Báo cáo khoa học: Enzymes in organic media Forms, functions and applications potx

REVIEW ARTICLE Enzymes in organic media Forms, functions and applications Munishwar N. Gupta and Ipsita Roy Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, India Enzyme catalysis in low water containing organic solvents is finding an increasing number of applications in diverse areas. This review focuses on some aspects which have not been reviewed elsewhere. Different strategies for obtaining higher activity and stability in such media are described. In this context, the damaging role of lyophilization and the means of overcoming such effects are discussed. Ultrasoni- cation and microwave assistance are two emerging approa- ches for enhancing reaction rates in low water media. Control of water activity and medium engineering are two crucial approaches in optimization of catalytic behaviour in nonaqueous enzymology. Organometallics and synthesis/ modification of polymers are two areas where nonaqueous enzymology can play a greater role in the coming years. The greater understanding of enzyme behaviour in nonaqueous media is expected to lead to larger and even more diverse kinds of applications. Keywords: antibodies in organic solvents; enzymes in organic solvents; high activity enzyme formulations; medium engineering; microwave assisted enzymatic reactions; synthesis and modification of polymers. Introduction The use of enzymes in organic media (with low water content) has been one of the most exciting facets of enzymology in recent times. Its importance can be appre- ciated from the fact that at least four books [1–4] and numerous reviews [5–7] are available on the subject. It is an area in which applications and phenomena preceded the understanding of catalysis at the molecular level. Also, it is an area in which enzymologists continue to discover unexpected and yet fundamental behaviour of biocatalysts. Thus, the excitement has not yet abated. The present review aims at two somewhat different objectives. The first one is to bring together both basic as well as applied aspects. In this respect, it is an update of the earlier review written by one of us [5]. The second aim is to cover some aspects which have not been generally covered by recent reviews. In some cases (e.g. ultrasonoenzymology and microwave-assisted non- aqueous enzymology), this is simply because these aspects are what the media calls Ôbreaking newsÕ. Some important features of this area which are covered or discussed in detail elsewhere are: designing enzymes by directed evolution [8,9], combinatorial biocatalysis [10], synthetic applications inclu- ding enantiomeric resolution [1,4,6,7,11,12], controlling enzyme specificity by medium engineering [13], solvent free systems and eutectic mixtures [1,3,14], smart biocatalysts in organic media [15], and bioanalysis and biosensors in organic media [2–5]. Forms and formulations While free enzyme powders continue to be used in nearly anhydrous media, a variety of forms and formulations have been described. There have been two major motivations behind evolving these designs. The first one has been the perception that enzymes are not stable in neat organic solvents. Unfortunately, a clear distinction between expo- sure to organic media and working in such media has not been made by many workers. When enzymes are exposed to such media (stress or denaturing conditions) for a limited time, and are recovered and checked for biological activity in water, early studies [16] indicate that full activity is observed. This implies that no irreversible denaturation has taken place. On the other hand, enzymes in different solvents display different k cat /K m , so operational stability is different in different organic solvents. A number of solvent parameters have been described; some have raised the question of nonpolar solvents not being the same as hydrophobic solvents [17]. The most acceptable, even if not totally satisfactory, parameter is log P, where log P is the partition coefficient of the solvent for the standard octanol/ water two-phase system [18]. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media. Chemical modification [19], chemical crosslinking [20], immobilization [21], protein aggregation [22,23] and protein engineering [24] have all been tried. Of these, immobilization has been used most often. In a review which dispels a lot of vagueness and myths (which are unfortunately associated with a lot of other papers and reviews), Halling [25] mentions that the most popular Correspondence to M. N. Gupta, Department of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India. Fax: + 91 11 2658 1073, Tel.: + 91 11 2659 1503, E-mail: mn_gupta@hotmail.com Abbreviation: HRP, horseradish peroxidase. (Received 28 February 2004, revised 31 March 2004, accepted 16 April 2004) Eur. J. Biochem. 271, 2575–2583 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04163.x immobilization method (adsorption on a support material like Celite) is, in fact, codrying or deposition rather than immobilization. The second kind of design has been prompted by the appreciation that k cat /K m in organic media (with low water content) is far less than the corresponding k cat /K m in aqueous media because drying by lyophilization causes reversible damage to the enzyme structure [26]. Thus, it is necessary to either prevent this damage or use other methods of drying. The use of additives (cryoprotectants, lyoprotectants, salts, etc.) during lyophilization falls in the former category. The latter approach consists of precipita- ting enzymes by organic solvents [27]. Some of the forms and formulations based upon both these approaches are: lyophilized powders containing cryoprotectants or/and lyoprotectants [28], propanol rinsed preparations [27], enzyme precipitated with and rinsed with propanol [29], enzymes obtained after three-phase partitioning [30], cross- linked enzyme crystals [22], crosslinked enzyme aggregates [31] and enzymes coated with ionic liquid [32]. Additives and activation Lyophilized enzymes are the most common forms of enzymes used in enzymology. Lyophilization involves [28,33] (a) initial freezing, (b) primary drying (in which ice separated from the protein phase is removed by sublimat- ion) and (c) secondary drying (in which the bound or trapped water is removed from proteins). Basically, lyo- philization (or freeze-drying) involves two denaturing conditions: freezing and drying. Cryoprotectants (very heterogeneous as far as chemical structures are concerned (e.g. sugars, amino acids, polyols, salts, etc.) prevent structural damage due to freeze stress. This protection may be due to Ôpreferential exclusionÕ [34]. During the drying stage (dehydration), one needs lyoprotectants. Lyoprotec- tants substitute water molecules, which are being removed, by forming H-bonds with the protein structure. Sucrose is a good example [33]. Evidence (most of it based upon FTIR) exists, which shows that the absence of lyoprotectant leads to both the random structure and the a-helical structure being partially converted to b-sheet structure (in which peptide bonds are linked to each other via H-bonds). In conventional enzymology (in aqueous media), rehydration reverses much of the structural damage during lyophiliza- tion. In nonaqueous enzymology, this becomes very crucial and is now considered as largely responsible for the low k cat / K m of enzymes in nearly anhydrous media. The k cat values for esterase (in water) and transesterification (in hexane) activities of subtilisin have been found to be 5.9 · 10 3 and 1.04 · 10 )1 M )1 Æs )1 , respectively [26]. Dabulis & Klibanov [35] showed that addition of a variety of substances (an amino acid derivative, sugars, polyethylene glycol) during lyophilization resulted in more active (in the range of 3–321-fold) preparation of seven hydrolytic enzymes. A few years later, Triantafyllou et al. [36,37] carried out a more intensive work on the effect of sorbitol and other additives on a-chymotrypsin and some lipases. While Dabulis & Klibanov [35] found that higher activity persists even if excipients are removed, Triantafyllou et al. [36] found that Ôwashing to remove sorbitol had a negative effect on the activityÕ of both lipase and a-chymotrypsin in their immobilized forms. One of the most remarkable results with additives has been the salt activation reported by Khmelnitsky et al. [38]. The 3750 times higher activity of subtilisin in the presence of 98% (w/w) of KCl was tentatively explained by either rigid salt structure protecting the enzyme in organic solvents or polar environment of salt helping in maintaining native structure of the enzyme. Soon after, Bedell et al. [39] provided evidence that it is not a result of reduced diffusional limitation. Subsequently, it was shown [40,41] that salt activation is the result of kosmotropicity and this stabilizing effect (during lyophilization) operates via prefer- ential hydration. Also, a combination of kosmotropic salt sodium acetate and sodium carbonate buffer had a cumu- lative effect. Hsu & Dordick [42] found that salt activation enhances enantioselectivity of subtilisin because the favoured reaction is more sensitive to the structural integrity of the enzyme. On the other hand, Altreuter et al. [43] found that salt activation is accompanied by expanded and unnatural regioselectivity of subtilisin. The latter feature is very valuable in the use of enzymes for developing combinatorial biocatalysis [10]. Recently, Lindsay et al. [44] reported the salt activation of a nonprotease, penicillin amidase and found that salt activation was dependent upon the water content in the solvent medium. However, the preparation lyophilized in the presence of trehalose behaved differently. The conclusion was that salt activation is mechanistically distinct from lyoprotection. On the other hand, Morgan & Clark [45] believe that the Ôpresence of salt protected enzymes from irreversible activationÕ in several cases. To sum up, the picture is still far from clear. That is not surprising as our appreciation of the role of lyophilization, while obtaining enzyme preparations for nonaqueous enz- ymology, is rather recent. That lyophilization, as a pheno- menon in itself is not simple, is not widely appreciated. Medium engineering It was Zaks & Klibanov [46] who reported that the activity of enzymes was higher in hydrophobic solvents than in hydrophilic ones. It was Laane & coworkers [18,47] who first addressed this issue in a comprehensive fashion and pointed out that log P, as a solvent parameter, correlates best with enzyme activity. Narayan & Klibanov [17] pointed out that it is hydrophobicity and not polarity or water miscibility which is important. As this may lead to some confusion among biochemists, it is best to quote ‘‘The log P parameter can be called a measure of solvent Ôhydropho- bicityÕ, which is an accurate description of what affects its value. This contrasts with other parameters such as dielectric, which measure the bulk ÔpolarityÕ. One illustration of the difference is to consider a homologous series of solvents. Adding extra methylene groups to an alcohol, for example, will cause a regular increase in hydrophobicity. Thus, decanol is more hydrophobic (higher log P) than hexane, but will be more polar by almost any measure of bulk properties’’ [25]. It is also pertinent to add that most often, the medium effects are explained in terms of more hydrophobic solvents having less tendency to strip water from enzymes. Such medium effects will not be seen if identical water activities in both media are used. Many 2576 M. N. Gupta and I. Roy (Eur. J. Biochem. 271) Ó FEBS 2004 solvent effects, in fact, arise from substrate solvation. If a substrate is more soluble in a solvent, the substrate molecules will be less available to the enzyme. Thus, higher K m values may lead to lower reaction rates [25]. The nature of the solvent media affects both the enantio- and regio- selectivity of the enzymes. Various hypotheses have been given to rationalize these important effects. No consensus seems to have emerged so far [7]. Finally, one must mention Ôsolvent-freeÕ media wherein one or more of the substrate(s) form the medium and no other solvent is required. In some cases, this results in more efficient conversion rates. A good example is the production of biodiesel with lipases [14]. Biodiesel consists of monoalkyl esters of long chain fatty acids. There is no petroleum or other fossil fuel in biodiesel; the diesel part of its name is based upon the fact that it can be substituted in place of petroleum diesel fuel. It is produced from vegetable oils or fats by lipase catalyzed transesterification with methanol or ethanol [48]. Importance of water activity in nearly anhydrous media It is now recognized that less than a monolayer of water is needed for an enzyme molecule to start showing biological activity. Beyond this, addition of more water molecules increases biological activity. However, in the case of hydrolases, beyond a threshold limit of water concentration, hydrolytic activity starts effectively competing with trans- ferase or synthetic activity [49]. The first clue that water content may not be the best parameter to look at was the work of Zaks & Klibanov [46]. Working with alcohol dehydrogenase in a variety of solvents, they determined the adsorption isotherm of water on the enzyme in a variety of solvents. It was observed that the water present on the enzyme correlated well with the reaction rates in different solvents. The higher the water content on the enzyme, the higher was the reaction rate. The rationale which emerged was that when water is added to the reaction system, it partitions among the enzyme, solvent, headspace of the reaction vessel and the immobilization matrix (if any). Soon after, Halling & coworkers made a seminal contribution in establishing water activity as a valid and useful parameter for carrying out biocatalysis in nearly anhydrous media [25,50–52]. Controlled addition of water continues to be used because of its simplicity. The relationship between mole fraction of water and activity in the case of several organic solvents has been reported [25]. Saturated solu- tions of different salts are known to release and absorb water at constant humidity [53]. Wehtje et al. [54] have described a method by which the water activity can be kept constant as the reaction proceeds. A saturated solution of the appropriate salt is pumped through silicon tubing that passes through the reaction mixture. As the water molecules can diffuse out of the tubing, the system remains equilibrated at the same water activity through- out. Another method, which is becoming increasingly popular, is the use of a pair of salt hydrates to maintain a constant water activity level throughout the reaction [49]. Here the lower hydrate or anhydrous form will absorb water and the higher hydrate will release it. A few years ago, suitable salt hydrates for different water activity levels were listed [55]. One area which needs more work is the effect of water activity on the stereoselectivity of the reaction. There are already a few reports which show that both rate and enantiomeric ratio change simultaneously when water activity is increased [56]. pH tuning and pH memory This is one dramatic observation [46] for which clear rationalization is now available [3]. The correct protonation state of side chains of amino acid residues of enzymes is important in nonaqueous media as well. Hence pH tuning (placing the enzyme in water at optimum pH of the enzyme, and lyophilizing) results in higher rates in organic solvents. However, this is valid only if the reaction does not change the acid/base concentration. Such changes have been mentioned by the same authors [57] who have also described useful protocols for pH tuning by the use of special buffers. Microwave-assisted reactions in nonaqueous enzymology The microwave region (0.3–300 GHz) falls between the infrared and radiofrequency regions of the electromagnetic spectrum. Chemical synthesis using microwave irradiation has been extensively reported. In this approach, microwave irradiation replaces conventional forms of heating [58]. Upon irradiation with microwaves, a polar molecule continually aligns itself with the fluctuating field. This converts electromagnetic energy into heat energy. It is not very clear whether these rate enhancements are purely due to thermal effects or whether some nonthermal effects are involved [59,60]. For example, Whittaker & Mingos [61] believe that Ôthe rate of a reaction in many syntheses is so high that it can not be accounted for by heating effects onlyÕ. Early work on microwave-assisted enzymatic reactions in water was naturally limited to the use of thermostable enzymes [62]. Considering that enzymes are highly thermo- stable in nearly anhydrous media [2,5,16], it is natural that microwave assistance is also explored for enzymatic cata- lysis in such media. Also, the commercial availability of microwave ovens, which are able to maintain constant temperature by remote IR feedback control, has also facilitated work in this area. Carrillo-Munoz et al. [63] used two commercially available lipases from Pseudomonas cepacia and Candida antarctica for esterification and transesterification reactions at a constant temperature of around 100 °C. The enzymes were immobilized on matrices which did not absorb microwaves. The microwave assistance was found to enhance both initial rates as well as enantioselectivity as compared to the results obtained under conditions of classical heating. An interesting result was that the use of substrates with greater polarity led to greater effect by microwaves. The results of Parker et al. [64], on the other hand, emphasized the importance of optimization of water activity for obtaining maximum increase (two- to threefold) by microwave assistance. Lin & Lin [65] have also observed enhanced reaction rates (four- to sixfold) and increase in stereoselectivity (three- to ninefold) upon use of microwave Ó FEBS 2004 Enzymes in organic media (Eur. J. Biochem. 271) 2577 irradiations. The molecular mechanisms for these results are seldom discussed and are not well understood. Chen et al. [66] have provided a protocol for using microwave assist- ance for protease catalyzed peptide synthesis. Unfortu- nately, most of these protocols do not take account of temperature control during microwave irradiations. Recently, esterification by a-chymotrypsin and transeste- rification by subtilisin Carlsberg were accelerated (in the range of 2.1–4.7 times) in six solvents of differing polarities and at different water activities [60]. Interestingly, micro- wave irradiation could be used in conjunction with pH tuning and salt activation. For example, at the same level of water activity (0.3%, v/v) in n-octane, untuned subtilisin showed a transesterification rate of 0.5 mmolÆh )1 at 25 °C. The salt-activated and pH tuned subtilisin showed about 20 times increase in reaction rates when microwave irradiations were used at 25 °C. It was also observed that the choice of the reaction medium was a factor which dominated the microwave effect. Maugard et al. [67] have exploited microwave assistance for a somewhat different kind of application, i.e. synthesis of galactooligosaccharides from lactose by using Kluyvero- myces lactis b-galactosidase. Low water activity, high lactose concentration and cosolvents with higher log P value all favoured oligosaccharide synthesis. By optimizing conditions and using microwave irradiations, the synthesis of galactooligosaccharides could be increased 217 times. It is hoped that when more experience with different systems becomes available, microwave assistance will become a powerful approach in nonaqueous enzymology. Ultrasonication Ultrasound waves have frequencies beyond the normal range of hearing, which consists of 20 kHz to 100 MHz and beyond. High power ultrasound waves can generate cavitation within a liquid. Cavitation provides a source of energy which can facilitate chemical processes. In this case, cavitation is an induced bubble activity. Cavitation can generate very high local pressure (up to 1000 atm) and very high local temperatures (up to 5000 K) [68,69]. The relevant effects of this are: (a) reduction in particle/ molecule size, (b) generation of free radicals facilitating mechanisms/pathways which involve free radicals and (c) increasing the fluid velocity, which facilitates mass transfer during the reactions. While ultrasonochemistry [70–72] is now a well-established area, ultrasonoenzymology has been carried out less extensively [73]. In view of harsh local environments during ultrasonication, it is expected that enzymes in nonaqueous media would be able to withstand ultrasonication better (in view of their higher stability in such media). Thus, it is not surprising that attempts have been made to use ultrasonicators in nonaqueous enzymology. Two distinct types of approa- ches have been used. Either ultrasonication has been used as a pretreatment step (for the biocatalyst) or the reaction has been carried out in the presence of ultrasonic waves. Both ultrasonic probes and ultrasonic baths have been used in ultrasonoenzymology. Unfortunately, in many cases, temperature has not been controlled. In such cases, one does not know whether the effects on reaction rates are due to ultrasonication alone or due to the rise in temperature as well. It is also difficult for others to reproduce results obtained with such ill-defined condi- tions. In other cases, temperature has been controlled either by use of a water-cooled reactor or interrupting ultrasonication so that the temperature does not rise. In the latter cases, cycles of ultrasonication and cooling have been used. Vulfson et al. [73] carried out subtilisin-catalyzed inter- esterification and reported (a) that pretreatment of subtilisin suspensions by ultrasound in alcohols led to an increase in enzyme activity. This effect was more pronounced with long-chain alcohols, being 6–8 times more in octanol as compared to the effect observed in short chain alcohols. The effect was dependent both on sonication power and water content of the medium, (b) the enhancement of reaction rates was, however, much higher if the reaction was carried out in the presence of ultrasound. The authors speculated on the mechanism of these effects; the reduction in mass transfer constraint because of reduction in enzyme particle size and increased fluid velocity seemed to be the most probable factors. Sinisterra [74], in his review on the application of ultrasound to biotechnology, has men- tioned data on ultrasonication increasing the percent yield in protease-catalyzed peptide synthesis in organic solvents. In hydrocarbons, the more hydrophobic the solvent (greater log P value), the larger was the sonication effect. In the case of organic halides, the relative increases under ultrasonication were CCl 4 >CH 2 Cl 2 >CHCl 3 . The reviewer concluded that it was difficult to analyze these facts. Lin & Liu [75] have reported that the use of ultrasonication enhanced the lipase-catalyzed acylation of a naphthol derivative with vinyl acetate in benzene-ether mixture by 83-fold. No temperature control was applied and hence the enzyme activity decreased if the tempera- ture crossed 37 °C. Bracey et al. [76] have re-examined the issue of the importance of enzyme particle size in nonaqueous envi- ronments. Zaks & Klibanov [46] had categorically stated that ÔInternal diffusional limitations were dismissed because ultrasonication of a suspension of chymotrypsin in octane (resulting in a reduction of an average enzyme particle from 270 to 5 lm, as revealed by direct micro- scopic examination) had no appreciable effect on the enzymatic transesterification rateÕ. According to Bracey et al. [76], no enhancement in the reaction rate of subtilisin-catalyzed interesterification in hexanol by ultra- sonication could be observed. This was contrary to the earlier observation of Vulfson et al. [73]. The attempts by Bracey et al. [76] to reconcile the two contrary results involve discussions on the possible mechanism(s) of ultrasonication effects and thus, are worth describing. Transmission electron microscopy by Bracey et al. [76] found that ultrasonication changed the open honeycomb structure of freeze-dried subtilisin into a plate-like struc- ture. Thus, size reduction did not result in significant increase in surface area. However, the hydration state of the enzyme may be critical. According to Rozicwski & Russell [77], solvation of a subtilisin particle in a hydrophobic solvent swells it, increasing the diffusion path for the substrate. If these pores have water molecules, reactant diffusion is inhibited. In such cases, 2578 M. N. Gupta and I. Roy (Eur. J. Biochem. 271) Ó FEBS 2004 increased fluid velocity by sonication would overcome this inhibition. Bracey et al. [76] believe that their enzyme was drier than those of other workers and hence they failed to observe any increase in reaction rate by ultrasonication. It is also worth noting that it is the hydration level before transferring the enzyme to the organic media, which was found to be critical. Thus, this mechanism does not explain the correlation between water content of the medium and the effect of ultrasonication reported earlier [73]. We need more structural work before ultrasonication can be used in nonaqueous enzymology in a predictable manner. Synthesis and modification of polymers Dordick et al. [78] have used about 80–85% organic solvents as media for peroxidase-catalyzed polymerization of phenols. The use of predominantly nonaqueous media has two advantages: the substrate phenols can be solubilized easily, and unlike in water where dimers and trimers precipitate out of solutions thereby terminating chain growth, oligomers of 1500–2000 Da range could be obtained in quite a few cases. Interestingly, it was reported that Ôthe decline in the molecular weight of the polymers above 85% dioxane is presently unclearÕ. Margolin et al. [79] also reported the lipase-catalyzed polycondensation of reactions between racemic diesters and achiral diols to form optically active trimers and pentamers. The peroxidase-catalyzed reaction has been further exploited for the synthesis of polyaromatic compounds [80]. Electron paramagnetic resonance studies with spin- labeled heme in peroxidase have revealed that inactivation of horseradish peroxidase (HRP) in anhydrous media is due to the substrate partitioning away from the active site to the bulk solvent. Whereas aromatic polymers were formed in both monophasic and biphasic solvents systems, use of reverse micelles yielded microspheric particles of uniform size (micrometer range or lower). The size of the microspheres was dependent upon the water to surfactant ratio. These nanoparticles could be used for encapsulating a variety of chemicals and enzymes. Polyanilines have also been synthesized and derivatized using HRP in solvent-buffer systems. It has been pointed out that these polymers have potential applications in biosensors, optical and electroptical devices and chromatography [80]. Cellulase has been used in aqueous-cosolvent systems to synthesize crystalline cellulose [81]. Lipases and proteases have been used for the synthesis of polyesters in supercritical fluids [82,83]. Immobilized lipase has been used to catalyze ring- opening/polymerization of 12-dodecanolide and e-capro- lactone [84,85]. Enzyme-catalyzed polytransesterification of alkanedioates and butane-1,4-diol in organic solvents has also been successfully carried out [86]. Use of subtilisin (solubilized by formation of protein- surfactant ion-pair method) in iso-octane for transesterifica- tion of a thin layer of amylose (deposited onto zinc-selenide plates) has been described [80]. The modification was followed by FTIR and thermogravimetric analysis of amylose. 1 H-NMR showed that all available primary amino groups were acylated. Further work in this area will definitely be useful in obtaining novel carbohydrate mate- rials for a variety of applications. Enzyme modification by directed evolution This approach originated in the realization that an enzyme is designed or evolved for a particular function and works in vivo as a part of a complex metabolic network. In many situations, these two features are constraints for indus- trial biocatalysts, especially if they are to be used in unnatural environments such as organic media. The approach consists of: Random mutations [87–89] At the outset, it is clear that one has to limit the number of mutations to only one or two residues at a time, otherwise the number of sequences generated will be unmanageably large. In the next step, it is possible to recombine and accumulate such beneficial mutations. This approach has been successfully used by Moore & Arnold [88] for directing the evolution of an esterase with desirable specificity. The choice of a suitable screening procedure also requires careful consideration. The screening procedure has to be simple so that a high throughput screening device such as an ELISA reader can be used. In reality, one desires a combination of new or retained features in the evolved enzyme: high expression levels, suitable pH or temperature optima, enantioselectivity and of course, high activity towards a particular substrate. While Ôdirecting evolutionÕ one has to make sure that one does not evolve one desirable property at the expense of other(s). A screening method can normally evaluate only one or at the most, a few, features. The overall strategy is then to carry out many generations of random mutagenesis by introducing errors while per- forming PCR. At each generation, the candidate having the best feature (which is being screened) is subjected to the next generation. The final enzyme, the result of this evolutionary progress, will be a more suitable biocatalyst for predecided function and milieu. In vitro combination In this alternate way of accumulating desirable changes (and features), the useful mutations identified during a generation can be recombined by ÔDNA shufflingÕ or Ôsexual PCRÕ [90,91]. ÔWhen combined with screening, DNA shuffling allows the discovery of the best combi- nations of mutations and does not assume that the best combination contains all the mutations in a populationÕ [92]. This form of recombination can simultaneously remove mutations associated with wrong features which may not have been screened out. Arnold & her coworkers have combined the two approaches (of random mutagenesis and in vitro recombi- nation) to obtain an esterase for hydrolyzing p-nitrobenzyl ester in the presence of polar organic solvents [88,93]. The application was to selectively remove p-nitrobenzyl groups during the synthesis of cephalosporin type antibiotics. The starting enzyme was from Bacillus subtilis with low p-nitro- benzyl esterase activity, especially in the polar organic solvents required to solubilize the esters. The clone obtained Ó FEBS 2004 Enzymes in organic media (Eur. J. Biochem. 271) 2579 by directed evolution showed more than 150-fold greater total activity than the wild type clone. It was also more stable in 15% (v/v) dimethyl formamide. Unlike site-directed mutagenesis, directed evolution can be applied without much structural and mechanistic infor- mation about an enzyme. Using antibodies in organic solvents Using antibodies in organic media provides a good oppor- tunity to carry out immunoassays of water insoluble antigens [94]. One major application of this is in environ- mental control and analysis [95]. Russell et al. [96] showed that antigen–antibody interaction is possible in low water containing organic solvents. Stocklein et al. [97] studied binding of triazine herbicides to antibodies in nearly anhydrous media. The work of Matsuura et al. [98] on screening monoclonals for okadaic acid in the context of shellfish poisoning is worth mentioning. Okahata & Yamaguchi [99] described a lipid-coated catalytic antibody which was soluble in organic cosolvents and had higher k cat as compared to the native molecule in such media. Recently, catalytic antibody, 38C2 with aldolase activity, has become commercially available. It has been found that 38C2 can catalyze a retroaldolization reaction in both aqueous and organic solvents [100]. Hydrolysis and esterification of organometallic substrates Suitable substrates for this type of reaction include orgnosilicon and organotin compounds and P-bonded complexes of iron, chromium and manganese [7,101]. A critical feature is allowing stereoselective reactions of water sensitive compounds to be carried out. Kinetic resolution of a-hydroxystannanes by lipases is described [102]. a-Hydroxystannanes are valuable synthetic reagents, notable as precursors to a-alkoxy lithium species, which react with a number of electrophiles, with retention of configuration at the a-center. Several applications include simple quenching with CO 2 to access a-hydroxy acid [103] and double bond transmetallation to generate a function- alized organocopper reagent for use in conjugate addition reactions [104]. The first use of an enzyme for the resolution of organotin compounds was reported by Itoh & Ohta [105] who examined the enatioselective hydrolysis of racemic a-acyloxystannanes catalyzed by Pseudomonas sp. lipases. Conclusion Finally, one can conclude by mentioning that few trends in nonaqueous enzymology can be clearly seen. The first trend is that although lipases and proteases continue to dominate vis-a ` -vis applications in the nonaqueous media, other enzymes like oxidoreductases (alcohol dehydrogenase, catalase, peroxidase) [3], penicillin amidase [106], b-glucosi- dase [2], b-galactosidase [68], catalytic antibodies [2] and whole cells [2] are also beginning to be used in organic media. Given the large number of lipases and proteases available with a wide range of specificities, this picture is unlikely to change significantly. The second trend is that greater understanding (at the molecular level) of the way enzymes behave in such media is emerging. The focus right now is on creating formulations/designs with greater activity. The control of water activity during the reaction is crucial. Microwave assistance and ultrasonoenzymology in such media have not yet been extensively studied, therefore one is likely to see more work in this area in the coming years. The iterative process of application fi basic understanding fi more applications is likely to continue for a while in this area. Acknowledgements The financial assistance from Department of Science and Technology, Department of Biotechnology, Council for Scientific and Industrial Research (both Extramural Division and Technology Mission on Oilseeds, Pulses and Maize) and National Agricultural Technology Project (Indian Council for Agricultural Research), all of which are Government of India organizations, is gratefully acknowledged. References 1. Koskinen, A.M.P. & Klibanov, A.M. (1996) Enzymatic Reac- tions in Organic Media. Blackie, London. 2. Gupta, M.N. (2000) Methods in Nonaqueous Enzymology. Birkhauser-Verlag, Basel. 3. Vulfson, E.N., Halling, P.J. & Holland, H.L. (2001) Enzymes in Nonaqueous Solvents, Methods and Protocols. Humana Press, New Jersey. 4. Drauz, K. & Waldmann, H. (2002) Enzyme Catalysis in Organic Synthesis, Vol. I Wiley-VCH, Weinheim. 5. Gupta, M.N. 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