Biotechnol. Prog. 1995, 11, 270-275
270
Surfactant-Coated Lipase Suitable for the Enzymatic Resolution of Menthol as a Biocatalyst in Organic Media Noriho Kamiya, Masahiro Goto,*,+and Fumiyuki Nakashio Department of Chemical Science & Technology, Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka, 812, Japan
Enantioselective esterification of menthol with fatty acids using a surfactant-coated lipase was carried out in organic media. The surfactant-coated lipase originating from Candida cylindracea appeared to be highly enantioselective and good biocatalyst for the resolution of racemic menthol. The enzymatic activity of the lipase in organic media was significantly increased by a coating with a nonionic surfactant. The reaction rate of the coated lipase was more than 100 times that of the powder lipase. In order to investigate the effect of the organic solvent on enantioselectivity, 19 kinds of solvents were employed. The nature of the organic solvent strongly affected the efficiency of the biocatalyst and the enantioselectivity. Among them, isooctane was the best organic solvent from the viewpoint of reaction rate and enantioselectivity. The effect of reaction temperature on esterification was also investigated. The optimal reaction temperature was around 35 "C. The enzymatic activities using n-saturated fatty acids with different alkyl chain lengths were compared, and long-chain fatty acids we e found to be better substrates than shorter ones. The relationship between the initial rate of the esterification and the carbon number of the fatty acid was not linear. These results suggest that there are inherent K, values for each fatty acid.
Introduction It is well-known that lipases catalyze the hydrolysis of ester bonds in an aqueous solution and in some cases show a specific selectivity for the substrates. Furthermore, if the reaction medium (i.e., the solvent) is changed from water to a nonaqueous medium, lipases can also catalyze the opposite reaction and enable the synthesis of ester compounds in organic media (Klibanov, 1986; Dordick, 1989; Borzeix et al., 1992; Stamatis et al., 1993a,b). On the basis of their high enantioselectivity compared to chemical catalysts, in recent years, lipases have attracted much interest as biocatalysts for the enzymatic resolution of racemic substances (Cambou and Klibanov, 1984a,b;Kirchner et al., 1985; Hedstrom et al., 1993; Tsai and Wei, 1994). (-)-Menthol is a commercially useful chiral compound in which only one of the enantiomers and diastereomers is biologically (or otherwise) active. It has been widely used in foods, cosmetics, and pharmaceutics. However, if we synthesize menthol by organic synthesis, racemic compounds are obtained. Therefore, an efficient separation method for the racemic compounds (i.e., optical resolution) is needed. In this paper, we focus attention on the enzymatic resolution of menthol because the enzymatic method is highly selective compared with other resolution methods, such as column chromatography or crystallization. When a hydrophobic substrate such as menthol is used in the enzymatic reaction, however, the enzyme should be utilized in a dry organic solvent from the viewpoint of the solubility of menthol and a favorable equilibrium shift to the ester synthesis reaction. The resolution of racemic menthol has been investigated using powder lipase originating from Candida cylindraAuthor t o whom all correspondence should be addressed. ' Present address (through June 1995): Department of Chemical Engineering, Massachusetts Institute of Technology, 66-325, Cambridge, MA 02139.
cea in some organic solvents (Langrand et al., 1985,1986; Lokotch et al., 1989)or Penicillium simplicissimum lipase in a water-in-oil microemulsion (AOThsooctane) (Stamatis et al., 1993). Furthermore, the microbial esterification of racemic menthol has also been investigated in a culture broth of Pseudomonas sp. NOF-5 (Inagaki and Ueda, 1987). Recently, novel enzyme materials have been developed by coating an enzyme with surfactants (Okahata and Ijiro, 1988; Okahata et al., 1988; Tsuzuki et al., 1991; Goto et al., 1993,1994,1995). Among them, surfactantcoated lipase is soluble in anhydrous organic solvents and shows high catalytic activity compared with other enzymatic systems, especially in esterification (Goto et al., 1994). That is why we chose this modified lipase as a preparative tool for the resolution of racemic menthol. So far, only the surfactant-coated lipase originating from Pseudomonas fiagi 22 has been applied to the esterification of menthol, but it did not catalyze the reaction, probably because of the steric hindrance near the OH group (Okahata et al., 1988). However, we have found that the synthesis reaction of menthyl ester proceeds smoothly using a surfactant-coated lipase in an organic solvent if we choose the proper origin of lipase. This work was aimed a t investigating the reactivity and enantioselectivity of a novel surfactant-coated lipase as a resolution biocatalyst for menthol in dry and homogeneous organic media. We selected the lipase originating from Candida cylindracea because this lipase has been applied to the resolution of racemic mixtures (Kirchner et al., 1985; Hedstrom et al., 1993),particularly menthol resolution (Langrang et al., 1985, 1986; Lokotch et al., 1989). In the present study, the effects ofthe origin of lipase, reaction temperature, reaction medium, and alkyl chain length of the fatty acid on the resolution of menthol were investigated, and optimal conditions are discussed.
8756-7938/95/3011-0270$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
Biofechnol. Prog., 1995, Vol. 11, No. 3
271
active rite of enzyme
-
E- surfactant (2Cj8AQGE)
Figure 1. Structure of the nonionic surfactant used to coat lipase and schematic illustration of the surfactant-coated enzyme.
Experimental Section Reagents. Six different lipases were used in this study: these were from porcine pancreas (specific activity of 46 olive oil unitdmg), which was purchased from Sigma Co., Ltd., and from Candida cylindracea (commercial name: lipase AY, specific activity of 12 olive oil unitdmg), Rhizopus sp. (commercial name: lipase F, specific activity of 150 olive oil unitdmg), Pseudomonas sp. (commercial name: lipase PS, specific activity of 30 olive oil unitdmg), Mucor jauanicus (commercial name: lipase M, specific activity of 10 olive oil unitdmg), and Aspergillus niger (commercial name: lipase A, specific activity of 60 olive oil unitdmg), which were kindly supplied by Amano Pharmaceutical Co., Ltd., and were used as received. All lipases were assayed by hydrolysis of olive oil around pH 7 and 37 "C. Optically pure (+)and (-)-menthol (purity of more than 99%)as substrates were purchased from Tokyo Chemical Industry Co., Ltd. All other chemicals used in this work were obtained commercially and were of analytical grade. All solvents were of the highest purity commercially available and were dried with 3 A molecular sieves prior to use. Nineteen kinds of organic solvents and eight kinds of fatty acids were employed; however, isooctane (2,2,4trimethylpentane) and lauric acid, respectively, were used mainly. A nonionic surfactant, glutamic acid dioleyl ester ribitol amide (abbreviated as 2CleA9GE),for coating the lipase was synthesized as described in a previous paper (Goto et al., 1987). Figure 1 shows the detailed structure of the surfactant and a schematic illustration of the surfactant-coated enzyme. Preparation of Surfactant-Coated Lipase. Figure 2 indicates the flowchart of the preparation method of the surfactant-coated enzyme. A typical preparation of surfactant-coated lipase was as follows: 1 g/L lipase in 0.1 M phosphate buffer solution (500 mL, pH 6.9) and 0.5 g of surfactant were mixed and sonicated in an ultrasonic bath (Sharp UT-204) for 20 min. The translucent solution, after incubation for 24 h at 4 "C, was collected by centrifugation and dried under reduced pressure. A white powder was obtained and the yield was about 20%. The enzyme content of the surfactantcoated lipase was calculated from the results of elemental analysis of the lipase and the lipase-surfactant complex.
lg4 Lipase solution 500ml (0.1M Phosphate buffer pH6.9)
I
0.5g Surfactant
(80W,20 min.)
Incubatingfor a day
1 Centrifugationfor 10 min.
(Vacuum drying)
Figure 2. Flowchart of the preparation method of surfactantcoated enzyme. Surfactant-coated lipases were prepared using six different lipases mentioned in the preceding section. The enzyme content of the surfactant-coated lipase greatly depended on the origin of the lipase (Okahata and Ijiro, 1988; Goto et al., 1995). The number of surfactant molecules attached around one lipase molecule was ca. 200,which means that the enzyme content in the lipasesurfactant complex was 28%, when lipase AY (from Candida cylindracea) was used. Enantioselective Esterification of Menthol. The reaction mixture for the enantioselective esterification was composed of 5 mM (+I- or (-)-menthol and 10 mM
Biofechnol. Prog., 1995, Vol. 11, No. 3
272
A
Surfactant-coated
A
A
(-)-Menthol
Rhizopus sp.
/\ (+)-Menthol
Candida cylhdracea-4
(-)-Menthylester
(+pMenthol
Figure 3. Schematic diagram of menthol resolution by surfactant-coated lipase.
fatty acid in a total volume of 10 mL. The concentration of the surfactant-coated lipase AY was 0.4 g/L (enzyme content: 0.1 g/L). A control experiment was carried out with the same enzyme content of the other surfactantcoated or native powder lipase. The enzymatic esterification of menthol with a fatty acid having different chain lengths was started by adding a surfactant-coated lipase in various organic solvents at 35 "C. The best reaction medium was selected by considering both the enantioselectivity and the conversion rate. When the effect of reaction temperature on the esterification was investigated, the temperature of the mixture was maintained at 10,22,28,35,40,50,or 60 "C via a thermostated water bath. Figure 3 shows a schematic diagram of the enantioselective esterification of menthol by a surfadantcoated lipase. Actually, chiral analysis of enantiometric mixtures, as well as quantitative analysis of kinetic resolution reactions (Chen et al., 1982),is needed. In this study, however, all experiments were conducted with homochiral substrates ((+I- or (-)-menthol) to simplify the methods, because we would like to focus attention on the search for optimal conditions for the resolution of menthol. Analytical Procedure. After the esterification was started, a 2 pL sample of the reaction mixture was withdrawn periodically. The sample was immediately analyzed by gas chromatography (HP 5890) with an FID detector and a 15 m capillary column (J&W Scientific DB1). The instrument was equipped with a cold on-column injection system. Decane was used as internal standard. The decrease in the concentrations of both unesterified menthol and free fatty acid was determined quantitatively. The difference between the amount of menthol at time zero and that at different reaction times was used to quantitate the produced ester. Initial reaction rates were determined by linear regression computer calculations, because curves of substrate consumption as a function of time were linear over several hours ((-1 5 h, (+) 20 h).
Results and Discussion Effect of the Origin of Lipase. The effect of six kinds of lipase on the reactivity of the esterification of (-)-menthol and lauric acid was investigated in isooctane a t 35 "C. The enzyme content was constant (0.1 g/L) in all lipases. Figure 4 shows the conversion to (-)-menthyl laurate by various surfactant-coated or powder lipases at 48 h. We found that the enzyme activity depended strongly on the origin of the lipase. Only the lipase originating from Candida cylindracea effectively catalyzed the synthesis reaction of (-)-menthyl laurate. Moreover, by coating the lipase, lipase AY showed much higher catalytic activity than the powder material. The lipase from Pseudomonas sp. did not show as a high conversion as previously attempted (Okahata et al., 1988b). In subsequent experiments, we selected lipase AY (from Candida cylindracea) as biocatalyst for the menthol esterification. Comparison of Reaction Systems. Figure 5 shows the time course of menthyl laurate formation using
-I
Pseudomonas sp.
-
Mucor javanicus at 48h
Aspergillus niger Porcine pancreas 0
20
40
60
80
100
Conversion[%] Figure 4. Effect of the origin of lipase on the enzymatic esterification of (-)-menthol. 100 1
80
-
1
0
20
0
(+)-Menthol by coated
A
(-)-Menthol by powder
0
0
5
10
15
20
25
30
Time[h] Figure 5. Time courses of menthyl laurate formation in isooctane.
surfactant-coated or powder lipase AY in isooctane a t 35 "C. The conversion rate of the (-)-menthol in the surfactant-coated system is much higher than that in the powder system. Also, we found that (-)-menthyl laurate was preferentially produced compared to the (+)-menthyl ester in the surfactant-coated system. After 24 h, more than 90% of the (-)-ester compound was produced, while the yield of the (+)-ester compound was only 9%. On the basis of these results, one may say that surfactant-coated lipase AY can perform the enantioselective esterification of menthol. These results mean that the surfactantcoated lipase can be a good biocatalyst for the resolution of racemic menthol. Another advantage of the surfactant-coated system is the high reaction rate in comparison with interesterification from triacetin and (-)-menthol with powder Candida cylindracea lipase in isooctane, where reaction equilibrium was attained after about 80- 100 h (Lokotch et al., 1989). Stamatis et al. (1993) reported that the reaction plateau of esterification from oleic acid and (-1menthol was reached in about 40 min with Penicillium simplicissimum lipase in AOTIisooctane microemulsions. The enantioselectivity of P. simplicissimum lipase was, however, relatively low compared to surfactant-coated lipase AY in this work. Therefore, surfactant-coated lipase AY can be an effective catalyst for menthol resolution with respect to both enantioselectivity and catalytic activity. Effect of Temperature. Figure 6 shows the effect of reaction temperature on the initial reaction rate of
Biotechnol. Prog., 1995, Vol. 11, No. 3 125
I
I
I
273 I
1
Table 1. Effect of Aromatic Solvents on Catalytic Activitv and Enantioselectivitv
I
v(-P
solvent (log P) benzene (2.0) toluene (2.5) 0 - , m-,p-xylene (3.l)*
v(-)Iv(+)
v(+) 1.94 2.28
27.1 71.3
14.0 31.2
a v: initial reaction rate Gm0Vmin.g of lipase). Xylene did not show enzymatic activity.
a
120 100
I
I
'
-
0
(-)-Menthol (+)-Menthol
-
0 0
10
20
30
40
50
60
70
3 60
TemperaturePC] Figure 6. Effect of reaction temperature on initial rate in menthyl laurate formation.
surfactant-coated lipase AY for (-1- and (+)-menthol. The reaction kinetics of the esterification was carried out at temperatures ranging from 10 to 60 "C. As is shown in Figure 6, the enzymatic activity depends strongly on the reaction temperature, and the optimal temperature region is considerably narrow. The optimal reaction temperature was found to be around 35 "C; hence the reaction temperature of 35 "C was chosen for use in further experiments. Effect of Organic Solvent. In order to investigate the effect of the nature of organic solvents on enantioselectivity, 19 kinds of solvents having different hydrophobicities, expressed by log P (Laane et al., 19871, were employed. When hydrophilic solvents (logP),such as 1,4-dioxane (-1.11, methanol (-0.781, acetonitrile (-0.331, and ethanol (-0.24) were tested, the reaction mixtures were suspended and surfactant-coated lipase AY did not catalyze the esterification reaction of menthol. On the other hand, in tetrahydrofuran (0.491, diethyl ether (0.851, and chloroform (2.0)) surfactant-coated lipase AY did not show catalytic activity, although it was soluble and clear solutions were obtained. These results suggest that the enzymatic activity is independent of the solubility of the lipase-surfactant complex in hydrophilic solvents, which might be due to the essential water around the enzyme being stripped into the bulk solution (Klibanov, 1986; Tsai and Wei, 1994). This observation agrees with the results obtained in di- and triacylglycerol syntheses catalyzed by surfactant-coated lipase D (from Rhizopus delemar) (Okahata and Ijiro, 1992) and in the esterification of benzyl alcohol with lauric acid catalyzed by surfactant-coated lipase originating from Pseudomonas sp. (Goto et al., 1994). The initial rates and the enantioselectivities ( u ( - ) h ( + ) ) obtained in aromatic solvents are listed in Table 1. The surfactant-coated lipase showed fairly high initial rates in benzene and toluene and was about twice as active with (-)-menthol in toluene than in benzene. The slower esterification of (-)-menthol in benzene resulted in a relaxation in enantioselectivity. We can choose toluene as the organic solvent when using an aromatic solvent from the viewpoint of the solubility of substances. Furthermore, when using 0-,m-, and p-xylene, the surfactant-coated lipase did not catalyze the reaction a t all, which might be explained by inhibition of the solvent. However, a detailed study is now under way. It is a surprising result that such a small change in the structure of the solvent causes quite different results.
2
3
4
5
6
7
logP
hexane4
4
30
CI
25
+ c %
20
L CI
>
isooctane
-
+
-
15
-
10
'
2
1
cyclohexane
%bne
dodecane
4 1
I
I
I
I
3
4
5
6
7
logP Figure 7. (a) Effect of aliphatic solvents on the catalytic activity of surfactant-coated lipase AY. Solvents (log P) used are as follows: pentane (3.01, cyclohexane (3.21,hexane (3.5),heptane (4.0),octane (4.51,isooctane (4.5),and dodecane (6.6). (b) Effect of solvent hydrophobicity on the enantioselectivity of surfactantcoated lipase AY.
Figure 7a shows the effect of aliphatic solvents on the catalytic efficiency of surfactant-coated lipase for the esterification of (-1- and (+)-menthol with lauric acid. These results are very interesting because the structure of the solvent is very sensitive to the activity of the enzymatic reaction. The initial rate increased with the enhancement of hydrophobicity of the organic solvents, except for cyclohexane and isooctane. In a previous paper (Goto et al., 19931, we pointed out the importance of the hydrophobic structure of the surfactant in obtaining high enzymatic activity for the surfactant-coated lipase. High initial rates in cyclohexane and isooctane can also be explained by the structural differences in the organic solvents. When an organic solvent having a branched or cyclic group was employed, the solubility of the surfactant-coated lipase might increase due to a double bond in the hydrophobic tail of the surfactant used in
Biotechnol. Prog., 1995, Vol. 11, No. 3
274 180
-
160 140
2 120 0)
2
100
E"
80
N
0
E
'E 60 : 5
40
>
20
Y -L
-
0
2
4
6
8 10 12 14 16 18 20
Carbon number of fatty acid
Figure 8. Effect of fatty acids on the initial rate of the menthol esterification.
this study. Therefore, the surfactant-coated lipase showed higher activity than in the solvents having a straight chain. We can see from Figure 7b that strong enantioselectivity is obtained in hexane and heptane. The decrease in enantioselectivity in pentane is due to a low reaction rate of the (-)-isomer. In octane and dodecane, enantioselective relaxation occurred due to an increase in the reactivity of the (+)-isomer. These observations are similar to the results obtained in the Candida cylindracea lipase-catalyzed esterifications of (R,S)-2-hydroxycaproic acid and (R,S)-2-hydroxyisocaproicacid in organic solvents (Parida and Dordick, 1991). High catalytic activity was observed in cyclohexane (Figure 7 a); however, the enantioselectivity was low, because the initial rate of the (+)-isomer was relatively high. This might be partly due to the increase in the solubility of menthol in cyclohexane, which has a structure similar to that of the substrate. On the basis of the results, isooctane gave the best result with regard to the reaction rate and enantioselectivity. Thus, isooctane will be selected as the organic solvent for the resolution of racemic menthol by surfactant-coated lipase AY. Effect of Fatty Acid on Enzymatic Activity. Various fatty acids with carbon chain lengths between 4 and 18 were used as substrates for the esterification of menthol under optimal conditions. The enzymatic activities as a function of the carbon number of the fatty acids are shown in Figure 8. A fatty acid having a long alkyl chain showed a higher reaction rate than a short chain acid as previously reported (Goto et al., 1994). The results suggest that a more hydrophobic substrate is preferred at the active site of the lipase. Because the result for the fatty acid of carbon number 14 seems rather strange to us, we tried the experiment several times. However, the reproducibility of the data was quite good. Because the plots of the initial rate are not proportional to the carbon number, we can speculate that there would be an inherent K, value for each fatty acid. Further study on determining each K, value is now under investigation to elucidate the difference in catalytic activity, that is, of the structure of the hydrophobic binding pocket for a fatty acid in Candida cylindracea lipase.
Conclusion A novel surfactant-coated lipase AY (from Candida cylindracea) was prepared with a nonionic surfactant and applied to the enantioselective esterification of menthol
in organic media. This lipase-surfactant complex could preferentially produce the (-)-menthyl ester and be a preparative tool for the resolution of racemic menthol. The reaction rate with surfactant-coated lipase AY was much higher than that with the native powder lipase. The optimal temperature was around 35 "C. The catalytic activity and enantioselectivity of the surfactantcoated lipase depend strongly on the organic solvents and the length of the fatty acids. Aromatic and aliphatic hydrocarbon solvents provided higher catalytic activity than hydrophilic solvents. Isooctane gave the best result with regard to the initial rate and enantioselectivity. Long-chain fatty acids showed higher enzymatic activity than did the short-chain acids. To clarify the substrate specificity of Candida cylindracea lipase, kinetic studies on the effect of fatty acids of different chain lengths are required.
Acknowledgment We are grateful to Amano Pharmaceutical Co., Ltd., for generously providing lipases AY, F, PS, M, and A. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Chemical Materials Research & Development Foundation, and the Shorai Foundation for Science and Technology. Literature Cited Borzeix, F.; Monot, F.; Vandecasteele, J.-P. Strategies for enzymatic esterification in organic solvents: Comparison of microaqueous, biphasic and micellar systems. Enzyme Microb. Technol. 1992,14, 791-797. Cambou, B.;Klibanov, A. M. Preparative production of optically active esters and alcohols using esterase-catalyzed stereospecific transesterification in organic media. J. Am. Chem. SOC. 1984a,106, 2687-2692. Cambou, B.; Klibanov, A. M. Comparison of different strategies for the lipase-catalyzed preparative resolution of racemic acids and alcohols: asymmetric hydrolysis, esterification, and transesterification. Biotechnol. Bioeng. 198413, 26, 14491454. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. Quantitative analyses of biochemical kinetic resolutions of enantiomers. J. Am. Chem. SOC. 1982,104,7294-7299. Dordick, J. S. Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol. 1989,11, 194-211. Goto, M.; Matsumoto, M.; Kondo, K.; Nakashio, F. Development of new surfactant for liquid surfactant membrane process. J . Chem. Eng. Jpn. 1987,20,157-164. Goto, M.; Kameyama, H.; Goto, M.; Miyata, M.; Nakashio, F. Design of surfactants suitable for surfactant-coated enzymes as catalysts in organic media. J. Chem. Eng. Jpn. 1993,26, 109- 111. Goto, M.; Kamiya, N.; Miyata, M.; Nakashio, F. Enzymatic esterification by surfactant-coated lipase in organic media. Biotechnol. Prog. 1994,10, 263-268. Goto, M.; Goto, M.; Kamiya, N.; Nakashio, F. Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media. Biotechnol. Bioeng. 1995,45,27-32. Hedstrom, G.; Backlund, M.; Slotte, J. P. Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase. Biotechnol. Bioeng. 1993, 42,618-624. Inagaki, T.; Ueda, H. Enantioselective esterification of racemic terpene alcohols with fatty acids by Pseudomonas sp. NOF-5 strain. Agric. Biol. Chem. 1987,51, 1345-1348. Kirchner, G.; Scollar, M. P.; Klibanov, A. M. Resolution of racemic mixtures via lipase catalysis in organic solvents. J . Am. Chem. SOC. 1986,107,7072-7076. Klibanov, A. M. Enzymes that work in organic solvents. CHEMTECH 1986,16,354-359. Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30, 81-87.
Biotechnol. Prog., 1995, Vol. 11, No. 3 Langrand, G.; Secchi, M.; Buono, G.; Baratti, J.; Triantaphylides, C. Lipase-catalyzed ester formation in organic solvents: An easy preparative resolution of a-substituted cyclohexanols. Tetrahedron Lett. 1985,26, 1857-1860. Langrand, G.; Baratti, J.; Buono, G.; Triantaphylides, C. Lipase catalyzed reactions and strategy for alcohol resolution. Tetrahedron Lett. 1986,27,29-32. Lokotch, W.; Fritsche, K.; Syldatk, C. Resolution of D,L-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea. Appl. Microbiol. Biotechnol. 1989,31, 467-472. Okahata, Y.;Ijiro, K. A lipid-coated lipase as a new catalyst for triglyceride synthesis in organic solvents. J . Chem. SOC., Chem. Commun. 1988,1392-1394. Okahata, Y.;Ijiro, K. Preparation of a lipid-coated lipase and catalysis of glyceride ester synthesis in homogeneous organic solvents. Bull. Chem. SOC.Jpn. 1992,65,2411-2420. Okahata, Y.;Fujimoto, Y.; Ijiro, K. Lipase-lipid complex as a resolution catalyst of racemic alcohols in organic solvents. Tetrahedron Lett. 1988,29, 5133-5134. Parida, S.; Dordick, J. S. Substrate structure and solvent hydrophobicity control lipase catalysis and enantioselectivity in organic media. J. Am. Chem. SOC.1991,113,2253-2259.
275 Stamatis, H.; Xenakis, A.; Provelegiou, M.; Kolisis, F. N. Esterification reactions catalyzed by lipases in microemulsions: The role of enzyme localization in relation to its selectivity. Biotechnol. Bioeng. 1993a,42, 103-110. Stamatis, H.; Xenakis, A,; Kolisis, F. N. Enantiomeric selectivity of a lipase from Penicillium simplicissimum in the esterification of menthol in microemulsion. Biotechnol. Lett. l993b, 15,471-476. Tsai, S.;Wei, H. Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol. Biotechnol. Bioeng. 1994,43, 64-68. Tsuzuki, W.; Okahata, Y.; Katayama, 0.;Suzuki, T. Preparation of organic-solvent-soluble enzyme (lipase B) and characterization by gel permeation chromatography. J. Chem. Soc., Perkin Trans. 1 1991,1245-1247. Accepted December 15,1994.@
BP9401007 @Abstractpublished in Advance ACS Abstracts, February 15, 1995.
