Eur. J. Biochem. 203,25-32 (1992)

0FEBS 1992

Review Enzyme function in organic solvents Munishwar N. GUPTA Chemistry Department, Indian Institute of Technology, Delhi, India (Received June 13, 1993) - EJB 91 0779

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water spondt.nce to M. N. Gupta, Chemistry Department, 1IT Delhi, New Delhi, India 110 016

tent (Yamane et al., 1989) and microaqueous organic solvents (Yamane, 1988) are some examples of the phrases used to describe such systems. Halling also argues that, strictly speaking, the description of low water systems should be reserved for cases where ‘the thermodynamic water activity (a,) is significantly less than 1’ (Halling, 1987). In as much as activity measurements are seldom carried out or mentioned, this review will take a broader view and look at the total scenario when water content is low. As will be discussed below, even when the amount of water is kept low, in the short range considered the exact amount is still critical. Hence there is a need for precise methods to measure the amount of water present in such systems. The titrimetric Fischer method (Zaks and Klibanov, 1988a) and gas chromatography (Reslow et al., 1988a) are two convenient methods. More recently, membrane inlet mass spectrometry has been used for measurement of water activity in organic solvents (Lundstrom et al., 1990). Apart from being adaptable to continuous measurements, it is claimed to be at least 50 times more sensitive than the Fischer method. Correlation of enzyme function with the nature of the medium

The next important point to consider is the search for an ideal parameter in terms of which enzyme activity (and stability) can be correlated to the nature of the medium. It is now generally agreed that less polar solvents give higher activity (Klibanov, 1987; Laane et al., 1987a; Aldercreutz and Mattiasson, 1987). Laane et al. (1987a) have discussed this issue quite comprehensively and point out that of all the various parameters, such as Hildebrand solubility parameter, solvatochromism of the dye, dielectric constant, dipole moment and logarithm of partition coefficient (log p ) , log P gives

26 Table 1. Various possibilities for non-aqueous media. ~~

Low water systems

High water content


3 . Eniyrnes

i n nearly anhydrous solvent5 2. Reverse micelles

3 . Cosolvent systems

(water-miscible organic solvent)

2. Organic aqueous biphasic systems

Table 2. Reasons which favour the use of organic media. When substrate(s) have greater solubility in organic solvents Shift of reaction equilibria in desirable directions such as use of hydrolases for synthetic reactions Reduced risk of microbial growth Enhanced thermoslability Recovery and reusability of enzyme even without immobikation More energy efficient downstream processing when volatile solvents are used Convenient to use ‘moisture-sensitive’ substrates/reagents like acid anhydridcs Possible control of substrate specificity, regiospecificity and enantioselectivity

the best correlation with the enzyme activity. The partition coefficient P is the one corresponding to a standard octanol/ water two-phase system. Laane et al. (1987a) not only provide log P values for commonly used organic solvents, they also illustrate how this parameter can be calculated from hydrophobic fragmental constant. Aldercreutz and Mattiasson (1987) also agree that log P is more useful than other parameters for choosing the best solvent, i.e. higher log P values (more hydrophobic solvents) are associated with better enzyme performance in non-aqueous media. However, occasional discrepancies have been reported while trying to correlate log P with enzyme activity and rationalized by using an additional parameter, water solubility, which is not a direct function of log P (Reslow et al. 1987). Advantages of using enzymes in low water systems What are the basic reasons that enzyme catalysis in low water systems has interested biochemists working in different areas? The advantages of using enzymes in organic solvents are listed in Table 2 and have been discussed frequently at greater length (Klibanov, 1986, 1987; Khmelnitsky et al., 1988; Brink et al., 1988). Conformational rigidity, ‘memory’ and ‘protein imprinting’ Our understanding of the structure of solid enzymes suspended in organic solvents is indirectly derived from two approaches: (a) hydration of dry solid protein, in which experiments have largely been conducted with lysozyme (Rupley et al., 1983; Poole and Finney, 1983); (b) looking at protein structure in a limited water pool in reverse micelles (Waks, 1986). I will now summarize the conclusions possible from the available data. (a) The dry enzyme has more or less the same gross conformation as the fully hydrated enzyme. The fully hydrated

molecule (based upon heat capacity data) has 0.38g water/g protein (300 mol water/mol lysozyme). This water is barely enough for formation of a monolayer and is about half as much as is present in protein crystals. (b) Using the model of D’Arcy & Watt (1970), which distinguishes two primary hydration sites, it is possible to view the gradual hydration event in terms of three stages (Rupley et al., 1983): (i) between 0-0.07 g water/g protein: hydration of ionizable (charged) groups; (ii) between 0.07 - 0.25 g water/ g protein: clusters of water grow around these polar patches; (iii) between 0.25 - 0.38 g water/g protein: water covers ‘less interacting surface elements’, presumably non-polar atoms. (c) According to Careri et al. (1979), the first stage corresponds to about 40 molecules water/protein molecule. Rupley et al. (1983) say this must be about 60. (d) The biological activity of lysozyme becomes detectable at 0.2 g/g. The change in protein mobility is mostly complete at 0.25 g/g but the activity continues to increase even beyond the 0.38-g/g level; 0.25 gig corresponds to about 220 molecules water/lysozyme molecule (Rupley, 1983). (e) The onset of activity starts as the protein mojecule approaches total mobility. This is in agreement with Karplus and McCammon (1983) who indicate this mobility to be essential for enzyme activity. (f) Klibanov (1989) has reiterated his earlier data (Zaks and Klibanov, 1988a) that chymotrypsin and subtilisin require less than 50 molecules water/protein molecule. I think the Fact that this number lies in the range of 40 - 60 may be significant. Perhaps in organic solvents, the activity is observed as soon as hydration corresponding to first hydration event, i.e. hydration of surface charged groups, is complete. It has been pointed out (Zaks and Klibanov, 1988a) that the main role of water is to form bonds with functional groups present on the protein. Perhaps the most critical partners are the charged groups which, in the absence of even this minimum of water, interact with each other and produce an inactive ‘locked’ conformation. Additional water makes protein more flexible, just as it is reported to do during hydration process. Thus, at the water level corresponding to about 50 molecules/molecule of subtilisin or chymotrypsin, while active in organic solvents, these enzyme molecules are still very rigid. Undoubtedly, it is this rigidity which is responsible for some interesting consequences observed in anhydrous organic solvents (water < 0.02% by vol.). a) Porcine pancreatic lipase does not react with large substrates in anhydrous organic solvents (Klibanov, 1986). b) pH memory: when ‘transferred’ from water to anhydrous organic solvents, the ‘state’ in water is retained. Thus enzyme precipitated from aqueous solution at its optimum pH functions with greatest efficiency (Klibanov, 1986). While working in the laboratory of Prof. Klibanov at Massachusetts Institute of Technology, I observed that this ‘pH memory’ falters even if the enzyme subtilisin is stored in the dry state at 4°C (unpublished results). Perhaps gradual hydration erases this memory. Loss of pH memory with hydration is generally attributed to redistribution of charges on the protein surface. c) Russel and Klibanov (1988) have shown that lyophilization of subtilisin from aqueous solution containing a competitive inhibitor induced a conformation which gave better transesterification rates in anhydrous organic solvents. d) A logical development of this phenomenon has been the technique of molecular imprinting developed by Braco et al. (1990) and Stahl et al. (1990). The work of Braco et al. (1 990) showed that bovine serum albumin, when lyophilized

27 from an aqueous solution containing p-hydroxybenzoic acid or c-tartaric acid, develops specific affinity towards these ligands in anhydrous organic solvents. On the other hand, Stahl et al. (1990) showed that, using a similar approach, chymotrypsin can be made to accept D-amino acid derivative. The work with reverse micellar systems indicate that protein structures tend to be ‘more compact’ and ‘more rigid’ in limited water as compared to aqueous media (Waks, 1986).

Enhanced thermostabitity Another consequence of rigidity in low water medium is the enhanced thermostability of proteins/enzymes (Gupta, 1991). This has been observed with numerous enzymes: porcine pancreatic lipase (Zaks and Klibanov, 1984), terpene cyclase (Wheeler and Crotean, 1986), lysozyme (Klibanov and Ahern, 1987), chymotrypsin (Zaks and Klibanov, 1988a), mitochondrial cytochrome oxidase (Ayala et al., 1986) and F ATPase (Garza-Ramos et al., 1989). The following comments may be made on this. a) The thermostabilization achieved is quite impressive. The lipase remained stable at 100°C for many hours, so did lysozyme. Such dramatic thermostabilization is seldom possible by using other approaches such as chemical crosslinking (Kamra and Gupta, 1988; Khare and Gupta, 1990; Rajput and Gupta, 1988), immobilization (Khare and Gupta, 1988a,b) or even protein engineering (Nosoh and Sekiguchi, 1988). b) Apart from rigidity, another reason for enhanced thermostability is that a number of covalent processes involved in irreversible inactivation such as deamidation, peptide hydrolysis and cystine decomposition require water. These irreversible processes are also extremely slow in low water media (Gupta, 1991). c) Thcse experiments, like most of non-aqueous enzymology, are restricted to relatively non-polar solvents. Leaving aside the enhanced thermostability, biocatalyst stability in general is quite low in polar solvents. However, Reslow et al. (1988 a) have examined the therinostability of Celiteadsorbed chymotrypsin at the moderately high temperature of 50°C and shown that the heat stability was better in solvents having log P values larger than 0.7. This kind of information is useful and ought to be collected for other systems. The same work also provides an interesting comparison of stability of Celite-adsorbed chymotrypsin in organic solvents with various immobilized chymotrypsin preparations in water at 50 “C for 60 min. The stability in hydrophobic solvents is far better than most of the immobilized preparations in water. d) While the previous work had been restricted to enzymes with relatively non-polar substrates and products, recent work of Garza-Ramos et al. (1990) indicates that enzyme catalysis with enhanced thermostability is possible with water-soluble substrates and products. In their system, mitochondrial ATPase hydrolyzes ATP at temperatures as high as 91 “C. It must be added that, in this innovative work, the authors providc evidence that the enzyme is localized in a compartment different from that of the substrates and products. Thus the work suggests some interesting possibilities regarding enzymatic catalysis in the case of water-soluble substrates and products (or that of hydrolytic reactions) in low water media. e) The above work (Garza-Ramos et al. 1990) also examines the interesting question of optimum temperature for catalysis in low water. The interesting finding is that the optimal temperature of ATP hydrolysis by mitochondrial ATPase is

the same (i.e. around 58°C) in aqueous medium as in low water medium. This means that, while we have the advantage of higher thermal stability of the catalyst in low water media, the rates in fact decrease beyond the optimal temperature. These findings have far reaching implications. There has been considerable excitement in the literature (Zaks and Klibanov, 1984; Klibanov and Ahern, 1987) about the enhanced thermostability of enzymes in anhydrous organic solvents. If the other systems behave like ATPase, this enhanced thermostability would be of little use since it would not have the main advantage of higher conversion rates. It may be added that earlier data had indicated that temperature effects on reaction rates were quite small in many systems (Zaks and Klibanov, 1984; Halling, 1987). f) In many cases, it has been observed that thermostabilization of enzymes/proteins is reflected in the accompanying stabilization towards other denaturing conditions such as proteolysis (Gupta, 1991). For the first time, Arnold (1990) has recently pointed out that there may be correlations between enhanced thermostability and stability in non-aqueous solvents. She, in fact, suggests ‘concepts that have proved useful in engineering protein thermostability may be applied to accomplish the goal of rational enzyme stabilization in nonaqueous solvents’. While this may be too sweeping a statement, it is probably worth investigating this correlationship between stability at higher temperatures and in non-aqueous solvents. For example, are thermostable enzymes from thermophilic organisms more stable in non-aqueous solvents than their mesophilic counterparts? At least in the case of malic enzyme and alcohol dehydrogenase from Sulfolohus solfatosicus (an extremophile), the correlation between thermostability and ‘organic solvent-resistance’ has been observed (Bartolucci et al., 1990).

Medium engineering There seems to be general agreement that enzymes in predominantly non-aqueous environment or low water environment can function provided the essential water layer around them is not stripped off (Klibanov, 1986; Laane, 1987a; Aldercreutz and Mattiasson, 1987; Khmelnitsky et al., 1988). Thus the golden rule of non-aqueous enzymology (Zaks and Klibanov, 1988a), that non-polar solvents are better than polar solvents, can be rationalized by pointing out that polar solvents, being water-miscible, strip off the essential water layer of the protein. Zaks and Klibanov (1988b) have examined the effect of water on alcohol dehydrogenase, alcohol oxidase and polyphenol oxidase in a variety of organic solvents. Their data indicate that the effect of organic solvents on an enzyme is due to interactions with the water shell around the enzyme rather than with the enzyme itself. They also found that the enzyme activity increased rapidly upon increasing the water content of the medium. It should be emphasized that this generalization is in the context of anhydrous media when the water content was below its solubility limit in the organic solvent. Whereas biocatalyst engineering aims at optimizing biocatalyst function by modifying the biocatalyst structure, medium engineering in the context of biocatalysis in nonaqueous solvents involves the modification of immediate surroundings of the biocatalyst (Laane, 1987). Thus the first gross rule of medium engineering is that already implied: non-polar solvents are better than polar since the former provide a better microenvironment for the protein/enzyme. Further refined, this rule can be restated as follows (Laanc, 1987; Laane et

28 al., 1987a, b). While the solvents having log P c: 2 would constitute poor choice, those with log P > 4 are most suitable. Solvents in the intermediate range of log P between 2 - 4 are unpredictable and are likely to require biocatalyst engineering. Laane et al. (1987a, b), as a further step in medium engineering, have stated that the properties of the substrate(s) and product(s) should also be considered. According to these rules, if the immediate microenvironment of the biocatalyst favors solubility of the substrate and has low product solubility, the reaction rates would be higher. No experimental model in support of these data is yet available in the case of enzymes in low water media. However, the results with reverse micelles in the case of 20-hydroxysteroid dehydrogenase (Hilhorst et al., 1984), cholesterol oxidase (Laane et al., 1987b) and enoate reductase (Verhaert et al., 1989) tend to support these rules. It should be realized that the distinction between medium engineering and biocatalyst engineering sometimes tends not to be sharp. For example Laane (1987), in an excellent review on medium engineering, discusses how the microenvironment of the biocatalyst is controlled by the nature of matrices to which the biocatalyst may be linked. What are the other lessons which are rapidly being learnt in medium engineering - an approach barely in its infancy? Khrnelnitsky et al. (1988), based upon a number of studies, have pointed out that glycerols and other polyols are by far the best additives. The reasons are that, apart from the virtue of maintaining solvophobic interactions essential for the native structure, they preserve (or actually enhance) the water shell around the protein molecule. Two comments may be added to this. a) Polyols are known to be useful additives for enhancing thermostabilization (Gupta, 1991). So Arnold’s suggestions (Arnold, 1990) vi.r-a-vis protein design in non-aqueous solvents can probably be extended to medium engineering as well. b) While Khmelnitsky et al. (1988) are correct that these polyol additives are of little practical utility since they increase medium viscosity considerably (and decrease rates), it is here that the advantage of enhanced thermal stability in organic solvents would become useful. Combine this kind of medium engineering (addition of polyols) with catalysis at high temperature (Zaks and Klibanov, 1984) and we may obtain some viable technologies. More recently, Mozhaev et al. (1989) and Khmelnitsky et al. (1991) have studied the stability of enzymes/proteins in mixed aqueous media. These investigations indicate that there is a critical concentration of organic cosolvent at which there is an abrupt change in catalytic and spectroscopic properties of the enzyme. The group of B. Mattiasson has recently carried out some excellent work in the area of medium engineering (Reslow et al., 1987,1988b; Reslow, 1989; Clapes et al., 1990a, b). Their system was a-chymotrypsin-catalyzed esterification and peptide synthesis. The results indicate that log P , as the solvent-associated parameter, gave best correlations vis-a-vis Km,,pp,Vappand enzyme stability. This work (Reslow et al., 1987; Reslow, 1989) goes beyond ‘non-polar solvents are better than polar solvents’ and points out, backed by experimental results, that one should search for the best medium and this choice will be dictated by other considerations which are specific to that particular system, e.g. very non-polar solvents may give very poor solubility for the substrates. This work (Reslow et al., 1988b; Reslow, 1989) also underscores the importance of determining the optimum amount of water for

the particular solvent, biocatalyst form and the reaction being catalyzed. The finding (Reslow et al., 1988a; Reslow, 1989) that the most hydrophobic solvent used, viz. toluene, required the minimum amount of water for the highest reaction rate can be easily rationalized by realising that added water becomes partitioned between the solvent and the catalyst. For the same reason, the amount of water required for the best biocatalyst performance will also be dictated by the nature of support (if any). That is where, as already pointed out, the distinction between biocatalyst engineering and medium engineering becomes blurred. As pointed out by these workers (Reslow et al., 1987), there is little information on the dependence of operational stability on water content. Such data, hopefully, will soon become an integral part of any serious medium engineering work. As a final remark on this topic (Reslow et al., 1987), the reaction catalyzed is an esterification which involves production of water. It will be interesting to carry out a transesterification using identical conditions and identical biocatalyst form to assess the role of this factor. Wherever this factor is important, different workers have used different strategies to remove the product water, e.g. using a loop reactor (Knox and Cliffe, 1984) or carrying out the reaction under reduced pressure (Miller et al., 1988). In this context, it may also be added that some innovative strategies to supply the required water contents to the biocatalyst, in systems where water needs to be replenished, have been used by Macrae (1985), Hansen and Eigtved (1985) and Reslow et al. (1988~). Yet another exciting finding by Reslow (1989) is that part of the necessary water can be replaced by polar solvents. Not only has this led to increased chymotrypsin-catalyzed transesterification, in the case of lipase such a strategy even changed the stereoselectivity. It may be recalled that Zaks and Klibanov (1988 b) had earlier shown that polar solvents can indeed substitute for water since such solvents interact with the protein molecule like water and provide the necessary flexibility to the protein molecule. The work of Reslow (1989) not only extends this to a practical domain but also demonstrates the immense possibilities in medium engineering. Another medium engineering work, again on chymotrypsin (suspended as a powder), has examined the medium effect on the esterification of aromatic amino acids by a-chymotrypsin, including the effect of water content variation up to 10% (Kise and Shirato, 1988). The work was restricted to polar organic solvents and emphasised the importance of the nature of the solvent (unfortunately correlation of product yield with a polarity parameter like log P was not reported) and water content in the medium. Yamane et al. (1989) have examined the effect of water content on intramolecular esterification by crude lipase in benzene. These workers have mathematically expressed the effect of water content on the initial rate. Recently, Halling (1990) has shown that predictions can be made for the influence of solvent choice on the equilibrium position of the enzyme-catalyzed reactions in liquid/liquid biphasic systems. These predictions are reliable only for dilute systems and are based upon the partition coefficients. Biocatalyst engineering

Just as immobilization of enzymes and protein engineering has been tried for improving catalysis efficiency/stability in water, similar efforts for improving catalyst performance in organic solvents have been made. An ‘umbrella’ term ‘biocatalyst engineering’ has been used by Laane (1987) for this and seems quite appropriate.

29 Table 3. Forms of enzymes used in low water systems.

free enzyme. Comparative stability data in the case of a-chymotrypsin has been reported by Nilsson and Mosbach Form Reference (1984) but their medium was 50% dimethylformamide. Earlier, Pliura and Jones (1980) had compared the esterolytic 1. Enzymes dissolved in concentrated Yamane, 1988 activities of native chymotrypsin and immobilized substrate solutions (e.g. fructochymotrypsin on Sephadex in the presence of a number of oligosaccharide production from organic solvents, but again this was done in a predominantly 50% sucrose by Aspergillus niger inaqueous medium. It is obvious that the extrapolation of results vertase) of such systems to low water systems is not valid. 2. Solid enzyme powder suspended in Cambou and Klibanov, The next important question to be addressed is what kind organic solvents 1984 of support is best for immobilization? A generally accepted Makita et al., 1987 view has been that relatively hydrophilic supports should be 3. Solid enzyme adsorbed on support Grunwald et al., 1986 best. Laane et al. (1987b) pointed out that the use of such particles Reslow et al., 1988a supports would alter the log Plactivity curve to lower log P 4. Poly(ethy1ene glycol)-modified en- Inada et al., 1986 values, making it possible to employ less hydrophobic solzymes soluble in aromatic vents. The recent work by Reslow et al. (1988a) unequivocally hydrocarbons demonstrates that, at least in the cases of chymotrypsin and 5. Enzyme entrapped within a gel Fukui et al., 1987 horse liver alcohol dehydrogenase, hydrophobic supports are better than hydrophilic supports. The authors explain that, 6 . Immobilized enzyme suspended in Tanaka and Kawamoto, organic solvents 1991 in the case of hydrophobic support materials, the enzyme competes successfully for available water and therefore shows higher activity. This work strikingly demonstrated the need for evaluation of various ‘concepts’ in this area by careful First, it may be worth listing the forms in which enzymes experimentation. It may also be added that the conclusions of have been used in low water systems (Yamane, 1988) (Table 3). Reslow et al. (1988a) are in conflict with the suggestion of Obviously, there is considerable scope in biocatalyst engi- Tanaka and Kawamoto (1991) (as discussed earlier) regarding neering for optimising the performance of an enzyme in low retention of water by the suitable support. It may also be water media. The forms 1 and 4 are special approaches. Form envisaged that, in the case of a non-polar substrate, a 1 is applicable to limited cases while form 4 has already been hydrophilic support may be unfavorable since it may restrict discussed adequately elsewhere (Inada et al., 1986). Thus these substrate diffusion. Arnold and her coworkers have contributed significantly two approaches will not be discussed further here. It may be pertinent to point out that no general guidelines can yet be to the application of the promising approach of protein engiprovided for choosing the most appropriate form of the en- neering to improving enzyme function in organic solvents. zyme for a specific purpose. Halling (1987) has given an excel- Arnold (1988, 1990) has advocated a set of rules for protein lent account of problems inherent in making meaningful rate design in non-aqueous solvents. These were largely based on comparisons. Hence, till such time as any comparative data is the work of this group with crambin (Arnold, 1988). Crambin available, it is difficult to compare the merits of any of the is a small plant protein which is soluble in organic solvents. The rules suggested by Arnold (1988, 1990) emphasized the approaches listed in Table 3. Forms 3, 5 and 6 can be considered together for this importance of increasing conformational stability and comdiscussion under the broad title of ‘immobilization on solid patibility of the enzyme surface with organic solvent. The supports’. Tanaka and Kawamoto (1991) have recently re- recent protein engineering work of this group (Chen et al., viewed immobilized enzymes in organic solvents. As they 1991) tends to confirm the wisdom of these design principles. pointed out, apart from the usual (i.e. in case of catalysis in Working with subtilisin E, two amino acid substitutions aqueous medium) advantages of (a) presumed stabilization Asp248-+Asn and Asn218+Ser, have been carried out. The and (b) reusability (if it is remembered that enzymes do have former diminishes the surface charge while latter is expected some, though limited, solubility in organic solvents), the ad- to improve H-bonding. Both mutations, individually and additional advantages of (c) favourable partitioning of available ditively were shown to result in improved stability in 40% water (and its retention) by the suitable support and (d) disper- dimethylformamide. The data with anhydrous dimethylsal of the enzyme molecules to a larger surface area with formamide is not given. Similarly, with cr-lytic protease, substitution of surface concomitant increase in accessibility of active sites to subpolar amino acids with hydrophobic amino acids was found strates may be mentioned. Rather than listing various instances where immobilized to improve the protease stability in 84% dimethylformamide. enzymes have been used in organic solvents, this review will It is believed that a more hydrophobic surface reduces the need be limited to mentioning general principles (or lack of them) for hydration for maintaining native conformation (Martinez and Arnold, unpublished results). in the area. Another group (Wong et al., 1990) have shown that First, limited data is available which proves in a quantitative fidshion the superiority of immobilized enzymes over free engineered subtilisin BPN variant was more stable than wildenzymes (in organic solvents) by comparing their respective type subtilisin in anhydrous dimethylformamide. As the origperformances under identical conditions. Halling (1987) has inal intention was to improve thermostability, the substimentioned that, in the case of lipases, immobilized lipases tend tutions carried out were aimed at enhancing conformational to give faster reaction rates as compared to simple enzyme stability. Thus in principle, the way is paved for engineering enzymes powders. Laane et al. (1987b) have listed several cases involving thermolysin, liver alcohol dehydrogenase, mushroom that are stable even in polar organic solvents. The work depolyphenol oxidase, lipase and carboxyesterase where the scribed above certainly shows that the approach is extremely immobilized enzymes were shown to perform better than the promising.

30 Table 4. Some important applications of non-aqueous enzymology. Application



Organic synthesis

Peptide synthesis lipase for synthesis of a penicillin G precursor peptide incorporation of D-amino acids into peptides by subtilisin isopeptide bond formation by protease and lipase lipase for dipeptide synthesis use of a-chymotrypsin and thermolysin use of thermolysin comparative study of water-miscible and water-immiscible solvents using chymotrypsin nucleophile specificity and medium engineering using chymotrypsin use of subtilisin use of trypsin use of chymotrypsin application of PEG-modified enzymes

Matos et al., 1987 Margolin et al., 1987a Kitaguchi et al., 1988 Margolin and Klibanov, 1987 Reslow et al., 1988a Ooshima ct al., 1985 Clapes et al., 1990a Clapes et al., 1990b Kise and Fujirnoto, 1988 Pugniere et al., 1986 Noritomi and Kise, 1987 Inada et al., 1986

Regioselectivelstereoselective synthesis

regioselective acylation of glycols by lipase regioselective acylation of sugars by lipase regioselective oxidation of phenols by polyphenol oxidase resolution of Racemic mixture o f acids by lipase asymmetric oxidoreductions by alcohol dehydrogenase for production of chiral alcohols and ketones synthesis of chiral cyanohydrins using mandelnitrile lyase regioselective acylation of carbohydrates by subtilisin in anhydrous dimethylformamide entantio- and regioselective synthesis of alcohols, glycerol derivatives, sugars and organometallics using lipase regiospecific interesterification of triacylglycerol by lipase

Cesti et al., 3985 Therisod and Klibanov, 1986 Kazandjian and Klibanov, 1985 Kirchner et al., 1985 Grunwald et al., 1986 Effenberger et al., 1987 Riva et al., 2988 Wang et al., 1988 Yokeseki et al., 1982

Synthesis uf hiosuyfactants using lipase

Chopineau et al., 1988

Interester ification of’ oils and,futs

Wisdom et al., 1984 Macrae, 1985

Lowering of stereoselectivity

Sakurai et al., 1988

Fat splitting

Lipolysis in organic solvents using lipases

Blain et al., 1976 Akhtar et al., 1980 Kim et al., 1984

Analysis (Gupta and Mattiasson, 1991)

Determination of cholesterol in organic solvents Thermal abuse sensors: lipase coupled with a pH indicator Horseradish peroxidase with a chromogenic substrate

Kazandjian et al., 1986 Swedish patent 7904320-4, 3980 Boeriu et al., 1986

Oligomerization and polymerization

Polymerization of phenols by peroxidase Stereoselective oligomerization of diesters and diols by lipase

Dordick et al., 1987 Margolin et al., 1987b

Applications As pointed out at the beginning of this review, enzyme catalysis in anhydrous or low water systems has resulted in a large number of varied applications. These applications have been frequently reviewed (Klibanov, 1986, 1987; Dordick, 1988; Tanaka and Kawamoto, 1991). The focus of this review is different and little will be gained by discussing these applications again. However, it may be worthwhile to provide an updated list of the nature of such applications with important illustrations (Table 4).

Conclusion The old picture of enzymes as fragile biocatalysts is surely and steadily changing. They can even be used in predominantly organic media. However, for the majority of enzymes, the organic solvents should be non-polar (water immiscible). An important issue, not yet resolved, is why only some of them, like xanthine oxidase and porcine pancreatic lipase, can function even in water-miscible organic solvents (Khmelnitsky et al., 1988). Subtilisin functions even in anhydrous dimethylformamide (Riva et al., 1988).


Another issue which still is not fully resolved is the way the medium influences enzyme function. Zaks and Klibanov (1988a) found that the amounts of water on chymotrypsin in octane (log P = 4.5) and in toluene (log P = 2.6) are almost same. Thus medium engineering rules still need further refinement. Nevertheless, medium engineering and biocatalyst engineering are likely to be used increasingly in a useful fashion for optimizing the performance of enzymes in low-water/organic media. This deceptively simple looking approach of non-aqueous enzymology has undoubtedly made enzymes even more powerful tools in the areas of organic synthesis and analysis. The preparation of this review was supported by project grants from the Department of Science and Technology (Government of India) and the Council of Scientific and Industrial Research, New Delhi. Thanks are due to the members of my research group, viz. Dr S. Ahmad and Dr R. Tyagi, for their help in the preparation of this manuscript. I am particularly grateful to Prof. Bo Mattiasson, Department of Biotechnology, University of Lund, Sweden for providing me with an opportunity to discuss this topic with him and members of his research group at Lund, Sweden. Thanks are also duc to Prof. F. H. Arnold (California Institute of Technology, USA) for her generosity in sending me preprints of her work.

REFERENCES Akhtar, M. W., Nawazish, M. N., Pasha, M. & Mirza, A. Q. (1980) Pak. J. Biochem. 13,56 - 62. Adlercreutz, P. & Mattiasson, B. (1987) Biocutalysis I, 99-108. Arnold, F. H. (1988) Protein Eng. 2, 21 -25. Arnold, F. H. (1990) Trend~sBiotechnol. 8, 224-249. Ayala, G., Tucna de Gomez-Puyou, M., Gomez-Puyou, M. & Darszon, A. (1986) FEBS Lett. 203, 41 -43. Bartolucci, S., Guagliardi, A,, Raia, C. A., Rella, R., Buckmann, A. F. & Rossi, M. (2990) Proc. 5th Eur. Congr. Biotechnol., p. 78. Blain, J. A., Akhtar, M. W. & Patterson, J . D. (1976) Pak. J. Biochem. 10,41- 53. Boeriu, C. G., Dordick, J . S. & Klibanov, A. M. (1986) BiolTechnology 4,997 -999. Braco, L., Dabulis, K. & Klibanov, A. M. (1990) Proc. Nut1 Acad. Sci. USA. 87, 274-277. Brink. L. E. S., Tramper, J., Luyben, K. Ch. A. M. & Van’t Riet, K. (1988) Enzyme Microb. Techno/. 10, 736-743. Cambou & Klibanov, A. M. (1984) Biotechnol. Bioeng. 26, 14491454. Careri, G., Giansanti, A. & Gratton, E. (1 979) Biopolymers 18,11871203. Cesti, P.. Zaks, A. & Klibanov, A. M. (1985) Appl. Biochem. Biotechn d . 11,401 -407. Chen, K., Robinson, A. C., Van Dam, M. E., Martinez, P., Economou, C. &Arnold, F. H. (1991) Biotechnol. Prog. 7, 125-129. Chopineau, J., McCaffery, F. D., Therisod, M. & Klibanov, A. M. (1988) Biotechnol. Bioeng. 31, 208-214. Clapes, P., Adlercreutz, P. & Mattiasson, B. (1990a) J . Biotechnol. IS, 323 -338. Clapes, P., Adlerceutz, P. & Mattiasson, B. (1990b) Biotechnol. Appl. Biochem. 12, 376-386. D’Arcy, R.L. & Watt, 1. C. (1970) Tram. Faraday Soc. 66, 12361245. Dordick, J . S. (1988) Appl. Biochern. Biotechnol. l Y , 103-112. Dordick, J. S., Marletta, M. A. & Klibanov, A. M. (1987) Biotechnol. Bioenx. 30, 31 - 36. Effcnberger, F., Ziegler, T. & Forster, S. (3987) Angew. Chem. Int. Ed. Engl. 26,458. Fukui, S., Tanaka, A. & Iida, T. (1987) in Biocuta[v.yis in organic media (Laane, C., Tramper, J. & Lilly, M. D., eds) pp. 21 -41, Elsevier, Amsterdam. Garza-Ramos, G., Darszon, A., Tuena de Gomez-Puyou, M. & Gomez-Puyou, A. (1989) Biochemistry 28, 3177-3182.

Garza-Ranios, G., Darszon, A., Tuena de Gomez-Puyou, M. & Gomez-Puyou, A. (1990) Biochemistry 29, 751 - 757. Grunwald, J., Wirz, B., Scollar, M. P. & Klibanov, A. M. (1986) J . Am. Chem. SOC.108, 6732 - 6734. Gupta, M. N. (1991) Biotechnol. Appl. Biochem. 14, 1 - 11. Gupta, M. N. & Mattiasson, B. (1992) Methods Biochem. Anal. 36, in the press. Halling, P. J. (1987) Biocatalysis I , 109- 115. Halling, P. J. (1990) Biotechnol. Bioeng. 35,691 -701. Hanscn, T. T. & Eigtved, P. (1985) in Proceedings of World Conference on emerging technologies infuts and oil industry (Baldwin, A. R., ed.) p. 365, American Oil Chemists Society, Champaign, I L . Hilhorst, R., Spruijt, R., Laane, C. & Veeger, C. (1984) Eur. J. Biochem. 144,459-466. Inada, Y., Yoshimoto, T., Matsushima, A. & Saito, Y. (1986) Trendy Biotechnol. 4, 68 -73. Kamra, A. & Gupta, M. N . (1988) Biochim. Biophys. Acta Y66, 181 187. Karplus, M. & McCammon, J. A. (1983) Annu. Rev. Biochem. 53, 263 - 300. Kawamoto, T., Sonomoto, K. & Tanaka, A. (1987) Biocatulysis I, 137-145. Kazandjian, R. Z., Dordick, J. S. & Klibanov, A . M. (1986) Biotechno[. Bioeng. 28,417-421. Kazandjian, R . Z. & Klibanov, A. M. (1985) J . Am. Chem. Soc. 107, 5448 - 5450. Khare, S. K. & Gupta, M. N. (1988a) Biotechnol. Bioeng. 31, 829833. Khare, S. K. & Gupta, M. N. (1988b) J. Biosci. 13,47-54. Khare, S. K. & Gupta, M. N. (1990) Biotechnol. Bioeng. 35,94-98. Khmelnitsky, Y . L., Levashov, A. V., Klyachko, N. L. & Martinek, K. (1988) Enzyme Microb. Technol. 10, 710-724. Khinelnitsky, Y. L., Mozhaev, V. V., Belova, A. B., Sergeeva, M. V. & Martinek, K . (1991) Eur. J. Biochem. 198, 31 -42. Kim, K. H., Kwoon, D. Y. & Rhee, J. S. (1984) Lipidr I Y , 975-977. Kirchner, G., Scollar, M. P. & Klibanov, A. M. (1985) J . Am. Chem. SOC.107,7072- 7076. Kise, H. & Fujimoto, K. (1988) Biotechnol. Lett. 10, 883. Kise, H. & Shirato, H. (1988) Enzyme Microb. Technol. 10,582-585. Kitaguchi, H., Tai, D. F. & Klibanov, A. M. (1988) Tetrahedron Lett. 29, 5487 - 5488. Klibanov, A. M. (1986) Chem. Tech. 6, 354-359. Klibanov, A. M. (1987) in Biocutalysis in organic media (Laane, C . , Tramper, J. & Lilly, M. D., eds) pp. 115- 116, Elsevier, Amsterdam. Klibanov, A. M. &Ahern, T. J. (1987) in Protein engineering (Oxender, D. L. & Fox, C. F., eds) pp. 213-218, Alan R. Liss, New York. Knox, T. & Cliffe, K. R. (1984) Process Biochem. I Y , 188-192. Laane, C. (2987) Biocutalysis 1, 17-22. Laane, C., Boeren, S., Kees, V. & Veeger, C. (1987a) Biotechnol. Bioeng. 30, 81 - 87. Laane, C., Boeren, S., Hilhorst, R. & Veeger, C. (1 987 b) in Biocatahsis in organic media (Laane, C., Tramper, J. & Lilly, M. D., eds), pp. 65 - 84, Elsevier, Amsterdam. Linhardt, R. J. (1986) Appl. Biochem. Biotechnol. 12,67-76. Lundstrom, B., Bohatka, S. & Degn, S. (1990) Ahstr 5th Eur. Congr. Biotechnol., p. 242, Munksgaard Publ., Copenhagen. Macrae, A. R. (1985) in Biocutulysis in organic synthesis (Tramper, J., Van der Plas, H. C. & Linko, P., cds), pp. 195-208, Elsevier, Amsterdam. Makita, A., Nihira, T. & Yamada, Y. (1987) Tetrohedron Lett. 28, 805 - 808. Margolin, A. L. & Klibanov, A. M. (1987) J. Am. Chem. Soc. IOU, 3802- 3804. Margolin, A. L., Tai, D. F. & Klibanov, A. M. (1987a) J . Am. Chem. SVC.109,7885-7887. Margolin, A. L., Crenne, J . Y. & Klibanov, A . M. (1987b) Biotechnol. L

Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium i...
944KB Sizes 0 Downloads 0 Views