203
Biotechnol. Prog. 1984, 70, 263-288
Enzymatic Esterification by Surfactant-CoatedLipase in Organic Media Masahiro Goto,*Noriho Kamiya, Masaki Miyata, and Fumiyuki Nakashio Department of Chemical Science & Technology, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812, Japan
Surfactant-coated lipases have been prepared with a synthesized surfactant. Preparation conditions to obtain a suitable surfactant-coated lipase were investigated. The enzymatic activity of the lipase in an organic solvent significantly increased with the coating of the surfactant. The esterification rate from the surfactant-coated lipase was much higher than that from the powder lipase. An aliphatic solvent showed higher activity than did alcohol, aromatic, and chloric solvents. Among them, isooctane gave the highest activity. The reactivity of the surfactant-coated lipase depends on the p H of the aqueous solution in the preparation and on the buffer solution. Surfactant-coated lipase prepared in the middle pH range using phosphate buffer showed high enzymatic activity. The surfactant-coated lipase was thermostable at high temperature compared to the native lipase. A kinetic study enabled a ping-pong bi-bi reaction mechanism with alcohol inhibition to be suggested. From the kinetic analysis, it was found that an alcohol substrate inhibits enzymatic esterification by lipase. The reaction rate of the coated lipase was about 100 times that of the powder lipase.
Introduction The use of enzymes in organic solvents has attracted much interest during the last 10 years due to ita obvious advantages,such as conversion of hydrophobiccompounds and favorable shifts in reaction equilibria. Severalstudies have been carried out on enzymatic catalysis with many enzymes in nonaqueous solvents. These approaches are generally classified into two groups. In the first approach, enzymes are dissolved in water pools in reversed micelles (Shield et al., 1986, Martinek et al., 1986). In the second, powder enzymes dissolved directly in organicmedia (Zaks and Klibanov, 1988;Dordick, 1989). In reversed micelles, it is well-known that one can see the superactivity of enzymes or different substrate specifity under optimal conditions (Martinek et al., 1989; Luisi et al., 1988). However, this approach is not available for the synthesis reaction as water is produced, and a fair amount of water is needed to obtain high enzymatic activity. On the other hand, in the latter, enzymes are directly exposed to the solvent and, hence, exhibit some remarkable novel properties compared to those in water. For instance, the thermostability of enzymes in nonaqueous media may be greatly enhanced compared to that in water (Zaks and Klibanov, 1984). The prochiral selectivity of various hydrolytic enzymes can be regulated by the organic solvents (Terradas et al., 1993). However, there is a crucial defect in the powder enzyme system. It has a low reaction rate compared to that in water, because the enzyme does not dissolvein such organic media. Therefore, most enzymatic reactions using a powder enzyme proceed in a suspensionsystem. To utilize enzymatic catalysis effectively, it is necessary to dissolve the enzyme in an organic medium. At least two approaches to that goal have been successfullydeveloped. In the first method, a section of amino acids in an enzyme is modified with an amphiphatic synthetic polymer, poly(ethy1ene glycol) (Takahashi et al., 19881, and in the second, the surface of the enzyme is coated with surfactant (Okahata
* Author to whom correspondenceshould be addressed. 8756-7938/94/3010-0263$04.50/0
and Ijiro, 1988; Tsuzuki et al., 1991; Goto et al., 1993). These modified enzymes are almost entirely soluble in organic solvents, and they give higher reaction rates compared to the powder enzyme. Polymer-modified enzymes are somewhat more difficult to prepare than surfactant-coated enzymes. However, very few papers concerning the surfactantcoated enzymes have been published. Only three groups (including our group) have reported on the preparation and enzymatic reactions of these surfactant-coated enzymes. Okahataet d.(1988) first proposed the preparation method of the surfactant-coated enzyme. They have reported that the catalytic activity of the surfactant-coated lipase was highly efficient compared with other enzyme systems for a synthesis reaction in a dry organic solution. Tsuzuki et al. (1991a,b) have also prepared several surfactant-coated lipases in high yield using a synthetic surfactant of glucose derivatives. However, information on enzymatic esterification with the surfactant-coated lipase is limited, and in particular, no kinetic study has been performed. The aim of the present study is two-fold. One purpose is to establish the preparation method of surfactant-coated enzymes as catalysts in organic media, and the other is to clarify the characteristics of enzymatic esterifications catalyzed by the surfactant-coated enzyme. We selected lipase as the model enzyme, because lipase is most widely used and is easy to obtain. In the present study, several surfactant-coated lipases have been prepared with a synthesized surfactant under various conditions,and their properties in an ester synthesis reaction are investigated in various organic solvents.
Experimental Section Reagents. Five kinds of lipase originating from Pseudomonas sp., Aspergillus niger, Rhizopus sp., Mucor jauanicus, and Candida cylindracea were used. All enzymes were provided by Amano Pharmaceutical Co., Ltd. and were used as received. A nonionic surfactant glutamic acid dioleyl ester ribitol amide (abbreviated
0 1994 American Chemical Society and American Instiiute of Chemical Engineers
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264
2CleA9G E
H O I II C H 3( CH2)7CH=CH(CH2)8 -0 -C -C H -N -C -( CHOH)4CH20 H 0 II
I
CH3(CH2)7CH=CH(CH2)8 -0-C
II
(CH2)2
0
Figure 1. Molecular structure of the surfactant used in the preparation of surfactant-coated lipase.
100 L
-
80
60
-
40
-
20
-
Y
a >
0
1
1
-
0 Surfactant-coated
0
8
-e
0
0
0 Reversed micelle
A Dispersed powder
0 0
0
S
2C18A9GE),which shows the highest activity (Goto et al., 1993),was used. The surfactant was synthesized according to a procedure described in a previous paper (Goto et al., 1987). Figure 1 shows the structure of the nonionic surfactant used in this work. Ten kinds of organic solvents were used; however, 2,2,44rimethylpentane (isooctane) was used predominantly, because it gave the highest reaction rate. As standard substrates, benzyl alcohol and lauric acid of analytical grade were used. Several kinds of carboxylic acids and alchols were used to study the effect of substrate structure on enzymatic esterification. Experimental Procedure. A typical preparation of surfactant-coated enzyme is as follows. An aqueous solution (250 mL of phosphate buffer (50 mM, pH 6.8)) of lipase (500 mg) and an aqueous solution (250 mL) of surfactant (500 mg) were mixed in an ultrasonic cleaner (SHARP UT-204) for 20 min, and the solution was incubated in a refrigerator for 1 day. The precipitates were collected by centrifugation and dried under vacuum. White powder was obtained and the yield was about 20%. The surfactant-coated enzyme was insoluble in water, but almost entirely soluble in organic solvents. The hydrophobic tails of a surfactant solubilizethe enzyme in organic media. The enzyme content of the complex was determined by elemental analysis. The enzyme content was from 10 to 28 wt % and depended greatly on the origin of the lipase. The enzymatic esterification of lauric acid (3 mM) with an excess amount of benzyl alcohol (6 mM) by surfactant-coated lipase was investigated in an organic solvent. The experiments were carried out at a reaction temperature of 308 K, except for the investigation on reaction temperature. The lipase content is constant (0.2 g/L) in all experiments. The disappearance of benzyl alcohol and the production of the ester compound were followed by gas chromatography (HP 5890)witha capillary column of 15 m X 0.53 mm (J&W DB1). Analytical conditions are as follows: injection temperature, 50 "C; oven temperature is raised 20 "C/minup to 330 "C, followed by a 5-min isotherm; carrier gas, He; flow rate, 16 mL/ min; detector, FID (350 "C), with H2 flow rate 30 mL/min and dry air flow rate 400 mL/min.
Results and Discussion Comparison of Reaction Systems. Three reaction systems were compared with the esterification by lipase from Pseudomonas sp. Two were homogeneous reaction systems, (1)the surfactant-coated system and (2) an AOTisooctane reversed micellar system (50 mM, w o = [HzO]/ [surfactant] = lo), and one was a heterogeneous reaction system, (3) a liquid-solid (isooctane-powder lipase) system. The concentration of the lipase is the same in all systems. Figure 2 shows the comparison of the three reaction systems on the conversion to the ester compound. It is found that the reaction system of the surfactantcoated lipase is the most suitable for esterification from the viewpoint of reaction rate. The conversion in the reversed micellar system was almost constant after about 40 min. Although it takes a long time (about 3 days), the final conversion in the dispersed powder system attained more than 90 % under the present experimental conditions.
0
0
O d
20
40
60
80
100
120
140
Reaction timermin]
Figure 2. Comparison of reaction systems of the esterification by lipase (solvent, isooctane; [BA] = 6 mM, [LA] = 3 mM, [enzyme] = 0.2 g/L).
In both the surfactant-coated and the dispersed powder systems, the water content in the organic solvent was very low-less than 0.01% (v/v) (about 60-80 ppm). On the other hand, that in the reversed micellar system was about 1% (v/v). These results suggest that restricting the water content is necessary to obtain a high yield in the esterification. In the following experiments, we studied operation factors in the esterification reaction in the surfactantcoated system in detail. Effect of Surfactant on Esterification by Surfactant-Coated Lipase. The coating surfactant plays a key role in the enzymatic reaction of surfactant-coated lipase, because the activity and stability of the lipase strongly depend on the type and structure of the surfactant (Tsuzuki et al., 1991a,b; Goto et al., 1993). In a previous paper (Gotoet al., 1993),the esterification was carried out using five kinds of surfactant-coated lipases. It was found that a nonionic surfactant is the best surfactant. In the equilibrium state, the conversions were almost the same for all lipases; however, a complex prepared by the cationic or amphoteric surfactants shows a low reaction rate compared with that of nonionic surfactants. When an anionic surfactant was used, the enzyme complex could not be prepared by an electrostatic repulsion for lipase, because the PI (isoelectric point) of lipase is relatively low (about 5). When the carbon number was the same, surfactants having a branch or a double bond showed higher activity than did surfactants having straight chains, because the solubility of the enzyme complex in an organic solvent increases due to such a hydrophobic group. It was found that the surfactant-coated lipase prepared by the surfactant having two oleyl chains, as shown in Figure 1, was most suitable as a catalyst in esterification. From the elemental analysis of the complex, it was found that the complex prepared by 2C18A9GE contains 23 wt % lipase, that is, one molecule of lipase is coated by about 170 molecules of the surfactant. Okahata and Ijira (1992) investigated in detail the structure of the surfactant-coated lipase by gel-permeation chromatography and reported that the complex contains a lipase coated heavily with one layer of 150 f 30 surfactant molecules. In our experiment almost the same results were obtained. Subsequent experiments were carried out using 2C18AgGE, which is the most effective surfactant. Effect of the Origin of Lipase. The effect of five kinds of lipase on the reactivity of the esterification of benzyl alcohol and lauric acid was investigated in isooctane. Figure 3 shows the conversion to the ester compound by various lipases at 60 min. Regardless of their origin, all surfactant-coated lipases showed higher reactivity compared to the powder lipase. Both Aspergillus niger and
Bbtechnol. prog., 1994, Vol. 10, No. 3 Aspergillus niger Rhizopus sp.
Mucor javanicus
7 at 6Omin
Candida cylindracsa
1 I I
Pseudomonas SI).
0
20
40 60 Conversion [%I
80
100
Figure 3. Ester conversion by various lipases at 60 min (solvent, isooctane; [BA] = 6 mM, [LA] = 3 mM, [enzyme] = 0.2 g/L).
Rizopus sp. lipases did not show enzymatic activity in the powder state. However,the two lipases effectively catalyze the esterification when the surfactant is coated. In subsequent experiments, the lipase originating &om Pseudomonas sp. was used as a standard lipase. Effect of Organic Solvent. The effect of ten kinds of organic solvents on the reactivity of the esterification was investigated by using the surfactant-coated lipase. Figure 4 shows the relative activity of various organic solvents when the reaction activity of isooctane is 1unit. Aliphatic solventsshowed higher enzymatic activities than did aromatic, alcohol, and chloric solvents. The results suggest that nonpolar solvents show higher reactivity. It is well-knownthat water is essential for enzymatic activity. In a polar solvent, the essential water may be taken away from the surfactant-coated lipase. Among these, isooctane gave the best results. Therefore, isooctane was selected as the organic medium in subsequent experiments. Effect of Reaction Temperature. Figure 5 shows the effect of reaction temperature on esterification by the surfactant-coated and powder lipases. The reaction rate of the surfactant-coated lipase was about 2 orders of magnitude higher than that of the powder lipase. Futhermore, the surfactant-coated lipase was stable at temperatures higher than 50 “C.It is considered that coating of the surfactant protects the lipase from hazardous environments. Effect of Aqueous pH on the Preparation of Surfactant-Coated Lipase. Effect of aqueous pH on the preparation of surfactant-coated lipase was studied. Figure 6 shows the relationship between the reaction rate of the esterification and pH in aqueous solution in the preparation of a surfactant-coated lipase. The pH value in the aqueous solution of the preparation strongly affects on the enzymatic activity of the esterification. The surfactant-coated lipase prepared in a middle pH range (about 7) of the aqueous solution showed high enzymatic reactivity. At low pH (below 5), its activity decreased drastically. This dependency on pH in isooctane is almost the same as that in aqueous solution. Klibanov (1986) reported that the enzyme “remembers”the pH of the latest aqueous solution to which it has been exposed. The results obtained in this work agree with his idea. Further, the activity of the coated lipase depended on the kind of buffer solutions. The surfactant-coated lipase prepared by phosphate buffer gave a high activity. Effect of the Concentration Ratio of Surfactant and Lipase in the Preparation. The effect of the concentration ratio of the surfactant and lipase in the preparation was investigated. Two experiments were conducted to study the effect of concentration ratio. In the first one, the concentration of lipase is constant (1 g/L) and that of the surfactant is varied. In the second
Benzene Toluene Ethanol Acetonitrile Chloroform
r--&-J Pseudomonas sp.
0.0
0.2
0.6
0.4
0.8
1.0
Relative Activity[-]
Figure 4. Influence of organic solvent on the reactivity of esterification by surfactant-coatedlipase [BA] = 6 mM, [LA] = 3 mM, [enzyme] = 0.2 g/L). 10-1
n n al v)
I
Y
0
I
I
Surfactant-coated Lipase
n
.-
E -.
i
oooa 0
E >
0
01
0
Y
1
I
I
Figure 5. Effect of reaction temperature on the reaction rate of esterificationby lipase in isooctane (origin of lipase, Pseudomonas sp.; [BAI = 6 mM, [LA] = 3 mM, [enzyme] = 0.2 g/L).
A
phosphate-citrate
P
6
301
A 0
3
.
4
5
6
’
’
7
’
’
8
I ’ Im. 9
1
0
PH Figure 6. Effect of pH of aqueous solution in the preparation of surfactant-coated lipase (concentration of buffer solution, 50 m M [BAI = 6 mM, [LA] = 3 mM, [enzyme] = 0.2 g/L).
experiment, the reverse was performed. Figure 7 shows the relationship between the reaction rate of the esterification and the concentration ratio, assuming a molecular weight for lipase of around 30 OOO. The reaction rate was almost constant at more than 20 times the enzyme concentration, and the yield is about 20% in all experiments. In the following experiments, we mainly prepared the surfactant-coated lipase with a concentration ratio of 40, which corresponds to a 1:l ratio by weight. Effect of Substrate Structure on Esterification by Surfactant-Coated Lipase. When benzyl alcohol was used as the substrate, acarboxylicacid of another substrate was changed. On the other hand, when lauric acid was used, an alcohol was changed. These results are shown in Figure 8a,b. Carboxylic acids having a long alkyl chain
Biotechnol. Prog., 1994, Vol. 10, No. 3
266
0'
, a ' -
0
20
Cenz.=const.
> 10
- 0
-
Y
0.oP. ' 0
Benzyl Alcohol ([LA]=1OmM)
k.(
'
10
'
20
'
I
*
'
30
40
'
50
'
1
60
Concentration of Substrate [mM]
0 0
I
I
I
20
40
60
80
Csu rf ./Cenz.[-]
Figure 7. Relationshipbetween the reaction rate of esterification and the concentration ratio ([BA] = 6 mM,[LA] = 3 mM).
Figure 9. Influence of substrate concentration on the esteri-
fication rate.
rate. This result suggests that benzyl alcohol inhibits the reaction. These results agree with an assumed ping-pong bi-bi mechanism with dead-end inhibition by one substrate, as described in Figure 10. The lipase reacts with lauric acid to yield the lipase-lauric acid complex. The complex then transforms to a carboxylic-lipase intermediate and water is released. The equation for such an esterification by lipase is expressed as follows (Segel,1975):
--v
-
Vm, 0 ,
0
0
,
,
,
,
,
,
,
[LA1[BAI Km,,~)[BAI(l+ [BAl/KJ + Km,,,)[LA1
,
12 16 20 Alkyl-Chain Length of Carboxlic Acid [-] 0
4
8
L
10
.I
-
0 .
0
0
5 -
.
0
(1)
where Vm, is the maximum rate of the esterification, [LA] is the initial lauric acid concentration, [BAI is the initial benzyl alcohol concentration, Km(m) and Km(BA)are the Michaelis constants of lauric acid and benzyl alcohol, respectively, and Ki is the inhibition constant of benzyl alcohol. Equation 1can be changed into a more convenient form: 1 -
2-nonyl alcohol
+ [LAl[BAl
1 = %[LA] + s 2
3
0
" " e " . " 0
4
8
12
16
Alkyl-Chain Length of Alcohol
20
(3)
[-I
Figure 8. Effect of substrate structure on the enzymatic esterificationby surfactant-coated lipase: (a)effect of carboxylic acid; (b) effect of alcohol.
(4)
show a higher reaction rate compared to those with short chains. However, in the alcohol the rate does not depend its length. When 2-nonyl alcohol was used, the reaction rate decreased drastically. We found that the esterification of a secondary alcoholwas not catalyzed owing to the steric hindrance near the OH group. Kinetic Study of Esterification by Lipase in Organic Media. A kinetic study of the esterifications of benzyl alcohol and lauric acid was carried out. The effect of the concentrations of both substrates on the reaction rate was investigated separately. Figure 9 shows the relationship between the reaction rate and the concentration of substrates. In lauric acid, the reaction rate increased with an increase in the concentration of lauric acid. On the other hand, in benzyl alcohol, the reaction rate increased proportionally in the low concentration range; however, it reached a maximum at a critical concentration value. A subsequent increase in the alcohol concentration ultimately led to a decrease in the reaction
Experimental data are arranged according to eq 2, and the relationship between l/v and the reciprocalof the lauric acid concentration is shown in Figure 11. The slope and the intercept in the figure give the SI and SZ values, respectively. We can obtain Km(BA)and Vm, values from the relation between l/[BAI and Sz, and Km(LA)and Ki values can also be determined from the relation between [BAI and SI. A typical analytical result is shown in Figure 12. In both the coated and the powder lipases, all parameters were obtained. Table 1 shows the kinetic parameters obtained in the esterification by lipase. We found that the Vm, value of the surfactant-coated lipase was 100 times that of the powder lipase. However, the Ki value of the coated lipase is smaller than that of the powder. The results suggest that the influence of the inhibitor, benzyl alcohol, in the surfactant-coated lipase system is large compared with that in the powder lipase system. The Michaelis constant for lauric acid, K m ( ~of~the ) , coated lipase is one-half that of the powder lipase. These results
Biotechnol. Rog., 1994, Vol. 10, No. 3
287
Lauric acld
Water
Enz
,
'
Benzyl alcohol
Enz'water
Enz'
Ben@ alcohol
L. Enz'-Ben
Ben,La --t
Enz-BenLa
Enz
Enz-Ben Enz : Enryme(lipase)
Enz' :Complex of lipase and lauric acid
Enz-Ben :Complex of lipase and benzyl akohol BenLa : Lauric acid benzyl ester
Figure 10. Schematic representation of the ping-pong bi-bi mechanism with inhibition by alcohol. 200
1
E
EE
. .-z
VKmd (mol/min.g) (mM) (mM) powder lipase 1.37X 1 P 204 101 surfactant-coated lipase 1.01 X 10-8 60 134
21mM 0 32mM
100
E
Y
. >
r
-
n -U.O5
0.05
0.00
0.10
1/[LA] [ l / m M ]
Figure 11. Relation between l/u and the reciprocal of the concentration of lauric acid. 2000
4
.
4
'
(a)
.
=
r h
Table 1. Kinetic Parameters Obtained in the Esterification by Powder or Surfactant-Coated Lipase Ki
(mM) 13 2
factant, the origin of the lipase, the organic solvent, and the aqueous pH in the preparation. The coated lipase prepared by glutamic acid dioleyl ester ribitol was the most suitable as a catalyst of the esterification in an organic solvent. The lipase becomes extremely thermostable by coating the surfactant. The kinetics of esterification are suggested to agree with a ping-pong bi-bi mechanism in which inhibition only by excess alcohol has been identified. From the analytical results, it was found that the reaction rate of coated lipase was 100times that of the native powder lipase.
Acknowledgment 1000
This work was supported by a Grant-in-Aidfor Scientific Research from the Chemical Materials Research & Development Foundation. The authors are grateful to Amano Pharmaceutical Co., Ltd., for the supply of many of the lipase origins.
5; Vmax
n " -20
I
0
PA1
0
u.00
.
20
,
40
.
60
Literature Cited
"I
I
0.05 l/[BA] [IlmM]
1
0.10
Figure 12. Determination of kinetic parameters: (a) SIvs [BA]; (b) Sz vs l/[BA].
are considered to be influenced by the microenvironment on the surface of the lipase.
Conclusion Surfactant-coated lipases have been prepared with synthesized surfactants. Using the lipase-surfactant complexes, the esterifications of an alcohol and a carboxylic acid were investigated in organic media. The activity of the coated lipase strongly depends on the coating sur-
Dordick, J. S. Enzymatic catalysisin monophasicorganicsolvents. 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.; Miyata, M.; Nakashio, F. Design of surfactants suitable for surfactant-coated enzymesas catalysts in organic media. J. Chem. Eng. Jpn. 1993,26,109-111. Klibanov, A. M. Enzymes that work in organic solvents. CHEMTECH 1986,June, 354-359. Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Reverse micelles as hosts for proteins and small molecules. Biochim. Biophys. Acta 1988,947, 209-246. Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khmelnitski, Yu. L.; Berezin, I. V. Micellar enzymology. Eur. J.Biochem. 1986,155,453-468. Martinek, K.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Yu. L.; Levashov, A. V. Micellar enzymology: its relation to membranology. Biochim. Biophys. Acta 1989,981,161-172. Okahata, Y.; Ijiro, K. A lipid-coated lipase as a new catalyst for triglyceridesynthesis 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 syntheses in homogeneous organic Jpn. 1992,65,2411-2420. solvents. Bull. Chem. SOC. 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.
Biotechnol. Prog., 1994,Vol. 10,No. 3
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Segel, I. H. Enzyme Kinetics; Substrate inhibition in ping pongsystems; Wiley-Interscience: New York, 1975;p 826. Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A. Enzymes in reversed micelles as catalysts for organic-phase synthesis reactions. Ind. Eng. Chem. Fundam. 1986,25,6036l2.
Takahashi, K.; Saito, Y.; Inada, Y. Lipase made active in hydrophobic media. J. Am. Oil Chem. Sci. 1988,65(6),911916. Terradas, F.; Henry, M. T.; Fitzpatrick, P. A.; Klibanov, A. M. Marked dependence of enzyme prochiral selectivity on the 1993,115,390-396. solvent. J. Am. Chem. SOC. Tsuzuki,W.; Okahata, Y.; Katayama, 0.;Suzuki,T. Preparationof organic-solvent-solubleenzyme (lipase B)and characterization
by gel permeation chromatography. J. Chem. SOC.,Perkin Trans. 1 1991a,1245-1247. Tsuzuki, W.; Sasaki, T.; Suzuki, T. Effect of detergent attached to enzyme molecules on the activity of organic-solvent-soluble lipases. J. Chem. SOC.,Perkin Trans. 2 1991b,1851-1854. Zaks, A.; Klibanov, A. M. Enzymatic catalysis in organic media a t 100 O C . Science 1984,224,1249-1251. Zaks, A.; Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem. 1988,263 (7),3194-3201. Accepted January 7, 1994." @
Abstract published in Aduance ACS Abstracts, March 15,1994.
