminate most of the constraints faced with the freecell systems. The remarkable advantage is the freedom to determine the cell density prior to fermentation. It also facilitates operation of fermentation on acontinuous mode without cell wash out even at highdilution rates. The immobilized cell technology process also decouples microbial growth from cellularsynthesis of favoured compounds. In addition to microbial cells the fermentation technology using bioreactors is also gaining importance for the production ofhigh value compounds using plant and animal cellcultures . Immobilization of such cells have beenshown to offer them stability against shear force whenused in continuous stirred tank bioreactors (Doernen

burg Knorr , 1995; Liang et ai 2000; Shoji et ai2000).

Techniques or the Immobilization o BiocatalystsBiocatalysts can be immobilized either through ad

sorption, entrapment, covalent binding, cross-linkingor a combination of all these techniques (Bickerstaff,1997; D Souza, 1989 a; 1998; 1999 a) Covalentbinding is a commonly used technique for the immobilization of enzymes and antibodies. A variety oftechniques are now available for covalent binding of

enzymes to natural and synthetic polymers and inorganic supports. A number of reviews and books dealwith such techniques (D Souza, 1999 a; Hartmier,1988 ; Mosbach, 1987 a, b, c). The common approachis to introduce highly active electrophilic or nucleophilic groups through activation of the support matrixfollowed by the covalent bond formation with biological systems under mild reaction conditions. Whencovalent binding or cross-linking is used precautionneeds to be taken so as to bind the enzyme withoutsignificantly affecting its conformational flexibilityand 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 activesite from inactivation. Conformational flexibility canbe retained by covalent binding of the enzymes usingspac er arms (Bonnington et ai 1995; Sarfo et ai1995). Glycoprotein enzy mes like glucose oxidase,peroxidase and invertase can also be covalently boundvia their carbohydrate moiety (Husain Jafri, 1995;Melo D Souza, 1992) . Such an approach often

results in better retention of enzyme activity as itavoids the chemical modification of functional groupsin the protein moiety of the enzyme. Covalent bindinghowever 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 thusaffecting their viability. There may also be a lo ss inthe structural integrity of the cell during continuoususe, leading to loss of intracellular enzymes. Amongothers is the very low cell loading that is achieved ascompared to entrapment and other techniques . A fewrecent reports are, however , available on the covalentbinding of cells for specific applications. Jirku (1999)has covalently bound Saccharomyces cervisiae cellsto an epoxide derivative of hydroxyalkylmethacrylategel via glutaraldehyde-diamine spacers. Direct cova

lent binding on glutaraldehyde activated proteinc supports like wool has also been reported (Krastanov,1997). Covalent binding of cells by introduction ofactive aldehyde groups on cell surface using periodateoxidation has also shown promise (Abelyan, 2000).Useful techniques have been developed in our laboratory for the covalent binding of cells on Se ph arosefor use as affinity ligand for the purification of enzymes (D Souza Marolia, 1999). These include thedirect 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 forthe immobilization of cell free enzymes in view oftheir possible leakage from the entrapment matrix.Enzymes have been encapsulated in liposomes fortheir controlled release (Laloy et ai 1998) and alsoinside the reversed micelle (Das et aI, 1997) . Cellshave been immobilized in a variety of synthetic andnatural 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 orbeads) using gamma-ray polymerisation has been extensively investigated in our laboratory (Deshpande etai 1987; D Souza 1998; 1999 a; D Souza Nadkarni, 1980 a, b c; Ghosh D Souza, 1989; Godboleet ai 1983 a, b). Basic advantage of polymerizationusing gamma rays is that unlike the routinely usedchemical polymerization technique, radiation polymerization can be carried out even at -75°C. This not


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324 INDIAN J BIOTECHNOL, OCTOBER 2002

only prevents heat inactivation (Deshpande et ai1987; Gupte D Souza , 1999) but also the samplescan be frozen in any required geometry helping inobtaining entrapment system in bead, tube or membrane forms. Radiation polymerization technique hasalso been extended to entrap cells in ge latine. Thistechnique may be useful for immobilization of enzymes which are otherwise sensitive to glutaraldehyde (Deshpande et ai 1986 .

Entrapment in alginate by ionotropic gelation usinga variety of divalent and trivalent cations has foundextensive use in immobilized viable cell technology(Kierstan Bucke, 2000; Smidsord & Skjak-Braek ,1990). The major limitations of Ca-alginate gels istheir destabilization and subsequent solubi li sation bythe Ca-chelators present in the processing solution orwaste and their low mechanical strength and density.

The common approaches used for stabilizing alginategels include direct covalent cross-linking of the carboxyl groups and covalent grafting of alginate withsynthetic polymers. Some of these include crosslinking with polyvinyl alcohol and treatment withpolyethylenimine followed by cross-linking(Hertzber et ai 1995; Kokofuta et ai 1987; Smidsord

Skjak-Braek, 1990). A novel technique has beendeveloped at BARC, Mumbai for stabilizing the alginate beads towards Ca-'Chelators by reinforcing themwith gamma ray polymerised polyacrylamide (Gupte

D'Souza, 1999). Unlike the Ca salt; Ba-alginategels have been shown to have better mechanical compression and have been shown to have low oxygenpermeability for use under anoxic conditions like denitrification (Yamagiwa et ai 1997). Density andstrength of alginate gels can be enhanced by incorporation of inorganic material s like silica, sand and a lumina (Ramakrishna Prakasham, 1999) . Effect ofsterilization of alginate prior to its use as support hasbeen 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 etai 1987; Yang et ai 1997; Tag et ai 2000). Polyvinylalcohol is one of the most widely studied polymers, asit can form beads , membr a nes, fibres , etc. Enzymesand cells have been immobilized in them either byentrapment, covalent binding , cross-linking, freezingand thawing, y-irradiation, photo -cross linking or entrapment followed by c r o ~ s l i n k i n g(Uhlich t aL 1996) . A technique has also been reported usingpolyvinyl alcohol crosslinked with sodium nitrate.

This new technique can simultaneously eliminate theagglomeration of PV A beads and the toxicity of boricacid caused by the PV A-boric acid and PV Aorthophosphate methods (Chang T se ng, 1998) .Photo-cross linkable polyvinyl alcohol bearing styrylpyridinium groups has been shown to entrap cellularorganelles and cells under very mild conditions retaining their biological activity (Rouillon e t 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 toxicityretaining survival rates of microorganisms greater

than 99% (Wilke et ai 1994; Lehmann et ai 1999 .Highly stable immobilized lipase pr epara tions havebeen obtained by entrapment in poly (N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacr ylate) hydrogel, with divinylbenzene as the cross linking agent(Basri et ai 1999). The use of a novel immobilizationtechnique utilizing an oil-in-water macroemulsion ,termed as colloidal liquid aphron has been developedfor the entrapment of enzymes for use in non-aqueousmedia (Lamb Stuckey, 1999). Microbial cells especially fungal cells have been immobilized by pa ss iveentrapment in polyurethane or vegetable sponges(Federici et ai 1996; Manohar et aL 200 I; PinheiroCabral, 1992; Slokoska Angelova, 1998). n addition to microbial cells entrapment techniques havealso been used for the immobilization of viable animal cells and cellular organelles (Bugarski et ai1993 ; Lee Palsson, 1990; Shen t aI 1993). Majorlimitation of entrapment technique is the additionaldiffusional 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 fibremodules (Ju et ai 2000; Lloyd et ai 1999; PiretCooney, 1991; Rucka Sroka, 1989) A highly porous sponge type proteinic matrix has been developedin our laboratory which allows for the diffusion ofeven 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


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on a variety of commercial ion exchange resins andgels . The basic advantage is the reversibility of binding which also helps in economic recovery of the support. This has been successfully adapted in industryfor the resolution of racemic mixtures of amino acidsusing amino acid acylase. In addition to ion exchangeresins, enzymes have also been immobilized throughadsorption on hydrophobic supports as well as affinitysupports (0 Souza, 1999 a).

Adsorption or more appropriately termed as adhesion is perhaps the oldest method of immobilizingcells . Many cells have natural tendency to adhere tosolid surfaces. Naturally adhered cells have played animportant role in many biotechnological applicationssuch as wastewater treatment and fermentations likevinegar (Marshall, 1984) . Techniques for the adhesionof whole cells on polymeric surfaces has gained considerable importance (0 Souza, 1990). A variety ofapproaches are being applied. The most common isthe passive adsorption of cells on surfaces such that anatural biofilm is obtained (Ho et al 1997; LewisYang, 1992; Yang Huang, 1995). The major limitation of this approach is the long time ranging fromdays to number of weeks required for the formation ofthe biofilm. Under natural p conditions most of thecells have a net negative charge and can hence be adsorbed on ion exchangers (Bar et al 1986). Howeverion exchangers often possess poor binding capacity.

These limitations have been reduced by adhesion ofcells using metallic ions like Al 3 or by coating thesupport or the cells with colloidal particles which actas binding agents between the cell surface and thesupport 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 havebeen developed in our laboratory for immobilizingviable 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'So uza Kamath, 1988; D Souza Melo,2001; Kamath D Souza, 1992; Melo D Souza,1999). The adhesion is found to be rapid and the cellsadhere as a monolayer . Adhesion being very strong,the high ionic concentrations and extreme p 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 simultaneousfiltration and immobilization of cells from a flowingsuspension, thus integrating downstream processingwith bioprocessing (Melo 0 Souza, 1999). Thesestudies were recently extended for immobilization ofinvertase containing yeast cells through adhesion onjute fabric for use in an annular column reactor for theinversion of concentrated sucrose syrups (D SouzaMelo, 2001). The PEI technique developed in ourlaboratory has been applied by others for the adhesionof cells and proteins (Nandkumar Mattiasson,1999; Senthuran et al 1997; Tampio n Tampion ,1987; Guilbault, 1989).

Cross-linking using bifunctional reagents likeglutaraldehyde has been successfully used for theimmobilization of enzymes and cells in varioussupports . 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 beenextensively used. Novel techniques have beendeveloped at BARC, Mumbai for immobilizingenzyme 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 typecross-linked proteinic matrix has also been developed(Marolia D Souza, 1993; 1994; 1999). The uniquefeature of this support is the large concentration oflysozyme naturally present in hen egg white whichgets co-immobilized thus imparting the bacteriolyticproperty to the support (Kaul et al 1983; MaroliaD Souza, 1993; 1999). The technique of cross-linkingin the presence of an inert protein can be applied toeither enzymes or cells. The chemical cross-linkingreagents used, often affect the cell viability. Thuscross-linking technique will be useful in obtainingimmobilized non-viable cells. The technique can alsobe used for the immobilization of enzymes by crosslinking the cell homogenates (D Souza 1989 a; 1999a). Enzymes from halophilic organisms have beenimmobilized by cross-linking the crude homogenatein the presence of an inert protein (0 Souza et al1997). Osmotic stabilization of cellular organeJles(0 Souza, 1983) or halophilic cells (D Souza et al


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1992) prior to immobilization using cross-linkers hasalso shown promise.

Adsorption followed by cross-linking has been extensively used in the immobilization of enzymes(D Souza 1999 a; Hartmier, 1988; Mosbach, 1987 ab 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 ai1988; Kamath et ai 1991) have been bound throughadsorption 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 throughdevelopment of simultaneous purification and immobilzation approaches. Recent studies from our laboratory have shown the possibility of simultaneous purification and reversible imrnobilization of D-aminoacid oxidase from Trigonopsis variabilis on hydrophobic support using the crude cell extracts (D Souza

Deshpande, 2001). Reversibility of immobilizationprocess helps in the reuse of the expensive supportmaterial and should find applications for the eco

nomic utilization of otherwise labile enzymes like Damino acid oxidase. Extraction and immobilization inone step of g a l a c t o s i d a s ereleased from a strain De-baromyces hansenii using hydroxyapatite (Riccio etai 1999) and maltose phosphorylase and trehalosephosphorylase from the crude extract of a strain ofPlesiomonas on an anion-exchange resin has beenreported (Y oshida et al 1998). A simple approach forthe simultaneous isolation and immobilization of invertase using crude extracts of yeast and jack beanmeal has been reported from BARC (MeloD Souza, 2000). rDNA technology is gaining impor

tance in tailoring enzymes (fusion proteins) by introducing recognition sites (e.g. streptavidinlbiotin) intoa specific protein. This approach is currently gainingimportance in one-step purification and simultaneousimmobilization of enzymes from crude cell lys a te(Clare et al 200 I; Huang et al 1996). Utilisation ofmolecular recognition ability of biomolecules likeavidin-biotin or streptavidin-biotin in conjunctionwith a lithographic technique is being investigated forthe micro immobilization of enzymes on silicone wa-

fers for biosensor applications (Koyano et ai 996 .Immobilization of enzymes on silicone supports hasattracted attention in biosensor chip technology and avariety of classical techniques have been proposed(Subramanian et ai 1998). Other approaches in thisdirection include immunoaffinity technique usingspecific anti-enzyme antibodies immobilized onpolymeric supports (Farooqui et al 1999) and techniques for oriented immobilization of biologicallyactive proteins as a tool for revealing protein interactions and function (Turkova, 1999). Enzymes havealso been immobilized on reversibly watersolublepolymers like Eudragit S-100 (Sardar et ai 1997). Inaddition to enzymes submitochondrial particles prepared from beef liver mitochondria have been immobilized on Fractosil, a porous form of silica, throughadsorption in order to stabilize their enzymatic activ

ity (Habibi Nemat, 1998). Human cells have beenimmobilized in macroporous microc arriers for the onsite evaluation of environmental waters (Soji et al2000).

Stability Characteristics Protective Effects ndPhysiological Alterations

Immobilization, in general, has been shown to stabilize the enzymes as well as the cells. Most of thestabilisation efforts have involved either limited intramolecular chemical cross-linking or protein engi

neering. In this respect useful strategies have beendeveloped for immobilization-stabilization of enzymes by multi point covalen t attachment to gels(Blanco et al 1988; Femandez et al 1995; Mateo etai 2000). Such approaches have been proposed toshow 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 verycareful control of these enzyme-support multiinterac

tion processes is necessary in order to get derivativeswith promising activity and stability characteristics .Amorphous enzyme aggregates prepared by chemicalcross-linking with glutaraldehyde have shown enhanced stability under stress conditions like temperature, and exposure to organic solvent (Tyagi et al1999). Formation of such aggregates is generally attributed to both intramolecular and intermolecularcross-links introduced in the protein molecule. Horseradish peroxidase which has applications in a variety


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of analytical systems has been extensively investigated in this respect to enhance the stability eitherthrough chemical modification or immobilization(Miland et al, 1996; Ryan et al, 1994). Immobilization has also been shown to enhance the stability ofenzymes by preventing the change in the ionizationstate of active site Fe in metalloenzyme like lipoxygenase by modulation of the ligand environment inthe active site (Chikere et al, 200 I). Such stabilizationdue to changed microenvironment in the vicinity ofthe 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 becarried out in organic solvents. Immobilization in

general has been shown to enhance their stability inorganic solvents (Barros et ai, 1999; Bouwer et ai,1997; Cabral et ai, 1997) . n this direction crosslinked enzyme crystals, microcrystals grown fromaqueous solution and cross-linked with a bifunctionalreagent such as glutaraldehyde have shown promise.Cross-linked enzyme crystals help in obtaining highlyconcentrated immobilized enzyme particles exhibitingbetter stability at elevated temperatures, in nearanhydrous organic solvents and a variety of otherconditions including attack by proteases (St Clair ,1992) . Methods have been developed to introducehighly hydrophilic nano-environment surroundingimmobilized enzymes like penicillin acylase leadingto its dramatic stabili.zation in organic solvents (Fernandez et ai 1998 a) .

n addition to protein stabilisation there is also aninterest in the stabilisation of cells and cellular organells. The animal and plant cellular organells can exhibit highly specific biological activity. However themajor 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 hasshown promise (D Souza, 1983). Halophilic cells aregaining importance in biochemical conversions underhigh salt conditions where the normal microbial cellsfail to grow or function. The major limitations ofthese cells which have potentials in processing undersaline conditions is their lysis with slight changes inthe external salt concentration from the optimum .Cross-linking techniques developed in our laboratorycan obviate these problems (D Souza et ai, 1992 .

Cross-linking technique can also be used for the stabilization of halophilic enzymes towards denaturationunder low salt concentration (D Souza et ai, 1997and for the stabilization of microbial cells towardslysis by lytic enzymes (D Souza Marolia, 1999) .Immobilization has been shown to improve the stability of hybridoma antibody productivity in serum freemedia (Lee Palsson, 1990) .

One of the other important advantages of immobilization is the protection of the enzyme/cells fromexternal environmental perturbations like abioticstresses such as freeze thawing, wet dry cycles, toxicchemicals and organic solvents (Cassidy et al, 1996;Joshi D Souza, 1999) and other biotic stresses likephage 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 alterationin the membrane composition of the immobilizedcells (Cassidy et al, 1996; Doran Bailey , 1986).Immobilization of cells in alginate has been shown toprovide protection to cells against a variety of organicsolvents, viz . esters, phthalates, alkanes, alcohols,phenols and perfiuorochemicals (Buitelaar et al, 1990 ;Cassidy et al, 1996; Joshi D Souza, 1999). Theseattributes have special significance in the effective useof microbial cells in fermentation and especially indeveloping newer strategies for introduction of theorganisms into soil for agricultural and other environmental applications as discussed in the later part ofthis review.

Microbial metabolism of immobilized viable cellshas been shown to be different than cells in their freestate of fermentation. Metabolic rates especially withrespect to final product and rate of respiration ha sbeen show to be enhanced (Doran Bailey , 1986) .No detailed studies are available, however, there is anindication that this may be due to the result of what is

termed as immobilization stress (Rao et al, 1994). Theenhancement phenomenon as a uniform and stableeffect of the whole cell immobilization is often discussed in relation to the effect of multi point cell-sohdsurface contact , potentially bringing positive modulation of complex cellular functions. Recently covalentattachment of Candida utilis cells, possibly simulatingnatural microbial immobilizations, stimulated stableand significant enhancement of extracellular production of alkaline protease as compared to other


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328 INDIAN J BIOTECHNOL , OCTOBER 2002

proteolytic enzymes (Jirku, 1997) . Covalent immobilization of Saccharomyces cerevisiae cells has beenshown to improve their tolerance to ethanol which hasbeen attributed to membrane compositional changesaccompanying immobili zation (Jirku, 1999). Studie sfrom our laboratory have shown the enhancement ofB-galactosidase activity in cells on immobilizationand use in the desugaring of milk (Rao et ai, 1994 .DeAlteriis et al (1995) reported subtle differences inthe electrophoretic mobility of external invertase fromfree and gel-immobilized yeast cells which has beenattributed to different levels of glycosylation of theprotein moiety . Immobilization has also been shownto alter the biosynthesis spectrum of pectin-degradingenzymes. The free cell cultures of Aspergillus nigerproduced four pectinolytic enzyme actIvIties, viz .polymethygalcturo nase (PMG), polygalactu

ronase (PG), pectinesterase, and pectinlyase, whileentrapped mycelium synthesized only PMG and PG(Pashova et al 1999) . Other attribute is the possibility of controlled growth of the immobilized viablecells through nutrient starvation. This interest hasstemmed from the increased use of genetically manipulated biological cells. One of the difficultieswhich arises from the insertion of foreign DNA intomicrobes is the reversion which can be overcome byplacing the cells in an environment in which cellularreplication can be minimized while cellular activity ismaintained at high levels. Immobil izati on of cells has

greatly helped in achieving this objective . (KumarSchugerl, 1990).

Immobilized Enzymes and Cells n Bio-processingand Fermentation

Immobilized enzymes and nonviable cells havebeen investigated for a variety of bioconversions bothin aqueous and organic solvents (Balcao, 1996 ;D Souza, 1999 a). Some immobilized preparationsbased 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 andresolution of recemic ami no acids using amino acidacylase. Studies from our laboratory have shown theirpossible potentials in the hydrolysis of sucrose (invertase) (D Souza Melo, 2001; D Souza Nadkarni, 1980 b; Ghosh 0 Souza, 1989; Godbole etal , 1990; Melo et al, 1992 ), preparation of keto acids

(D-amino acid oxidase) (Deshpande et al , 1987 ;D Souza Deshpande, 2001), removal of hydrogenperoxide using catalase (D Souza Nadkarni, 1980a; 0 Souza et al, 1987), hydrolysis of lactose in milk(B-galactosidase) (Kaul et ai, 1984), and removal ofglucose using glucose oxidase (Sankaran et al, 1989;Marolia D Souza, 1994). Enzyme cellcoimmobilizates have been investigated for the con-version of sucrose to fructose and gluconic acid (invertase, glucose oxidase and catalase) (0 Souza, 1989b; D Souza Melo, 1991; D Souza Nadkarni ,1980 c) and in the initiation of lactoperoxidase antimicrobial system (B-galactosidase an d glucose oxidase) (Kaul et ai, 1986) . Immobilized viable cellshave also been investigated in a variety of fermentation processes including antibiotics , organic acids,enzymes and alcohols (0 Souza 1999 b; R amakrishna

Prakasham, 1999). The most extensively studiedsystem has been the use of immobilized viable yeastcell s in the preparation of fuel ethanol and alcoholicbeverages. The extensive studies on immobilized cellcarriers, viability, vitality, mass tran sfer characteristics and bioreactor design indicate that an industrialscale 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 ofimmobilized viable yeast cells for the rapid fermentative removal of lactose from milk (Rao t al, 1988

and glucose from eggs (0 Souza Godbole, 1989) .In general, entrapment technique is being increasinglyinvestigated for the preservation of cultures as well asa source of continuous inoculum for a variety of fermentation applications (Broadent Kondo, 1993 ;Cassidy et al, 1996; Morin et al, 1992). Immobilizedviable cells have also gained importance in the continuous production of enzymes (Bagai Madamwar ,1997; Slokoska Angelova, 1998 . A few typicalexamples of immobilized enzymes and cells havebeen summariz ed in Table 1.

Agricultural and Environmental ApplicationsImmobilized cells are being investigated as an al

ternative technology for a variety of environmentalapplications in agriculture, biocontrol, pesticide application and pollutant (e.g. pesticide/xenobiotics) degradation in contaminated soils . Microbial inoculantshave been investigated for soil applications such a senhancement of symbiotic or assoc iative nitrogenfixation, biological control of soil-borne plant


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D SOUZA: TRENDS IN IMMOBILIZED ENZYME AND CELL TECHNOLOGY 329

Table I-Applications of Immobilized Biocatalysts

Biosystem Immobilization Application Reference

G Iucoamy Iase Cross-linking on cotto n cloth Hydrolysis of starch D Souza Kubal, 2002Glucose oxidase Cross- linkin g in raw hen egg Removal of g lucose from egg Marolia D Souza, 994

whiteTrypsin-streptavidin fusion Biotinyla ted porous glass Limited proteolysis of milk proteins Clare et ai, 2001proteinAspergi llu s niger Alginate Polymethylgalacturonase produc- Pashova et ai , 1999

tionHydantoinase and L-N- Eupergit-C Optically pure L-amino acids. Ragnitz et ai, 200 1carbamoy IaseProteases Agarose-glutara ldehyde Hydrolysis of whey proteins Lamas et ai, 200 IYeast Alginate Asymmetric ketone reduction Griffin et ai, 200 ILipase Algi nate Hydrolysis of blackcurrant oi l Vacek et ai, 2000Lipase Hydrophobic zeo lite Palm oi l hydrolysis Knezevic et ai , 1998Lipase Poly(methylmethacrylate Synthesis of fatty esters Basri et ai, 1996Tannase Chitosa n Hydrolysis of tannins Abdel et ai, 1999Psuedomonas dacullhae Carrageenan L-Aalanine Calik et ai, 1998

a- L-arabino- furanosidase Ch itosan Increase the aroma of wine Spag na et ai, 1998and P-D -glucopyronisidasep -g lucos ida se Hydroxyapatite Release of specific-bound aroma in Riccio et ai, 9

wine and fruit juicesMaltose phosphorylase and Anion-exchange resin Production of trehalose from mal- Y oshida t ai, 1998trehalose phosphorylase to seCa 2+-independen t micro- Chitosan Deamidation of food proteins No naka et ai, 1996bial transglu taminasePectinlyase Acrylic resin Fruit ju ice treatment Spagna et ai , 1995Pectinlyase Ny lon Fruit juice clarification Alkorta et ai, 1996Protease Liposome Cheese ripening Laloy et ai, 1998Invertase Affinity precipitation using Sucrose hydrolysis Melo D Souza, 2000

lectinsYeast ce lls (invertase) Ad hesion to cotton cloth o r Sucrose syrup hydrolysis Melo D Souza, 1999;

jute; entrapment in alginateD Souza

Melo, 2001;stabilized with polyacrylamide Gupte D Souza, 1999Inulinases Poro us glass beads Sucrose hydrolysis Ettalibi Baratti, 2001D-amino acid oxidase Adsorption on hydrophobic Preparation of a-keto ac ids D Souza Deshpande,

support 2001Basidomycetes cells Alginate or carrageenan Decolurisation of molasses Tamaki et ai, 1989Escherichia coli Copolymer of methacrylamide 6-Aminopenicillanic acid Hsiau et ai, 1997(penic illinG acylase) and

N N methylenebisacrylamideD-amino acid oxidase from Duolite A35-po lystyrene resin Deamination of cephalosporin c Golini et a i , 1995different yeastsPenicillin V acylase Alginate 6-Ami nopenicillanic acid a nd 7 - Shewale Sudhakaran ,

aminodesaacetoxy -tephalosporanic 1997acid

Psuedofl/onas sp . Alginate Salicylic acid production Manohar et ai , 1999D amino acid oxidase and Entrapment in porous supp ort D-Phenyla lanine to phenylpyruvic Femadez et ai, 1998 bcatalase acidMicrococcus lysodeikticus Cova lent binding to Sepharose Purification of lysozyme D Souza Marolia, 1999cellsLy sozyme Cross-linking of raw hen egg Bacterial cell lysis Marolia D Souza, 1999

whiteCoimmobi lized lactate de- Adsorption on Amberlite NAD+INADH recycling Le Means, 1998hydrogenase and g lut amate XAD-7 (non ionie polyacrylatedehydrogenase beads)Carboxypeptidase A Polyacry lamide C-Term in al amino acid ana lysis Vertesi et ai, 1999

Comd)


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Table I- Applications of Immobilized Biocataly sts-Contd

Bio system

Activated s ludgeMercury resi stant AzotobaCTer chroo coccIIIII mi

crob esEs cheri chia coli andDes lI/f viiJrio d eslI/filricallsDe IIII[oviiJrio de llIljuri clIlIs

Ureolytic cell s

PSlledolllOllas spBa cillll s spCatalase

Atoxige llic Aspergilllls/la-1 11.\'

Ch/or ella vulgaris

Azo lpirillulII brasi l ellseGluco se oxida seUrease

Gluco se oxida se. urea se,lipa seIs lets

Immobilization

Entrapment in alginateAlginate

Hollow tiber reactor

Biofilm on Pd -Ag membran esurfaceFlocculation and adh es ion oncottonPolyurethane foamPolyurethane foam or alginateCro ss linked on alumina carri er

Al g inate

Coimmobiliz ed in alginate

Cotton clothChito san beads

Elctropolymeri sed polyanilineconducting pol ymerAl g inat e microcap sules

pathogens, reduction of aflatoxins , and biodegradationof xenobiotic compounds (Cassidy et al 1996; Daigle

Cotty, 1997). One of the major limitations for theapplications of microbial consortium into soil is theirsurvival under biotic and abiotic stresses. Soil mois

ture content , heavy metal toxicity, temperature, p ,texture, oxygen availability, rate of oxygen diffusionand nutrient availability have been suggested as abiotic factors controlling survival of introduced bacteriain soils . Biological factors include predation by protozoans, phages, a lower level of starvation resistance ofthe introduced bacteria and lack of suitable soil nichesfor extended cell survival (Cassidy et al 1996). Immobilization of the microbes through encapsulation orentrapment in certain defined polymers has beenshown to afford protection to cells under such adverseconditions (Cassidy et aI, 1996 ; Joshi D ' Souza ,1999 ; Leung et i 1995 ; Morin e l aI , 1992; Smit et ai1996; Steenson et aI 1987; Trevors et aI 1993). Thephysical soil environment is heterogeneous andchanging environmental conditions can result in various alterations of the soil s. Encapsulation providesnot only protection, but a more stable microenvtronment for the entrapp ed microbial cells. For examplemicrobial cells entrapped in alginatL' remained stableafter a few drying/wetting cycle s in soil , wherea s freece lls under similar c o n d i t i ) n ~were reduced b y about

Application

Degradation of phenolVolatilization of mercury

Reduction of techn etium

Palladium rec ov e ry

Tr eatment of urea e ffluent

Degradation of naphthal e neDegradation of dim ethylphthalat eRemoval of H20 2 from textil ebleaching efflu entIntroduction into soil s

Eff ective mean s of incre asin g mi-

croalgal populati onGluco se biosen sorEstimation of urea

Glucose , urea , lipids bio sen sor

Artificial pancr eas

Referen ce

Jo shi D' Sou za. 1999Gho sh el a i 1996

Lloyd et a l . 1999

Yon g et al. 2002

Kamath D' Souza, 1992

Manohar et ai 200 INiazi Kare go udar , 200 ICo s ta el al . 2002

Dai g le Cotty , 1995

Gonzalez Bashan. 2000

Kumar el al. 1994Kayastha Srivastava ,2001Sukeerthi C ontractor ,1999Mullen el al . 2000

log 2 CFUlg (Cassidy et ai 1996 ; Trevors et aI 1993).

The entrapment techniques discussed earlier havebeen modified to include in addition to the microbialconsortia, nutritional amendments like milk proteins,

oil seed meal, etc. and fillers like clay (Cas s idy et ai1996). Such immobilization technique s can help indevising newer strategies in the future in the introduction of microbes into the soils . Using this approach it is now possible to provid e the microbialconsortium with a more defined microenvironment ,quite different from that encountered when directly incontact with soil environments. In thi s 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 o f plant growthrelated organisms like Rhi zobium into so ils. Microenvironment in the bead may initially protect cells fromthe soil microenvironmenl. Microorganisms are released after adaptation to prevailing environmentalconditions. This may enable cells to overcome thenumerous changing conditions in soil and increasemicrobial survival. Entrapment in gel-matrix offers astable, defined, consistent. protectiv e environment.without the immediate relea se of large number ofcells, where cells can survive and met abolic activity


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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 especiallyn terms of ease of storage and transportation and alson terms of biosafety features that limit contaminat ion

and bioaerosol formation. Coimmbilization of thefresh water micro-alga, Chlorella vulgaris and theplant-growth-promoting bacterium, Azospirillum bra-silense in alginate beads resulted in a significant increased growth of the micro-alga. Such an approachhas 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 alsobeen extensively investigated for pollutant degradation with an emphasis on in situ bioremediation ofchemically 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 foruse in the environment in varied soil systems.

Bioremediation is currently gaining considerableimportance as an economically alternative technologyfor the treatment of heavy metal and radionuclidewaste. Bacteria, yeast, fungi, algae and agro biomasscan remove heavy metals and radionuclides from

aqueous solutions in substantial quantities eitherthrough the process of biosorption or bioaccumulation(Bhainsa D Souza, 1999; D Souza 1999c; D Souzaet al 2001; Gadd, 2000; L10yd Macask ie, 2000 ;Sar D Souza, 2001; 2002; Veglio BeoIchini,1997) . One major requirement of any such process isa biosorbent of mechanical stability and integrity inaddition to its biosorption capacity, particularly formultiple adsorption-desorption cycles (VeglioBeoIchini, 1997). Other important criteria for the useof microbial biomass for bioremediation of heavymetal /radionuclides is their ability to be retained in abioreactor. This is mainly because unlike the organiccompounds which can be biodegraded heavy metalsare immutable at elemental level thus methods to retain them in the bioreactor, forms one of the importantstep in their utilization. Unlike the fungal pellets,which can be used directly, for the effective use in thebioremediation processes, microbial biomass including yeast and bacteria consists of very small particlewith low density, poor mechanical strength and littlerigidity . Hence these have to be pelletized. This can

be achieved by immobilization of the biomass. Immobilization imparts more operational flexibility andsolves the problems associated with solid-liquid separation in settling tanks. Immobilization also helps inthe 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 beinginvestigated for this purpose (D Souza, 1999c;D Souza et al 200 I; Leenen et al 1996; Macaskie etal 1995; Michael Reeves , 1997; Rus et aI 1995Siedel Jeffers, 1991; Tucker et al 1998). Studiesare under progress in our laboratory for the preparation of bioresins containing a variety of microbes foruse in the treatment of radionuclide and allied wastes .

Analytical Biosensor) and Medical Applications

Immobilized proteins, enzymes, cells and cellularorganelles have been widely used in the field ofanalysis and medicine. The use of immobilized biomaterials in these directions can be divided into twomajor categories: biosensors and artificial organs.Biosensors are gaining applications in a variety ofanalytical fields. Biosensor consists of a transducer inconjunction with a biologically active material thusconverting a biochemical signal into a quantifiableelectric response to yield a measurable signal. Thespecificity of the biosensor will depend on the selec

tion of the biomaterial. As biological sensing elements enzymes, antibodies, DNA , receptors, organelles and microorganisms as well as animal and plantcells or tissues have been used (D Souza, 1999b;2001a, b; MuIchandani Rogers, 1998; RogersMulchandani, 1998; Shanmugam et al 2001 ; Turneret al 1987). The basic requirements of a biosensor arethat the biological material should bring the physicochemical changes in close proximity of a transducer.

n this direction immobilization technology hasplayed a major role. Immobilization not only bringsabout the intimate contact of the biological catalystswith the transducer, but also helps in the stabilizationof the biological system thus enhancing its operationaland storage stability . The biological material has beenimmobilized directly on the transducer or in mostcases, in membranes, which can subsequently bemounted on the transducer. Most of the techniquesdescribed above have been used for the immobilization of biocatalyst for biosensor applications. Selection of a technique and/or support would depend onthe nature of biomaterial, nature of substrate and


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configuration of the transducer used . The choice o fsupport and technique for the preparation of membranes ha s often been dict ated by the low diffusionalresistance of the membr a ne (Kumar et al, 1992 ,1994) . Gentle techniques need to be applied whenviabl e cell preparations are to be used. The development of molecular device s incorporating a sophisticated and highly or g a niz ed biological informationproce ss ing function is a long-term goal of bioelectronics . For this purpo se, it is necessary in the futureto dev e lop suitable me thod s for micro immobilizingth e proteins /enzyme s into an organized array/pattern,(Farooqi et al, 1999 ; Koy a no et al, 1996 ; Subr amanian et ai, 1998 ; Yang et a i 1997). There is also acurrent interest in developing technique s for immobilization of biomolecule on electrode surfaces by entrapment or attachment to electrochemically polym

erized conducting or non-conducting films. (Cosnier,1999; Sukeerthi Contractor, 1999) . Som e of thecurrent developments and future potentials in biosenso r field has been recently reviewed by the authour(D Souza 2001a , b).

Ever since the feasibility of pre paring artificialcells wa s first demon strated in 1957 their importancein medicine especially in the fabrication of artificialorgans has gained interest. An increasing number ofapproaches to their preparation and use have becomeavailable (Chang , 1984 ; Liang et aI , 2000). Such en

trapment sys tem s can be obtained as bead s, microcapsules or membranes and can be formed u sing ava riety of synthetic or na tural pol y meric materialswith de sired variation in their permeability , surfaceproperties and biocompatibility. A variety of materi a lsincludin g enzymes, cells, cellular organ e lle s vaccines, antibodies, adsorbents, etc. have been tr appedespecially for use in medicine (Chang, 1984; Liang eta i 2000). One of the import ant current intere st insuch an approach is to form immunobarriers . Ba s i-cally low-molecular weight sub stances such as nutrients, electrolytes, oxygen, bioactive secretory prod

uct s and cellu la r waste products can diffuse freelyac ro ss the arti fIc ial membranes while immunoglobulin s and other immune effector mechanisms are excluded . This i s ga ining considerable interest in thecreation of bioartifical o rg ans like bioartificial pancreas using entrapped i slets (Jwata et aI, 1994; Lanzaet ai, 1995 a, b; Lim Sun , 1980; Mikos et aI 1994;Mull en et al, 2000). Tran splantation of tissue s andcells across discordinate barriers remains a challengeboth in experimental and clinical settings in view of

the immunolgical problems. Immobilization especially the entrapment technique has been propo se d toplay an important role in this direction (Iwata et al,1994; Lanza et aI, 1995 a, b; Mikos et a i , 1994 .

ConclusionThere are intere sting po ss ibilities w ithin the field

of immobilized enzymes and it is imm inent that in thefuture many applications will be repl ace d by immobilized systems and many more new syste ms will become tec hnic a lly as well as commercially fea s ible .Biotechnology in general is a highly interdisciplinaryarea of re sea rch. A fruitful fu s ion in the future ofvarious scientific, engineering and medical di sc ipline swill allow biotechnology to reali se its full indu strial ,environmental, analytical and medical p o te ntial

cknowledgementThe author is thankful to all collea g ue s who have

contributed to the work reviewed in thi s paper.Thanks ar e also due to Dr (Mr s) A M Samuel , Director Biomedical Group, BARC for her encouragement.

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

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