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Lipase-catalyzed reactions in organic media: competition and applications
Bioehimi~ et Biophysica Acta 911 (1987) 117-120 Elsevier
117
BBA Report
BBA30159
Lipase-catalyzed reactions in organic media: c o m p e t i t i o n and applications H . D e l e u z e a, G . L a n g r a n d a, H . M i l l e t a, J. B a r a t t i b, G . B u o n o a n d C. T r i a n t a p h y l i d e s a
a
" Ecole Sup~rieure de Chimie de Marseille, Centre de Saint-J~rgmw, rue Henri Poincar~, 13397 Marseille C~dex 13 (France) and h Laboratoire de Chimie Bactdrienne du C.N.R.S., B.P. 71, 13277 Marseille Cddex 9 (France) (Received 1 July 1986)
Key words: Lipase: Transesterification; Organic solvent; Enzyme kinetics: Reaction selectivity
Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.
Ester hydrolysis of emulsified substrates by lipases (EC 3.1.1.3) obeys complex kinetics [1-4]. In organic media ester synthesis and transesterification have been described [5-16] with an insoluble enzymatic preparation. As reported by others [17], and observed by us in this study, the reaction rate oA, in organic solvent, increased with the substrate concentration [A] according to a Michaelis-Menten equation: t'A
VA"[A] K A + [A]
(1)
Furthermore a ping-pong mechanism has been proposed including the formation of an acyl enzyme intermediate [17]. When two substrates (A and B) are present at the same time in the reaction mixture, a competitive factor a is then defined by the ratio of the catalytic powers according to Eq. 2
Correspondence: Dr. C. Triantaphylides, Ecole Sup~rieure de Chimie de Marseilles, Centre de Saint-J~rbme, rue Henri Poincar& 13397 Marseille C~dex 13, France.
[20]: VA/KA a= - -
VB/K.
(2)
This formalism was also used by Chen et al. [18] for two enantiomers of the same substrate. The competitive factor can be calculated from one of the following equations [20]: ~A [A] ,,-~ ~N
(3)
log ([A]/[A]o) = a log ([B]/[B]o)
(4)
=
In this paper we describe the use of the preceding model for reactions in organic media using different acyl donors and acceptors. For instance, when two different esters (AcX and AcY) of the same acid are used as acyl donor a competitive donor factor a D can be calculated (Fig. 1, full arrows). When two acceptors (X and Y) are used a competitive acceptor factor aA can be calculated (Fig. 1, dotted arrows). The competitive factors
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118 AcX
+ E
~.~X Z AcE
AcY
+ E
....
L
AcZ
+ E
1
J'~Y
Fig. [. Lipase competition reactions. AcX, AcY and AcZ = acyl donors (esters or acids). E = lipase, X, Y and Z, acyl acceptors (alcohols or water); AcE = acyl enzyme; full arrows, competition between two acyl donors (AcX and AcY); dotted arrows, competition between two acyl acceptors (X and Y).
were estimated from the variations of substrate c o n c e n t r a t i o n s (A a n d B) with time plotted according to Eq. 4. Fig. 2 shows a typical result for the competition between b u t a n o l (A) a n d water (B) with isopentyl b u t y r a t e as acyl donor. I n the same figure the competition between the R a n d S e n a n t i o m e r s of 2 - b u t y l b u t y r a t e is also shown. Table I shows the results for the competitive d o n o r factors a D for four different couples of acyl donor, three different acceptors a n d two lipases. It is seen that the competitive d o n o r factor a D was i n d e p e n d e n t of the alcohol used as acceptor when a given mixture of acyl d o n o r was used. F o r instance a D was 3.6 for the competition between cyclohexyl butyrate a n d isopentyl b u t y r a t e indep e n d e n t l y of the acceptor (geraniol, lauryl alcohol or ( - ) - m e n t h o l ) . This conclusion was true for every couple of acyl d o n o r tested (AcX a n d AcY) even when butyric acid or t r i b u t y r i n were used. The lipases from Candida Cylindracea (recently r e n a m e d Candida rugosa) a n d Mucor miehi showed the same property a n d identical results were observed using different substrate concentrations. Thus it can be concluded that this is a
i
0.5
O(
'
t
,
'
J
0.5 LOG
i
,
i
i
i
1
~--
(Bo/B)
Fig. 2. Estimation of competitive factor. A, competition between butanol ([A]0= 0.25 M) and water ([B]0 = 0.235 M) with isopentyl butyrate as acyl donor (2 M) in tetrahydrofuran. O, competition between R and S 2-butyl butyrate ([A]0 = [B]0= 0.25 M) with lauryl alcohol (0.5 M) as acceptor in hexane. Temperature was 40 °C and C. cylindracea was used.
general property of the lipase-catalyzed reactions in organic solvents. The competitive acceptor factors a A were also measured using the same method. The results are shown in Table II for three different donors, three couples of acceptors a n d two lipases. The competitive acceptor factor otA was i n d e p e n d e n t of the ester (AcZ) used as d o n o r and of the lipase tested. F o r instance, a A was 2.2 for the competition between geraniol a n d nerol i n d e p e n d e n t l y of the d o n o r (tributyrin, isopentyl butyrate a n d butyric acid). The same conclusion can be drawn for the
TABLE I ACYL DONOR COMPETITIVE FACTORS otD IN LIPASE-CATALYZED REACTIONS IN ORGANIC MEDIA The reaction conditions were: 40°C, acyl donors and acceptors 0.5 M in hexane. Crude commercial lipase preparations were used at a concentration of 1 to 6% (w/v). The activity of the lipases on tributyrin was 14000 I.U./g for C. cylindracea (lipase My, Meito Sangyo) and 120000 I.U./g for M. miehi (Novo); for definition of units see Ref. 22. Esters and alcohols were assayed by GLC using silica fused capillary columns SE52 or DB5, 25 m. Acids were assayed by titration and water by the Karl Fischer method. Acyl donor (AcX, AcY) Acceptor (Z): Cyclohexyl butyrate/isopentyl butyrate Butyric acid/butyl butyrate Butyl butyrate/isopentyl butyrate Tributyrin/butylbutyrate
M. Mielhi lipase
C. ~ylindracea lipase lauryl alcohol
(-)-menthol
geraniol
lauryl alcohol
3.64_+0.06 3.19_+0.08 2.66_+0.06 20.09_+2.7
3.67+0.07 3.14_+0.01 2.57-+0.06 -
3.02-+0.02 1.32+0.01 6.1 +_1.6
2.88+0.10 1.41+_0.01 6.1 +_0.4
geraniol
3.66+0.11 3.14-+0.04 2.58+0.10 20.09_+3.9
119 T A B L E II A C Y L A C C E P T O R COMPETITIVE F A C T O R a ^ IN LIPASE-CATALYZED R E A C T I O N S IN O R G A N I C M E D I A Experimental conditions were the same as in Table I. Geraniol and nerol were 0.25 M in pure tributyrin or isopentyl butyrate and in 1 M butyric acid in heptane. Butanol was 0.25 M, water 0.235 M and esters 2 M in tetrahydrofuran. The lipase concentrations were 0.25% (w/v).
C. cylindracea lipase
Donor (AcZ)
Acceptor (X, Y): geraniol/nerol Tributyrin Isopentyl butyrate Butyric acid
2.25 _+0.04 2.20 _+0.04 2.20 _+0.08
competition between butanol and water and for both lipases tested. From the results in Tables I and II it can be concluded that esters (or acids) and alcohols (or water) bind to different enzyme species which are, most probably, the free and acyl enzymes (E and AcE in Fig. 1). In organic solvent, the formation and the solvolysis of the acyl enzyme occur in two independent steps. This result for C. cylindracea and M. miehi is in agreement with a previous report for pancreatic lipase [17] in organic solvent. The use of competitive factors a D is a simple and easy way of describing the kinetics of the lipasecatalyzed reaction with two substrates. This is not the case in a biphasic system, where the kinetics are usually much more complex [3]. The domain of application of the model was further extended to comparison with equilibrium constants. Application of the Haldane [19-21]
M. mielhi lipase butanol/water
geraniol/nerol
butanol/water
3.86 _+0.03 3.58 _+0.02 -
1.93 _+0.02 1.57 _+0.02 1.99 _+0.03
1.08 _+0.01 1.08 + 0.01 -
equation to the following equilibrium: AcX+Y~
AcY+X
(5)
allows the derivatization of Eqn. 6 which correlates the equilibrium constant with the competitive factors:
Keq = aD/a
A =
VAcX/KAcX
V¥/Kv
VAcy/KAc Y "
Vx/K x
The competitive factors otD and a A were measured for different donors (AcX and AcY) with lauryl alcohol as acceptor (Z) and for different acceptors (X and Y) with tributyrin as donor. The results are shown in Table III. The equilibrium constant (Keq) was estimated by measuring the substrate and product concentrations (AcX, Y, AcY and X) at equilibrium and compared to the ratio OtD/OtA. The two values were identical within
T A B L E III R E L A T I O N S H I P BETWEEN COMPETITIVE F A C T O R S (a o A N D AA) A N D T H E E Q U I L I B R I U M C O N S T A N T OF T H E R E A C T I O N AcX + Y ~ AcY + X F O R C. C YLINDRA CEA LIPASE For experimental conditions see Table I. The enantiomeric excess was determined by G L C on the alcohols according to the method of Konig [23]. For 3,3-dimethyl-2-butanol derivatization was done with (R)-( + )-phenylethylisocyanate. (X, Y):
Acyl donor
Acyl acceptor
butyryl esters (AcX, AcY), lauryl alcohol (Z): tributyrin(AcZ), alcohols (X, Y): 0tD OtA Butyl/isopentyl Cyclohexyl/isopentyl Cyclohexyl/butyl ( R/S)-2-Butyl
(R/S)-3,3-Dimethyl-2-butyl
0tD/a A
Keq
2.58 _+0.10 3.66 +_0.11 1.39 + 0.01
3.91 _+0.07 1.13 _+0.07 0.30 +_0.29
0.66 3.2 4.6
0.56 3.1 4.9
1.18 _+0.01 26.0 _+3.5
1.13 _+0.01 26.9 _+1.4
1.04 0.97
120
experimental errors, for the three different equilibria tested. This is a strong confirmation of the validity of the kinetic model in organic media. An important application of the model is the classification of acyl donors (or acceptors) according to their competitive factors. For instance, from the results in Tables I and II with the C. cy6ndracea lipase, taking butyl butyrate as reference, the following scale of acyl donor can be deduced: tributyrin (20.9)> butyric acid (3.19) > cyclohexy|butyrate (1.39)>isopentyl butyrate (1/2.66). Then one can easily compare the reactivity of different acyl donors in transesterification reactions. If all the acyl donors are present at the same time in the reaction mixture the model allows the prediction of reaction rates for each of them. This will be very useful when natural mixtures of esters are used in transesterification reactions. For acyl acceptors, the same approach gives the following scale in the case of butyryl transfer with c. cylindracea lipase: butanol (3.7) > cyclohexanol (1.1) > water (1.0) > isopentanol (0.95). Some examples of kinetic separations are now given using C. cylindracea lipase. In the case of alcohol resolution, the two enantiomers are considered as competitors towards the enzyme active site [18]. According to the microreversibility principle one should expect that a D = e~A. The results are given in Table III for two alcohols. 2-Butanol could not be easily resolved ( a = 1.15) under our conditions. A different result with this alcohol has been reported by others [10,11]. In contrast the other alcohol, (R/S)-3,3dimethyl-2-butanol could be easily resolved ( a = 26.5). The R enantiomer was prepared in large amounts (50 g) in two steps. In the first step the butyryl ester was synthesized and then ester solvolysis was done in the experimental conditions given in Table III. The conversion yield was 40% for both reactions and the enantiomeric excesses were respectively 88 and more than 98%. Acid separations were demonstrated using butyric-lauric competition. When geraniol was used as acceptor the selectivity was higher for triacylglycerides (a D = 32 _+ 3) than for free acids (ai~ = 8.9 _+ 0.9). Thus the separation of butyric and lauric acid can be easily done using tributyrin and trilaurin. In these competitive reactions, two
different acyl enzymes are concerned, in contrast with alcohol separations, where the same acyl enzyme is involved. Both the leaving and the acceptor groups played a role in the reaction selectivity. This approach can be easily extended to acid resolutions for which a systematic search for better acceptor and leaving groups has to be carried out. References 1 Densnuelle, P. (1972) in The Enzymes 3rd Edn. (Boyer, P.D., ed.), Vol. 7, pp. 575-616, Academic Press, New York 2 Brockerhoff, H and Jensen R.G. (1974) Lipolytic Enzymes, pp. 34-90, Academic Press, New York 3 Verger, R. and De Haas, G.H. (1976) Annu. Rev. Biophys. Bioeng. 5, 77-117 4 Verger, R. (1980) Methods Enzymol. 64, 340-392 5 Bell, G., Blain, J.A., Patterson, J.D.E. and Todd, R. (1976) J. Appl. Chem. Biol. 26, 582-583 6 Bell, G., Blain, J.A., Patterson, J.D.E.~ Shaw, C.E.L. and Todd, R. (1978) FEMS Microbiol. Lett. 3, 223-225 7 Patterson, J.D.E., Blain, J.A., Shaw, C.E.L., Todd, R. and Bell, G. (1979) Biotechnol. Lett. 1,211-216 8 Seo, C.W., Yamada, Y. and Okada, H. (1982) Agric. Biol. Chem. 46, 405-409 9 Zaks,A. and Klibanov, A.M. (1984) Science 224, 1249-1251 10 Cambou, B. and Klibanov, A.M. (1984) J. Am. Chem. Soc. 106, 2687-2692 11 Cambou, B. and Klibanov, A.M. (1984) Biotechnol. Bioeng. 26, 1449-1454 12 Kirchner, G., Scollar, M.P. and Klibanov, A.M. (1985) J. Am. Chem. Soc. 107, 7072-7076. 13 Koshiro, S., Sonomoto, K., Tanaka, A. and Fukui, S.J. (1985) Biotechnology 2, 47-57 14 Langrand, G., Secchi, M., Buono, G., Baratti, J. and Triantaphylides, C. (1985) Tetrahedron Lett. 26, 1857-1860 15 Marlot, C., Langrand, G., Triantaphylides, C. and Baratti, J. (1985) Biotechnol. Lett 7,647-650 16 Langrand, G., Baratti, J., Buono, G. and Triantaphylides, C. (1986) Tetrahedron Lett. 27, 29-32 17 Zaks, A. and Klibanov, A.M. (1985) Proc. Natl. Acad. Sci. USA 82, 3192-3196 18 Chen, C.S., Fujumoto, Y., Girkaudas, G. and Sih, C.J. (1982) J. Am. Chem. Soc. 104, 7294-7299 19 Haldane, J.B.S. (1965) in Enzymes, p. 80, MIT Press Paperback 20 Laidler, K.J. and Bunding, P.S. (1973) in The Chemical Kinetics of Enzyme Action, 2nd Edn., pp. 173-175, Clarendon Press, Oxford 21 Segel, S.H. (1975) in Enzyme Kinetics, pp 34-37, Wiley Interscience, New York 22 Lavayre, J. and Baratti, J. (1982) Biotechnol. Bioeng. 24, 1007-1013 23 Konig, W.A., Francke, W. and Benecke, I. (1982) J. Chromatogr. 239, 227-231
117
BBA Report
BBA30159
Lipase-catalyzed reactions in organic media: c o m p e t i t i o n and applications H . D e l e u z e a, G . L a n g r a n d a, H . M i l l e t a, J. B a r a t t i b, G . B u o n o a n d C. T r i a n t a p h y l i d e s a
a
" Ecole Sup~rieure de Chimie de Marseille, Centre de Saint-J~rgmw, rue Henri Poincar~, 13397 Marseille C~dex 13 (France) and h Laboratoire de Chimie Bactdrienne du C.N.R.S., B.P. 71, 13277 Marseille Cddex 9 (France) (Received 1 July 1986)
Key words: Lipase: Transesterification; Organic solvent; Enzyme kinetics: Reaction selectivity
Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.
Ester hydrolysis of emulsified substrates by lipases (EC 3.1.1.3) obeys complex kinetics [1-4]. In organic media ester synthesis and transesterification have been described [5-16] with an insoluble enzymatic preparation. As reported by others [17], and observed by us in this study, the reaction rate oA, in organic solvent, increased with the substrate concentration [A] according to a Michaelis-Menten equation: t'A
VA"[A] K A + [A]
(1)
Furthermore a ping-pong mechanism has been proposed including the formation of an acyl enzyme intermediate [17]. When two substrates (A and B) are present at the same time in the reaction mixture, a competitive factor a is then defined by the ratio of the catalytic powers according to Eq. 2
Correspondence: Dr. C. Triantaphylides, Ecole Sup~rieure de Chimie de Marseilles, Centre de Saint-J~rbme, rue Henri Poincar& 13397 Marseille C~dex 13, France.
[20]: VA/KA a= - -
VB/K.
(2)
This formalism was also used by Chen et al. [18] for two enantiomers of the same substrate. The competitive factor can be calculated from one of the following equations [20]: ~A [A] ,,-~ ~N
(3)
log ([A]/[A]o) = a log ([B]/[B]o)
(4)
=
In this paper we describe the use of the preceding model for reactions in organic media using different acyl donors and acceptors. For instance, when two different esters (AcX and AcY) of the same acid are used as acyl donor a competitive donor factor a D can be calculated (Fig. 1, full arrows). When two acceptors (X and Y) are used a competitive acceptor factor aA can be calculated (Fig. 1, dotted arrows). The competitive factors
0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118 AcX
+ E
~.~X Z AcE
AcY
+ E
....
L
AcZ
+ E
1
J'~Y
Fig. [. Lipase competition reactions. AcX, AcY and AcZ = acyl donors (esters or acids). E = lipase, X, Y and Z, acyl acceptors (alcohols or water); AcE = acyl enzyme; full arrows, competition between two acyl donors (AcX and AcY); dotted arrows, competition between two acyl acceptors (X and Y).
were estimated from the variations of substrate c o n c e n t r a t i o n s (A a n d B) with time plotted according to Eq. 4. Fig. 2 shows a typical result for the competition between b u t a n o l (A) a n d water (B) with isopentyl b u t y r a t e as acyl donor. I n the same figure the competition between the R a n d S e n a n t i o m e r s of 2 - b u t y l b u t y r a t e is also shown. Table I shows the results for the competitive d o n o r factors a D for four different couples of acyl donor, three different acceptors a n d two lipases. It is seen that the competitive d o n o r factor a D was i n d e p e n d e n t of the alcohol used as acceptor when a given mixture of acyl d o n o r was used. F o r instance a D was 3.6 for the competition between cyclohexyl butyrate a n d isopentyl b u t y r a t e indep e n d e n t l y of the acceptor (geraniol, lauryl alcohol or ( - ) - m e n t h o l ) . This conclusion was true for every couple of acyl d o n o r tested (AcX a n d AcY) even when butyric acid or t r i b u t y r i n were used. The lipases from Candida Cylindracea (recently r e n a m e d Candida rugosa) a n d Mucor miehi showed the same property a n d identical results were observed using different substrate concentrations. Thus it can be concluded that this is a
i
0.5
O(
'
t
,
'
J
0.5 LOG
i
,
i
i
i
1
~--
(Bo/B)
Fig. 2. Estimation of competitive factor. A, competition between butanol ([A]0= 0.25 M) and water ([B]0 = 0.235 M) with isopentyl butyrate as acyl donor (2 M) in tetrahydrofuran. O, competition between R and S 2-butyl butyrate ([A]0 = [B]0= 0.25 M) with lauryl alcohol (0.5 M) as acceptor in hexane. Temperature was 40 °C and C. cylindracea was used.
general property of the lipase-catalyzed reactions in organic solvents. The competitive acceptor factors a A were also measured using the same method. The results are shown in Table II for three different donors, three couples of acceptors a n d two lipases. The competitive acceptor factor otA was i n d e p e n d e n t of the ester (AcZ) used as d o n o r and of the lipase tested. F o r instance, a A was 2.2 for the competition between geraniol a n d nerol i n d e p e n d e n t l y of the d o n o r (tributyrin, isopentyl butyrate a n d butyric acid). The same conclusion can be drawn for the
TABLE I ACYL DONOR COMPETITIVE FACTORS otD IN LIPASE-CATALYZED REACTIONS IN ORGANIC MEDIA The reaction conditions were: 40°C, acyl donors and acceptors 0.5 M in hexane. Crude commercial lipase preparations were used at a concentration of 1 to 6% (w/v). The activity of the lipases on tributyrin was 14000 I.U./g for C. cylindracea (lipase My, Meito Sangyo) and 120000 I.U./g for M. miehi (Novo); for definition of units see Ref. 22. Esters and alcohols were assayed by GLC using silica fused capillary columns SE52 or DB5, 25 m. Acids were assayed by titration and water by the Karl Fischer method. Acyl donor (AcX, AcY) Acceptor (Z): Cyclohexyl butyrate/isopentyl butyrate Butyric acid/butyl butyrate Butyl butyrate/isopentyl butyrate Tributyrin/butylbutyrate
M. Mielhi lipase
C. ~ylindracea lipase lauryl alcohol
(-)-menthol
geraniol
lauryl alcohol
3.64_+0.06 3.19_+0.08 2.66_+0.06 20.09_+2.7
3.67+0.07 3.14_+0.01 2.57-+0.06 -
3.02-+0.02 1.32+0.01 6.1 +_1.6
2.88+0.10 1.41+_0.01 6.1 +_0.4
geraniol
3.66+0.11 3.14-+0.04 2.58+0.10 20.09_+3.9
119 T A B L E II A C Y L A C C E P T O R COMPETITIVE F A C T O R a ^ IN LIPASE-CATALYZED R E A C T I O N S IN O R G A N I C M E D I A Experimental conditions were the same as in Table I. Geraniol and nerol were 0.25 M in pure tributyrin or isopentyl butyrate and in 1 M butyric acid in heptane. Butanol was 0.25 M, water 0.235 M and esters 2 M in tetrahydrofuran. The lipase concentrations were 0.25% (w/v).
C. cylindracea lipase
Donor (AcZ)
Acceptor (X, Y): geraniol/nerol Tributyrin Isopentyl butyrate Butyric acid
2.25 _+0.04 2.20 _+0.04 2.20 _+0.08
competition between butanol and water and for both lipases tested. From the results in Tables I and II it can be concluded that esters (or acids) and alcohols (or water) bind to different enzyme species which are, most probably, the free and acyl enzymes (E and AcE in Fig. 1). In organic solvent, the formation and the solvolysis of the acyl enzyme occur in two independent steps. This result for C. cylindracea and M. miehi is in agreement with a previous report for pancreatic lipase [17] in organic solvent. The use of competitive factors a D is a simple and easy way of describing the kinetics of the lipasecatalyzed reaction with two substrates. This is not the case in a biphasic system, where the kinetics are usually much more complex [3]. The domain of application of the model was further extended to comparison with equilibrium constants. Application of the Haldane [19-21]
M. mielhi lipase butanol/water
geraniol/nerol
butanol/water
3.86 _+0.03 3.58 _+0.02 -
1.93 _+0.02 1.57 _+0.02 1.99 _+0.03
1.08 _+0.01 1.08 + 0.01 -
equation to the following equilibrium: AcX+Y~
AcY+X
(5)
allows the derivatization of Eqn. 6 which correlates the equilibrium constant with the competitive factors:
Keq = aD/a
A =
VAcX/KAcX
V¥/Kv
VAcy/KAc Y "
Vx/K x
The competitive factors otD and a A were measured for different donors (AcX and AcY) with lauryl alcohol as acceptor (Z) and for different acceptors (X and Y) with tributyrin as donor. The results are shown in Table III. The equilibrium constant (Keq) was estimated by measuring the substrate and product concentrations (AcX, Y, AcY and X) at equilibrium and compared to the ratio OtD/OtA. The two values were identical within
T A B L E III R E L A T I O N S H I P BETWEEN COMPETITIVE F A C T O R S (a o A N D AA) A N D T H E E Q U I L I B R I U M C O N S T A N T OF T H E R E A C T I O N AcX + Y ~ AcY + X F O R C. C YLINDRA CEA LIPASE For experimental conditions see Table I. The enantiomeric excess was determined by G L C on the alcohols according to the method of Konig [23]. For 3,3-dimethyl-2-butanol derivatization was done with (R)-( + )-phenylethylisocyanate. (X, Y):
Acyl donor
Acyl acceptor
butyryl esters (AcX, AcY), lauryl alcohol (Z): tributyrin(AcZ), alcohols (X, Y): 0tD OtA Butyl/isopentyl Cyclohexyl/isopentyl Cyclohexyl/butyl ( R/S)-2-Butyl
(R/S)-3,3-Dimethyl-2-butyl
0tD/a A
Keq
2.58 _+0.10 3.66 +_0.11 1.39 + 0.01
3.91 _+0.07 1.13 _+0.07 0.30 +_0.29
0.66 3.2 4.6
0.56 3.1 4.9
1.18 _+0.01 26.0 _+3.5
1.13 _+0.01 26.9 _+1.4
1.04 0.97
120
experimental errors, for the three different equilibria tested. This is a strong confirmation of the validity of the kinetic model in organic media. An important application of the model is the classification of acyl donors (or acceptors) according to their competitive factors. For instance, from the results in Tables I and II with the C. cy6ndracea lipase, taking butyl butyrate as reference, the following scale of acyl donor can be deduced: tributyrin (20.9)> butyric acid (3.19) > cyclohexy|butyrate (1.39)>isopentyl butyrate (1/2.66). Then one can easily compare the reactivity of different acyl donors in transesterification reactions. If all the acyl donors are present at the same time in the reaction mixture the model allows the prediction of reaction rates for each of them. This will be very useful when natural mixtures of esters are used in transesterification reactions. For acyl acceptors, the same approach gives the following scale in the case of butyryl transfer with c. cylindracea lipase: butanol (3.7) > cyclohexanol (1.1) > water (1.0) > isopentanol (0.95). Some examples of kinetic separations are now given using C. cylindracea lipase. In the case of alcohol resolution, the two enantiomers are considered as competitors towards the enzyme active site [18]. According to the microreversibility principle one should expect that a D = e~A. The results are given in Table III for two alcohols. 2-Butanol could not be easily resolved ( a = 1.15) under our conditions. A different result with this alcohol has been reported by others [10,11]. In contrast the other alcohol, (R/S)-3,3dimethyl-2-butanol could be easily resolved ( a = 26.5). The R enantiomer was prepared in large amounts (50 g) in two steps. In the first step the butyryl ester was synthesized and then ester solvolysis was done in the experimental conditions given in Table III. The conversion yield was 40% for both reactions and the enantiomeric excesses were respectively 88 and more than 98%. Acid separations were demonstrated using butyric-lauric competition. When geraniol was used as acceptor the selectivity was higher for triacylglycerides (a D = 32 _+ 3) than for free acids (ai~ = 8.9 _+ 0.9). Thus the separation of butyric and lauric acid can be easily done using tributyrin and trilaurin. In these competitive reactions, two
different acyl enzymes are concerned, in contrast with alcohol separations, where the same acyl enzyme is involved. Both the leaving and the acceptor groups played a role in the reaction selectivity. This approach can be easily extended to acid resolutions for which a systematic search for better acceptor and leaving groups has to be carried out. References 1 Densnuelle, P. (1972) in The Enzymes 3rd Edn. (Boyer, P.D., ed.), Vol. 7, pp. 575-616, Academic Press, New York 2 Brockerhoff, H and Jensen R.G. (1974) Lipolytic Enzymes, pp. 34-90, Academic Press, New York 3 Verger, R. and De Haas, G.H. (1976) Annu. Rev. Biophys. Bioeng. 5, 77-117 4 Verger, R. (1980) Methods Enzymol. 64, 340-392 5 Bell, G., Blain, J.A., Patterson, J.D.E. and Todd, R. (1976) J. Appl. Chem. Biol. 26, 582-583 6 Bell, G., Blain, J.A., Patterson, J.D.E.~ Shaw, C.E.L. and Todd, R. (1978) FEMS Microbiol. Lett. 3, 223-225 7 Patterson, J.D.E., Blain, J.A., Shaw, C.E.L., Todd, R. and Bell, G. (1979) Biotechnol. Lett. 1,211-216 8 Seo, C.W., Yamada, Y. and Okada, H. (1982) Agric. Biol. Chem. 46, 405-409 9 Zaks,A. and Klibanov, A.M. (1984) Science 224, 1249-1251 10 Cambou, B. and Klibanov, A.M. (1984) J. Am. Chem. Soc. 106, 2687-2692 11 Cambou, B. and Klibanov, A.M. (1984) Biotechnol. Bioeng. 26, 1449-1454 12 Kirchner, G., Scollar, M.P. and Klibanov, A.M. (1985) J. Am. Chem. Soc. 107, 7072-7076. 13 Koshiro, S., Sonomoto, K., Tanaka, A. and Fukui, S.J. (1985) Biotechnology 2, 47-57 14 Langrand, G., Secchi, M., Buono, G., Baratti, J. and Triantaphylides, C. (1985) Tetrahedron Lett. 26, 1857-1860 15 Marlot, C., Langrand, G., Triantaphylides, C. and Baratti, J. (1985) Biotechnol. Lett 7,647-650 16 Langrand, G., Baratti, J., Buono, G. and Triantaphylides, C. (1986) Tetrahedron Lett. 27, 29-32 17 Zaks, A. and Klibanov, A.M. (1985) Proc. Natl. Acad. Sci. USA 82, 3192-3196 18 Chen, C.S., Fujumoto, Y., Girkaudas, G. and Sih, C.J. (1982) J. Am. Chem. Soc. 104, 7294-7299 19 Haldane, J.B.S. (1965) in Enzymes, p. 80, MIT Press Paperback 20 Laidler, K.J. and Bunding, P.S. (1973) in The Chemical Kinetics of Enzyme Action, 2nd Edn., pp. 173-175, Clarendon Press, Oxford 21 Segel, S.H. (1975) in Enzyme Kinetics, pp 34-37, Wiley Interscience, New York 22 Lavayre, J. and Baratti, J. (1982) Biotechnol. Bioeng. 24, 1007-1013 23 Konig, W.A., Francke, W. and Benecke, I. (1982) J. Chromatogr. 239, 227-231