Indian Journal of Biochemistry & Biophysics Vol. 39. August 2002. pp. 220-228

Applied Biocatalysis: An Overview

Munishwar N Gupta* and Ipsita Roy

Chemi stry Department. Indi an Institute of Technology. Delhi , Hauz Khas. New Delhi 110 0 16. India.

Received 12 Jllly 2002

Introduction

"Rather I prize the doubt. Low kinds exist without,

Finished alld finite clods. Untroubled by a spark "

Robert Brownillg

The title of thi s overv iew/mini-sympos ium may rai se a few eyebrows. Why applied biocatalys is? Why not si mply biocatalysis? ]s there a difference? The foll owi ng anecdote may give a clue. Michael Faraday presented hi s discovery of electricity to the Briti sh government. The Lord of Exchequer asked him the poss ible use of such a discovery. Faraday 's answer showed his brilliance. He repli ed: "My Lord, one day you will be able to tax it!" He was given the green signal for further work . It is a thin line whi ch di vide:; fundamenta l and applied sciences. If you put a biocatalys t to use for a spec ifi c purpose and the applicati on is economicall y viable (or at leas t shows potential ), we can call it appli ed biocatalys is. The adj ec ti ve keeps Ollt conventi onal enzy mology, e.g. purify ing an enzy me with 5-10 steps.

A few years bac k, Whites ides and assoc iates wro te a very good articl e on perceived advan tages and disadvantages of usi ng enzy mes l

. The laller included "expens ive, unstab le, restricted to predo minantl y aqueous environments and di fficult to manipul ate" . Thi s overview will show that these disad vantagcs have been more or less overcome. (I n fact , the various papers in thi s mini-sy mpos ium ill ustrate thi s with specific examples) . Much of the biotechno logy today deals with either prod ucing enzy mes/protein s or using ' them! Appli ed Biocatalysis is an are:.! wh ich is trul y multi-discipli nary in nature: mi crobiologists . tiss ue

;, Author to whom correspondence: Illay be add ressed. rc I.: 91 - 1 1-659 1503 rax:9 1-tt-658 tOn Ema il : mn_g upta@hotmail.co lll

culture ex perts, chem ical engineers, biomedical engineers and immunologists merely consti tute an illustrative list.

The hi story of app lied biocatalysis is nearly as old as human civili zation. Earliest applications were em pirical and were more of recipe than science. Moreover, these app li cati ons were there even before the concept of biocatalys t or enzy me was known to us. Table I summarizes these early applicati ons and discoveries and some correspond ing current perspecti ves.

P,-oduction of Enzymes The production of therapeuti c proteins, which has

been made possib le by new discoveries in biotechnology, has in 200 I, generated sales exceeding $25 bi lli on 1o This, combined with the commercial need of vaccines, industrial biocatalysts , enzymes for syn thesis of fine chemica ls by 'green' rou tes and di ag nostic kits, have necessitated a 'second look' at the cost factor in downstream processing of proteins. This stage also faces challenges from the fact th at the target product form s only a minor part of a hi ghly complex broth.

Apart from conventional methods of extracting enzy mes from anima l, plant and mi crobial sources, today one can produce enzy mes by cloning, ti ss ue cu lture or transgen ic technique ". All stages from the poi nt of production to obtaining the enzy me with "adeq ua te purity level" constitu te downstream proccss ing' c. In the case of intracellul ar enzymes, dow nstrea m process ing starts with cell d is ruptionl.1 by chemical, mechani cal or biochemical means. For all cnzymes. the next step is so li d/l iquid separation for re mov ing the su pended impurit ies and cell debri . Beyond thi s, there are no set rul es but the trend is to use met hods of increas ingly hi gher resol ution. Thus, vec ipitat ion ~ ch romatography ~ affinity chromato­graphy have, over the years, fo rmed the basic outl ine for the purification protocol.

GUPTA & ROY: APPLIED BIOCATALYSIS: AN OVERV IEW

Table 1- Hi storica l Perspective and Some Current Tre nds in Appli ed Bioc3talys is

• Produc ti on o f soy deri ved foods in Ch in a and Japan. Microbi al amylases and proteases were in volved2

• Homer described " the productio n of cheese by sti rring mil k with a twi g of fig tree". Fic in , obviously, was in vo lved2

Milk kept in a bag made from the sto mach o f a recently s laughtered calf gave a drier substance wh ich gained a fl avour after some time. Thus was born the art of cheese making with the he lp of re nnin2 Our more recent know ledge te ll s us that ca lf rennin catalyzes the proteolytic clea vage o f a s ingle peptide bond (Phe lo'_ Met I06

) in the K-casein mo lecu le, ini ti ating protein precipita tion).

We now also know that changes in tex ture and fl avour o f cheese are associated with the furth er hydro lys is of milk . ~

pro te ins . Proteins -7 Peptides -7 Amino ac ids -7 Volatile fl avours and aromas

Occasional shortages of ca lf re nnin (chymosin ) have prompted the use of fungal proteases from Elldothill pa/'Gsitica. Mucor meihei and M. puci/lus as substitutes . G lobal demand for chymosin is estimated to be worth about $ 100 milli on a year. O n March 23, 1990, chymos in became the first genetica lly engineered protein to be used in food process ing'.

Rennet (a mi xture of rennin and pepsi n) inc identally was also the first enzyme prepara tion to be marketed, by a company called Chritian Hansen (Copen hagen, Denmark ) in 18752

.

• Otto Rohm obtained trypsin-in-detergent patent in 19 13. Thi s led to the introduction o f the first enzymatic detergent ' Burnus' consisting of pancreatin and sodium carbonate6

. However, it was qui te some time before enzy me-based detergents became commonl y acceptable. Like many o ther app lications of enzy mes, thi s story is linked with the iss ues of stabi lity and stab ili zati on of enzy mes. The enzy mes incorporated in the detergents should be stable at alkaline p H, at fairl y high temperatures and in the presence of chelati ng agents and surfac tants . Around the early s ixties, "everybody wanted Biotex , the protease contai ning detergent ,,2 However, there was the scare of a ll e rgic responses in the la te sixti es. Thus, it is interesting to note that by the ea rl y seventies, enzymes were withdrawn fro m detergents3

. Granu lat ion techniques a llowed the ir reintroduction in the detergents as it was found that it was the inhalation of dust formed in the enzy me powder which was responsible for the a lle rg ic responses3

Prote in engineered subtili sin addressed the aspect of operat iona l stab ili ti. Today, c nzy mc-based detergents constitute a major application of biotechno logy. T he detergent industry accounts for - 30 % o f to tal sales of enzymes. Apart from pro teases and lipases, amy lases and cellul ases are also used.

Apart from detergents for tex tiles, enzy mes are also used in clean ing agents for o ther purposes, e.g. lipases in contact lens cleaners and windshi eld washing fluids, e tc.2

• Starch processing : Today, po lysaccharide-degrading enzy mes constitute a major component o f the market for industrial e nzymes . Wi thin th is , starch-degrading enzymes represent the biggest porti on. a- Amylase and g lucoamylase y ie ld sugars. The latter can e ither be fe rmented to alcohol (an energy source) or converted into prod ucts like high fructose corn syrup (HFCS ) (industri al sweetener) by glucose isomerase. The enzy mo logy o f starch degrada ti on is dep ic ted in Fig. I.

A major constra int has been that whil e industrially usefu l (thermo tab le enzy mes fo r starch liquefac tion) a-amy lases are uns table be low p H 6.0, g lucoamy lase requires p H of around 4.2-4.5 for furth cr hydrolysi s to g ive g lucose . pH adjusting means additional cost, more so if g lucoamylase ac tion has to be fo llowed by glucose iso merase (with pH

opti mum around 7-8). Few years back, Shaw et al. ( 1999) have rev iewed protei n engineeri ng of a-amylase for fun ct ioning at low p H7. Thi s perhaps g ives a good idea of what applied biocata lysi s is a ll abo ut!

• PeClill hydrolysis: It was in 1930 that Kertesz tiled a patent for "enzy matic clarificati on of unfermented ap~le juice"S Today, en zy mes

are used in a variety of ways : in extracti on. clarificat ion and modification of frui t juices . While pectinase is a key en zyme (see Fig. 2), man y other enzy mes are a lso employed in specific cases . For example, add ition of cellulases helps in releasing desired colou red compounds in the case of black currants and red grape. Some fruits like pear are ri ch in arabans and arabanase is required for c larifica tion of pear juice. Naringi nase is requi red fo r debinering, espec ially in cases like grape fru it ju ice. Another interesti ng requi rement is that of g lucose ox idase for removal of O2 from "headspace" in the case of wine. beer and fruit j ui ce. Thi s cuts down non-enzymatic browning of these beverages. Thi s is one area where know ledge of biocatalysis has become an acceptable practi ce at industrial level. For example. while technology fo r production of French cider has esse nt ia ll y remained unchanged since 1909, in about 30 % cases, the industry has switched ove r to the use o f RapiJ ase Pomaliq (a mix ture of pcctinases, heillicellulases and cel lulases by Gi st-Brocades, Nctherlands) fo r c utting down the process time by enzymaticall y hydro lyz ing pec ti c material in appless

• Today, there is hardly any industry whcre enzy mes are not used. Ani mal feed. producti on of low lactose milk and whey hydrolysi s. cdib lc oil ex trac ti on. textiles, leather and paper indus tries are some other notab le examples.

221

222 INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, AUGUST 2002

Starch

1 a -amylase

J Oligosaccharide l

Pullulanase Glucoamylase ). p-amylase

Gl ucose Maltose

11 Glucose isomerase

Fructos(

Fig. I- Schematic diagram of enzy mati c degradation of starch. I a-A mylase hydro lyzes internal a- l ,4 linkages (endohydrolase) . p-A mylase removes maltose units from the non-reduc ing end (exohydrolase). Glucoamylase hydrolyzes a- l,4 glycos idi c bonds and removes glucose units from the nonrcducing end of the oligosaccharide. Pullulanase, on the other hand . hydrolyzes a-I,6 glycosidic bonds. Glucose isomerase converts glucose to fru ctose and is used for producing industrial sweeteners as fru ctose is much more sweet than glucose].

00B-0-v> E,Opolyg,Lumn", 1 COOCH) cleaves links between non-reducing ends of chains Endopolyga!acturonase and

pectate lyase cleave bonds between non-esterifi ed galacturonic acid residues

Pectin lyase cleaves bonds between esterified galacturonic acid residues

Fig. 2- Schematic diagram of enzymatic degradati on of pectin

Modern downstream processing is based upon the realization that a limited number of unit processes is an essential feature of an economical protocol. Thi s has resulted in two broad approaches :

(a) To deal with crude suspensions directly and skip the solid/liquid separation step. This has been possible by using two-phase extractions, expanded bed chromatography and in recent years, three phase partitioning.

(b) To bring up affinity-based processes ri ght in the beginning and not at the polishing stage. Thi s has been made possible by interfacing affinity

interactions with prectpltati on, me mbrane separati ons, fluidized beds, two-phase and three­phase partitioning methods.

Both broad approaches coincide in shaping many modern bioseparati on techniques. Thi s is one area of bi ocata lys is where much has been happening in the las t decade or so. Hence this wi ll be dea lt with in somewhat grea ter detail. A rev iew of some of the important developments fol lows .

Expanded Bed Chromatography In this approach, the use of tai lor-made media

a llows one to operate chromatography in a fluidi zed bed mode. Particulate adsorbents, if packed in a column, can not dea l with crude suspensions as clogging of adsorbent beds will take place. Batch adsorption in stirred tank is an opti on in such cases, but shows poor resolution and capacity. A fluidized bed in whi ch inter-parti cle di stance in adso rbent is increased, creating voids th rough which suspended impuriti es pass th rough, is best suited to capture the protein from the crude extract. The bes t way of fluidizing the adsorbent particles in practice is by a liquid flow directed upwards. Such flui dized beds can combine clarifi cati on, concentration and (varying ex tent of) fractionation in a simple un i t process 14 .

More sophi sticated designs and more deta i led di scuss ion can be found in some of the reviews I5.1(i.

Operationally, an expanded bed chromatography protocol fo llows the standard sequence (of packed beds): equilibration, sample application, wash, elution and c1eaning-in-place (CIP) . Expanded bed chromatography has already been succes fully applied in many cases. Some illustrative examples from our laboratory are g iven in Table 2.

Affinity Precipitation Affinity precipitation involves preCIpitati on of the

target protein in free solution, mostly using a reversibly soluble-insoluble macroaffinity ligand I7.IS.

Thi s macroaffinity li gand consists of a polymer which is so designed that its solubility is contro lled by altering some solvent parameter such as pH, temperature, addition of metal ions, etc. In some fortuitous cases, the polymer itself has an affinity for the protein. In other cases, a ligand is coupled to the polymer. Since the target protein has affini ty for the macroaffinity ligand, it is se lectively precipitated along with the macroaffinity " li gand upon the necessary change in the controlling parameter. It can be recovered us ing specific or non-spec ific elue nts

GUfYfA & ROY : APPLIED BIOCATALYSIS: AN OVERVIEW

Table 2- Some examples of the use of the technique of expanded bed chromatography (Adapted from Ref. 12)

Enzyme/ Protein

a-A mylase

Cellulase

Amy lase inhi bitor

Phospholipase D

Alkaline phosphatase

Pullulanase

Polyphenol ox idase

Source

Bacillus amylo/iqllefac iens Porcine pancreas

Wheat germ Scyralidiull1 thennophihllll

Aspergillus niger

Wheat germ

Peanut

Chicken intestine

Bacillus acidopullulyticlis

Duranta plulllieri

Matrix

Alginate

Cellulose beads Chitosan

Cu2+- linked agarose

Cross linked alginate

Dye-linked cellulose beads

Alginate

Streamline DEAE

Yield (%)

92.5 99.7 89.8 80.2

91 80

83

78

70

97

77

Table 3-- Some examples of proteins purified by affin ity precipitation

223

and the macroaffinity ligand is ava ilable for reuse. The process thus results in conce ntrati o n and purification of the target protein and is amenable to scale-up. It can also be used direc tl y with crude culture broth, unlike chromatographic techniques where fouling of the column (especially in the packed bed mode) is a recurring problem when using ex tracts containIng particulate matter. Some illustrative examples are tabul ated in Table 3.

Polymer Enzy me/Protein Reference

Three Phase Partitioning Precipitation wi th salts and org::lIlic so lvents are

well-establi shed techniques for concentration of crude protein extracts . Three phase part itio ni ng (TPP) was first developed as a bridge between upstream and downstream processes32

. The basic protocol involved is outlined in Fig. 3. The mechanism of how partitioning operates is not very c learly understood. It is believed to be the result of a co llective operation o f pri nciples in vo lved in numerous techniques such as conventional salt ing o ut. Morton 's Il-butano l extraction method , isoi onic precIpItati on, cold cosolvent precipitati ~n and osmolyte and ko", motropi c precipitation of proteins33

. In an effort to understand the mechan ism involved , proteinase K was subj ected to three phase partitioning. The X-ray d iffracti o n patterns of crystal of pure nati ve proteinase K and TPP-treated prote inase K (TPK) were compared.l l

. As a resul t of this treatment, the sPecific ac ti vity of TPK had gone li p by 2. 1 times. The attenti on was thus focussed o n the binding reg Ion o f the enzy me. The

Eudragit S- IOO

Alginate

Chitosan

Copolymer of I-viny limidazole and N-viny lcaprolacltim

Poly (N-i sopropyiacryl­amide)

Copolymer of I-vinylimidazo le and N- isopwpylacryl ­ami de

puri fied

Xylanase IgG-type monoclonal anti body

Pectinase a-Amylase Phospholipase D Lipase

Wheat germ lectin Lysozy me

Soy bean trypsin inhibitor

Alkaline protease

Amylase inhibitor

Gal actomannan Human IgG

Poly (N-isupropylacry l- Avi din amide)

19 20

21 22 23 24

25 26

27

28

29

30

31

224 INDI A J. BIOCHEM. BIOPHYS., VOL. 39, AUGUST 2002

Crude extract

Add ammonium sulphate and t-butanol

Lower Aqueous phase

Interfacial protein precipitate

~

Upper organic layer

Recover and dissolve in aqueous buffcr

Fig. 3-- Flowsheet for three phase partit ioning

most striking change in TPK was found in the conformation of the side chains of a number of residues. The residues indicated more than one conformational state for their side chains. This structure (TPK) corresponds to the highest observed B-factor for proteinase K. Thus, the increased flexibi lity of the molecule is responsible for hi gher activity of the enzyme.

Some exa mples from our laboratory , listing the success of thi s technique, are shown in Table 4. It has been shown that interfacing a metal affinity based step with TPP protocol makes the latter a hi gh ly selec ti ve technique. Metal affinity based separati ons (of which immobilized metal ion affin ity chromatography (IMAC) is the most frequently used version) exploit the affinity of surface hi st idine, cysteine and tryptophan residues in the protein molecule for metal ions like Cu2

+, Zn2+ and Ni 2

+.

Soybean tryps in inhibitor (ST!) has surface hi stidine residues and has been purifi ed via metal affinity. The selectivity and usefulness of meta l ion-ass isted TPP has been evaluated by purifying STI from soybean meal39.

A modification in this process has been introduced by macroaffinity ligand facilitated three phase partitioning (MLFTPP). As discussed above, there are many polymers which show affinity for the enzy mes. Using these polymers to capture the target enzymes in the TPP mode, enhances the selectivity of this simple process. This has been used to purify xy lanase usi ng Eudragi t S-IOO and the polymer is ava ilable for reuse

, after the end of one purification cycle4o.

Other Emerging Techniques Simulated Moving Bed Technology

Continuous chromatographic separat ion processes based on the si mulated mov ing bed (SMB) techno-

Table 4-- List of enzymes/proteins purifi ed by three phase partitioning

Source EnLy melProtei n Reference

Chicken intestine A I kal i ne phosphatase 35

Dacus carota Phospholipase D 36

Wheat germ a-Amy laselProteinase K 37 inh ibitor

Tomato Pect inase 38

Soybean Trypsin inhibitor 39

Aspergillus lIiger Xylanasc 40

logy have been gatntng increasing importance since the beginning of the last decade for app licat ions in the fine chemical and pharmaceutical industries, in particular for the resolution of enantiomers~'. The foc us has shi fted from sugar and petrochemical industries for very large scale fractionation when the technology was introd uced in the late 1950s. The technology has evo lved out of true moving bed (TMB ) concept. The so lid adsorbent particles flow along the column countercurrent to the fluid stream at a velocity that pushes mixture co mponents (wh ich are to be separated) in oppos ite directions. The mi xture is fed in the middle and componen ts A and B can be co llected from the top and bottom portions. Apart from the most critical choice of flow rates, design of a TMB (or 5MB) also has to cons ider feed concentrati ons, number of co lumns per zone, column length, column diameter and particle size.

Nicoud has described the use of moving bed for removal of ammonium sulphate from a protein~2 . Purification of trypsin from porcine pancreatic ext racts, purificat ion of human serum albumin, separation of myoglobin and lysozyme have shown the potential of thi s approach42 . Gottschli ch and Kasche have purified a monoclonal antibody from a cell culture supernatant with yie ld exceedi ng 90%43. SDS-PAGE of the feed and the pooled stream confirmed the removal of more than 99% of the contaminating proteins. 5MB technology offers several advantages over conventional preparative chromatographic techniques: a continuous process, it can be allowed to run unattended and ensures stable quality of the product. Productivity per unit mass of the stationary phase is high (the column works in the non-linear region of the adsorption equilibria of the

GU PTA & ROY : APPLI ED BIOCATALYSIS: AN OV ERVI EW 225

components to be separated, i.e. under overload conditi ons, thus ensuring optimal use of the stati onary phase) and the recycling of the fluid phase implies that the solvent requirement is low. Al so, the technique is more robust since hi gh purity of the product can be attained with a smaller number of theoretical pl ates. At present , however, thi s technology is limited to effi cient and complete separati on of a mi xture of two proteins.

Perfusion Chromatography

rn contrast to conventional chromatography medi a, perfusion chromatography medi a particles have two di screet classes of pores. Large "throughpores" allow convecti on fl ow to occur through the parti cles themselves, quickly carrying sample molecules to short' "diffusive" pores inside. By reducing the distance over which diffusion needs to occur, the time required for sample molecules to interact with interi or binding sites is reduced. Diffusion is no longer limiting and fl ow rates can be dramaticall y increased - without any loss of resolution or capacity. Separati ons can be achieved at I ,000 to 5,000 cm h( l compared to 50 to 360 cm h( l for conventional medi a. The technique is associ ated with high throughput and hi gh dynamic capacity . Fahrner and Blank have shown that an on-line chromatographic assay can reli abl y control antibody loading in real-time protein A affinity chromatographi c puri fication of a recombinant antibody from clarified Chi nese hamster ovary cell culture f1 uid44 . Hizel et al. have described a new procedure fo r the iso lation, puri fica tion and quantification of the product of the oncosuppressor gene brcal in tumour cells45

. It invo lves esS] methi onine labelling of intrace liular proteins followed by two perfusion chromatographies. DNA-binding proteins were isolated by heparin affinity chromatography on Poros 20 HE. BRCA I, which inc ludes a zinc fin ger, could be eluted along with all the nucleic acid-binding proteins using a salt grad ient.

Monoliths

Monoliths are continuous stati onary phases that are cast as a homogeneous column in a single pi ece and prepared in various dimensions with agglomerati on­type of fi brous mi crostructures. They exhibit hi gh effi ciencies even at high flow rates due to their fast convective mass transfer and can therefore be used at very high mobile phase velocities (up to 400 mlmin-1), leading to high producti viti es46.47.

Monoliths have been applied fo r the fas t separat ion and purificati on of proteins, DNA, smaller molecules like organi c acids48, hydroxybenzoates,

I· I 'd d 'd 4950 Q .. o Igonuc eotl es an peptl es ' . uantltatlve analysis of affinity interactions between antibodi es and immobilized group specifi c li gands (protein A, G and L) has been attempted using hi gh performance monolith affinity chromatography (HPMAC)51. Monoliths have been used' for in situ solid phase peptide synthesis52 and immobili zation of polynucleotide phosphoryl ase53 . The benefits of using monolithic columns include miniaturi zati on leadi ng to extremely fast separation (within seconds) and easy sca le-up. Further applicati ons of thi s upcoming technology include the areas of capillary electrochromatography, chip technology and scale-up in purifications4 .

Tailoring Enzyme Properties or Biocatalyst Engineering

Early efforts in tailoring enzymes to suit spec ific needs utilized chemical modifications and chemical crosslinking55. The key issue has been of enhancing stability (especially thermal stabili ty)56. Immobilization techniques brought in reusability and possible stabilizati on57 . Both noncovalent and covalent methods of immobili zation have proved use ful. Two relatively recent developments in this area have been:

(i) Introduction of cross li nked enzy me cry. ta ls, CLEC™, by Altus Biologicals. These micro­crysta lli ne preparati ons are reported to be stable in both aqueous and nonaqueous media58.

(ii) Design of smart biocatalysts. This has mostly consisted of creating bioconjugates of enzymes with smart polymers59. This enables the biocatalys t to be in free so luti on and one can transform macromolecular and insol ubl e substrates. After use, the biocatalyst can be recovered by app lying suitable stimulus which is chosen depend ing upon the smart pol ymer. Temperature, p H and add ition of chemicals are some of the stimuli whi ch have been used. An attrac ti ve des ign is to build in a smart domain with in the enzy me structu re60.

Protein engineering has been a more powerful tool for altering specific properti es of enzymes61 . While earl y applications again focussed on enhanci ng stability, more innovative approaches like changi ng pH optima (for effi cient biodegradati on of starch) are beginning to emerge7. Directed evolution is the

226 INDIAN J. BIOCHEM. BIOPHYS .. VOL. 39, AUGUST 2002

lates t tool which attempts at creating enzymes with suitab le reg io- and stereo-selec ti vity and suitab le kineti c properti es62. Thi s continues to use recombinant methods bu t takes advantage of hi gh throughput screen ing methods to reach a pre-selected property.

Bioconversions, Organic Synthesis and Non­Aq ueous Enzymology

Converting biomass to va lue-added products has been a major class of bioconvers ions. Some of these have already been menti oned in Table I. Hydro lys is of cellulose and ligni n to sugars and finall y to bioethanol has been an act ive area which has recentl y been reviewed by Himmel et al. 63 . Starch degradation has already been mentioned. Starting with corn starch, production of high fructose corn syrup (HFCS) is one of the top few applications of enzy mes in the industryl. Enzymes are increasingly being employed to develop 'green technology' for replac ing processes based upon aggressive chemi cals64

. A less-known example in thi s category is the extracti on of edible oil s by aqueous enzymatic oil ex tracti on process65

.66.

The process essentially degrades macromolecul ar struc tures entrapping oil bodies and thus facilitates the oil recovery.

Development of methodology for use of enzymes under low water conditi ons constitutes a milestone for applied biocatalysis. Both "nearly anhydrous systems" pioneered by the groups of Klibanov67, Mattiasson68 and Halling69 and "reverse micell ar systems" developed by the group led by Levashov and Martinek7o, Luisi71 and more recently by Cabral 72

,

have made it possible to use hydrolases for synthesis. Lipases, undoubted ly, have been used more often, fo llowed by proteases and carbohydrases. Several interesting phenomenon like pH memory, unusually high thermal stability and molecul ar imprinting have been observed with the use of enzymes in "neat organic solvents,,73. Again the major issue here has been that of stabili zing the enzymes against inactivation by organic solvents: Polar so lvents are known to be more damaging as compared to non­polar solvents74 . Again, a variety of techniques like chemical modification , immobili zat ion and protein engineering (used for enzyme stabilization in aqueous media against denaturing conditions) have been used with varyi ng success75. Recent work with directed evolution is especially relevant to the use of enzymes for obtaining drug intermediates76. This is an area where much is go ing on and it is likely to prove

immensely useful for the overall purpose of synthesis of fine chemicals and pharmaceuticals.

Ex posure to organic solvents as such can have some unusual and interesting con eq uences . It has been shown that in the presence of about 5- 10% organic solvent, vari ous enzy mes show hi gher

. . 77 I h acti vity . t as also been observed that heating enzy mes in the presence of organic so lvents at moderately hi gh temperatures results in obtaining enzymes with higher turnover numbers78

.

Biosensors and Diagnostics

Biosensors are basicall y created by interfac ing a biocatal ys t with a transducer. The fo rmer may be an enzy me, ti ssue sli ce, or whole organi sm. The purpose of the biocatalys t component is to detect the presence of a chemica l substance and amplify the 's ignal' via its turnover number. The transducer converts it into an observab le parameter. Depending upon the latter, we have enzy me electrodes79, fibre optic sensors (optodes)8o, field effect transi stors (FETs)81 and thermistors82. The applications of biosensors extend to diverse areas like clinical chemistry and health care, veterinary, agriculture and food sciences, fermentation and pharmaceutical production and environmental control and pollution monitoring. With the advent of smart materi als, the last few years have seen des ign of 'molecular gates ' and 'valves ' which sense and respond to the presence of certain metabol i tes83 .

ELISA, based upon antigen-enzyme or antibody­enzy me bioconjugates is the best example of use of biocatalyst diagnostics84. On-line analyzers using flow injection (FIA) in combination with expanded beds and perfusion chromatography are relatively recent examples85

.86.

Proteomics and Beyond

Proteomics represents the most recent and still fashionable face of applied biocatalysis. Genomics has given us the information. Proteomics has to tell us the "action part". Which enzyme/protein occurs where, how much , along with what other proteins and doe what, are the questi ons which define the contours of this emerging science. It may be interesting for the reader to refer to a recent debate on what proteomi cs should mean87! Ri ght now, for most of the people, it means mass spectrometry (electro spray and occasionally MALDI-TOF) or 2-D electro­phoresi s. There are some more sensible views ex pressed, which foresee that more sophisticated

GUPTA & ROY : APPLIED BIOCATALYS IS: A OVERVIEW 227

bioseparation strategies at the semi-preparati ve level will have to be deve loped88

. Thus, once again, we may come back full circle. The old protei n chemistry . uddenly became fashionable and was reborn as protein fo ldi ng. May be, we will see renaissance of downstream processing as a part of proteomics.

Thus, the area of applied biocatalys is, which perhaps started by stirring milk wi th the twig of a fi g tree, has come a long way . Today it in vo lves more sophi sticated too ls and strategies .

" Where is the knowledge we have lost in ill/ormation ? "

T. S. Eliot

Acknowledgement The publicati on of thi s arti cle and the work quoted

herein from the authors' laboratory were supported by funds from Council of Scientifi c and Industri al Research (Extramural Di vision and Technology Mission on Oil seeds, Pulses and Maize), Department of Science and Technology, Department of Biotechnology and Nati onal Agri cultural Technology Project (lndian Council fo r Agricultural Research).

References Akiyama A, Bednarski M, Ki m M J, Simon E S, Wa ldmann H & Wh ite ides G M (1988) Chell/tech Oct 627-634

2 Tramper J ( 1994) in Applied Biocatalysis (Cabra l J M S, Best D, Boross L & Tramper J, eds), pp. 1-45. Harwood Academic Publi shers, Chur.

3 Walsh G & Headon D R ( 1994) (eds) Proteill Biotechllology, pp. 302-336, John Wiley, Chichester

4 Walker J M & Gingold E B (1988) MoleClila r Biology alld Biotechllology, pp. 168- 193, The Royal Soc iety of Chemistry, London

5 Glazer A N & Nikaido H (1995) Microbial Biotechllology: FUlldall/clllals of Applied Microbiology, pp. 209- 240, W H Freeman & Co., New York

6 Eriksen N (1996) in Industrial Ell zYlllology (God frey T & West S. ed), 2nd edn, pp. 189-200, Stockton Press, New York

7 Shaw A, Bott R & Day A G ( 1999) Curl' Opill Biotechllol 10, 349-352

8 Grassin C & Fauquembergue P ( 1996) in III dust rial Ell zymology (Godfrey T & West S. ed), 2nd edn, pp. 225-264, Stoc kton Press, New York

9 http:// 134.225. 167.1 14/NCBE/FEATURES/juice.ht ml 10 http://bvt.gbf.de/pdp/dpd.html II Wright G & Noble J ( 1998) in Bioseparatioll alld

Bioprocessing (Subramani an G, ed), Vol. 2, pp. 67-79, Wiley VCH, Weinheim

12 Roy I & Gupta M N (2002) Proc Illdian Natl Sci Acad (B) (In press)

13 Middelberg A P J (1998) in Bioseparation and Bioprocessing (Subramanian G, ed), Vol. 2, pp. 131- 164, Wiley VCH, Weinheim

14 Chase H A (1994) Trends BioteciJnol 12,296-303

15 Hjort h R, Lcijon P. Frej A K B & Jagerstell C ( 1998) in Biosepa/'{/Iioll all d Bioprocessillg (Subramanian G, ed), Vol. I, pp. 199-226, Wiley-VC H. Weinheim

16 Anspach F B, Curbelo D, Hartmann R, Garke G & Deckwcr W D ( 1999) J ChrolllalOgr A 865. 129- 144

17 Gupta M N & Mattiasson B ( 1994) in High/v Selectil'e Separat iolls ill Biotec!lIwlogr (Street G. ed), pp . 7-33. Chapman and Hall , London

18 Roy I & Gupta M N (2002) in Methods of Affill ity-based Separatiolls of EII ZYlll es alld Proteills (G upta M N. ed.), pp. 130- 147 , Birkhauser Verlag, Basel

19 Gupta M N, Guoqiang D, Kaul R & Malti asson B (1994) BiOlechllol Tech 8, 11 7- 122

20 Taipa M A. Ka ul R H. Mattiasson B & Cabra l J M (2000) Bioseparalioll 9, 29 1-298

21 Gupta M N, Guoqui ang D & Maltiasson B ( 1993) Biotechllol Appl Biochelll 18, 32 1 - 324

22 Sharma A, Sharma S & Gupta M N (2000) Proteill Expr Purij I8, 111- 11 4

23 Sharma S, Sharma A & Gupta M N (2000) Bioseparalioll 9, 93-98

24 Sharma S & Gupta M N (200 I) BiOlechllol Appl Biochelll 33 , 16 1-165

25 Senstad C & Malti asson B ( 1989) Biotechllol Bioellg 33 , 2 16-220

26 Tyagi R. Kumar A, Sardar M. Kumar S & Gupta M N (1996) 1.1'01 Puri(2 , 2 17-226

27 Gal aev 1 Y, Kumar A, Agarwal R, Gupta M N & Mattiasson B ( 1997) Appl Biochelll Biotechllol 68, 12 1- 133

28 Nguyen A L & Luong J H T ( 1989) Biotechnol Bioellg 34, 11 86- 11 90

29 Kumar A, Ga laev I Y & Mattiasson B ( 1998) Biolec!m ol Bioellg 59, 695-704

30 Bradshaw A P & Sturgeon R J ( 1990) Biolec!lIIol Tech 4 , 737-743

3 1 Garret-Flaudy F & Freitag R (2000) Biolec!lIIol Bioellg 7 1, 223-234

32 Tan K & Lovrein R ( 1972) J Bioi Chelll 247, 3278-3285 33 Dennison C & Lovrein R ( 1997) Proleill Expr Purij II, 149-

16 1 34 Si ngh R K, Gourinath S, Sharma S, Roy I, Gupta M N.

Betzel C, Srini vasan A & Singh T P (200 I) Prolein £lIg 14. 307-3 13

35 Sharma A, Sharma S & Gupta M (2000) Bioseparalioll 9 , 155- 161

36 Sharma S & Gupta M N (2001 ) Proleill Expr Purij 21, 3 10-3 16

37 Sharma A & Gu pta M N (2001 ) Process Biochelll 37, 193-196

38 Sharma A & Gupta M N (200 I) Biolechllol Lell 23, 1625-1627

39 Roy I & Gupta M N (2002) Allal Biochelll 300, 11 -14

40 Sharma A & Gupta M N (2002) BiOlechnol Bioellg (In press) 4 1 Juza M, Mazzotti M & Morbidelli M (2000) Trellds

BioleciJllol 18, 108- 1 18 42 Ni coud R M (1998) in Bioseparalion and Bioprocessing - A

Halldbook (S ubramani an G, ed) Vol. I, pp. 3-39, Weinhei m, Wiley-VCH.

43 Gottschli ch N & Kasche V ( 1997) J Chromalogr A 765,201-206

228 INOI AN J. BIOCHEM. BIOPHYS .. VOL. 39, AUGUST 2002

44 Fahrner R L & Blank G S ( 1999) J ChrolllaTog r A 849, 191 -196

45 Hi zel C, Mauri zis 1 C, Ri o P, Communal Y, Chassagne J, Favy 0 , Bignon Y J & Bernard-Gallon 0 J ( 1999) J Chrolllatogr B 72 1, 163- 170

46 Podgornik A, Bawt M, Strancar A, Jos ic 0 & Koloini T (2000) Anal Chell1 72, 5693-5699

47 Berreux L G & Freitag R (2002) in Methods oj Affin ity-based Separations oj Enzymes and Proteins (Gupta M N, ed.), pp. 82- 1 [4, Birkhauser Verlag, Basel

48 Wistuba 0 & Schurig V (2000) Electrophoresis 2 1, 3 152-3 159

49 Xi e S, Allington R W, Svec F & Frechet J M ( 1999) J Chroll1atogr A 865,1 69- 174

50 Xu B, Zhu Z, Tang Y, Huang Q & Wang X ( 1999) Protein Expr Purif 16,22 1-223

5 1 Berruex I G, Frei tag R & Tennikova T B (2000) J Pharlll Biomed Anal 24, 95- 104

52 Tripp J A, Svec F & Frechet J M (2001) J COll1b Chelll 3, 604-6 11

53 Platonova G A, Surzhik M A, Tenn ikova T B, Vlasov G P & Timkovskii A M (1999) Russ Bioorg Chelll (English ) 25 , 166- 171

54 Ten ni kova T & Strancar A (2002) LabPllls IntemaTl 16, 10-13

55 Tyagi R & Gupta M N ( 1998) Biochemislly (Mose.) 63, 334-344

56 Gupta M N (1993) (ed) Th erlllostability oj EnZYll1es, Springer Verl ag, Heidelberg

57 Tischer W &Kasche V (1999) Trends Biotechnol 17, 326-335

58 Lalonde J J, Navia M A & Margolin A L ( 1997) Methods Enzymol 286, 443-464

59 Roy I, Sharma S & Gupta M N (2002) Adv Biochelll Eng Biotechnol (In press)

60 Petka W A, Harden J L, McG rath K P, Wirtz 0 & Tirrell 0 A ( 1998) Science 28 1, 389-392

6 1 Arnold F H & Moore 1 C ( 1999) in New En zymes Jor Organic Synthesis: Screening, Supply and Ellgil/.eerillg (Scheper T, ed), pp. 1-1 4, Springer Verlag, Berlin

62 Mehta P K & Chri sten P (2000) Adv Ell zYlllol Relat Areas Mol Bioi 74, 129- 184

63 Himmel M E, Ruth M F & Wyman C E (1999) CUlT Opill Biotechnol 10,358-364

64 Liese A & Filho M V ( 1999) Cll rr Opin Biotechnol 10, 595-603

65 Kali a V C, Rashmi , Ut i S & Gupta M 1'< (200 I) J Sci Indlls Res 60, 298-3 10

66 Sharma A, Kh are S K & Gupta M N (200 I) J Allier Oil Chelll Soc 78, 949-95 1

67 Zaks A & Klibanov A M ( 1988) J Bioi Chelll 363, 3194-320 1

68 Adlercreutz P & Mattiasson B (1987) Biocatalysis I, 99- 108 69 Hall ing P J ( 1987) Biocatalysis I, 109-1 15 70 Khmelnitsky Y L, Neverova I N, Gedrov ich A V. Polyakov

V A, Levashov A V & Marti nek K ( 1992) Ellr J Biochelll 2 10,75 1-757

7 1 Walde p, Han 0 & Lui si P L ( 1993) Biochemistry 32, 4029-4034

72 Melo E P, Aires-Barros M R & Cabral J M (200 I) Biotechllol Anllll Rev 7, 87 -129

73 Klibanov A M (200 1) Natllre (Lo lldoll) 409, 24 1-246 74 Gupta M ,Batra R, Tyagi R & Sharma A (1997)

Biotecllllol Prog 13, 284-288 75 Gupta M N (2000) (ed) Methods ill NOll aqlleolls EnzYlllology

Birkhauser Verl ag, Basel 76 Chartrain M, Salmon P M, Robinson 0 K & Buck land B C

(2000) Cllrr Opin Biotecllllol 11 ,209-2 14 77 Batra R & Gupta M N ( 1994) Biotechllol Lell 16, 1059 -

1064 78 Gupta M N, Tyagi R, Sharma S, Karthikeyan S & Singh T P

(2000 ) Proteills 39, 226-234 79 Shi G, Liu M, Zhu M, Zhou T, Chen J, l in L & Jin 1 Y

(2002) Allalyst 127, 396-400 80 Xiao 0 & Choi M M (2002) Allal Chell1 . 74, 863-870 8 1 Yeung C K, Ingebrandt S, Krause M, Offenhausser A &

Knoll W J (2001) Phanllacol Toxicol Methods 45,207-2 14 82 Ramanathan K & Oanielsson B (200 I) Biosens BioeleClroll

16,4 17-423 83 Stayton P S, Shimoboji T, Long C, Chilkoti A, Chen G.

Harri s J M & Hoffman A S ( 1995) NaTure (Lolldon) 378, 472-474

84 Hoc k B, Seife rt M & Kramer K (2002) Biosens Bioelectron 17,239-249

85 Nandakumar M P, Lali A M & Mattiasson B ( 1999) Bioseparatioll 8, 229-235

86 Fahrner R L & Blank G S (J 999) Biotecl1ll01 Appl Biochelll 29, 109- 11 2

87 Baum R M (2002) Chelll Eng NelliS 80, 38-39 88 Lesley S A (200 I) Proteill Expr Purif 22, 159-164