Ai

XD

a

ARRA

KERRL

1

ocas

tbiitoti[ovba−i

o

0d

Enzyme and Microbial Technology 48 (2011) 454–457

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

novel control of enzymatic enantioselectivity through the racemic temperaturenfluenced by reaction media

in Jin, Bokai Liu, Zhong Ni, Qi Wu, Xianfu Lin ∗

epartment of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 1 December 2010eceived in revised form 27 January 2011ccepted 31 January 2011

a b s t r a c t

The influence of reaction media on the racemic temperature (Tr) in the lipase-catalyzed resolution ofketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantios-electivity and the prediction of the increasing tendency was developed based on the Tr influenced by

◦

eywords:nzymatic enantioselectivityacemic temperatureeaction mediumipase

reaction media. The Tr for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 C inaqueous and S-ketoprofen was obtained predominantly at 40 ◦C. However, CRL showed R-selectivity at40 ◦C in diisopropyl ether because the Tr was changed to 56 ◦C. CRL, lipase from AYS Amano® and Mucorjavanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane,DIPE, isooctane and their mixed media with water. The effects of the reaction medium on Tr could berelated to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between

lipase

the enantiomers and the

. Introduction

Since enzymes have been used as efficient catalysts for therganic synthesis, much attention has been drawn towards theontrol of the enzymatic enantioselectivity. The control is gener-lly related to the reaction media and temperature due to theirimplicity and reliability [1–4].

The study on the control of enzymatic enantioselectivityhrough varying the temperature as a thermodynamic method haseen interesting at present [1,5]. The racemic temperature (Tr)

s an especial temperature at which enzymes show no discrim-nation between the enantiomers [6–8]. Tr was first observed inhe reaction of 2-butanol in aqueous solution catalyzed by sec-ndary alcohol dehydrogenase [9–11]. It has been applied to exhibithe changes of the enzymatic enantioselectivity and predict thencreasing/decreasing tendency as a key parameter since then12–16]. The enantioselectivity of the lipase-catalyzed resolutionf 3-phenyl-2H-azirine-2-methanol can be increased up to 99 (Ealue) by lowering temperature, as the reaction temperature iselow the Tr (425 ◦C) [16]. In an acyl-transfer reaction catalyzed byCal-B mutant, the reaction temperature is higher than Tr (about

30 ◦C) so that a high enantioselectivity can be obtained through

ncreasing the temperature [17].The Tr has been calculated in some researches for the analysis

f the enzymatic enantioselectivity and some results have shown

∗ Corresponding author. Tel.: +86 571 87953001; fax: +86 571 87952615.E-mail address: llc123@zju.edu.cn (X. Lin).

141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2011.01.009

.© 2011 Elsevier Inc. All rights reserved.

that different enzymes and mutants may have different Tr for dif-ferent substrates [14,16,18]. For example, compared with the wildenzyme, the Tr of a mutant of secondary alcohol dehydrogenasechanged from 111 ◦C (WT) to −66 ◦C [19]. However, less atten-tion has been paid to the influence of reaction media on the Tr inenzymatic reactions and thus a further control on the enzymaticenantioselectivity can be achieved by the change of Tr.

Since organic solvents were introduced into the enzymatic reac-tion, it has been an effective method to control the properties of theenzymatic reactions, such as the conversion and enantioselectivity[20–23]. Although the influence of the nature of reaction media onenzyme enantioselectivity has been investigated and interpreted interms of various solvent parameters, such as log P, dielectric con-stant and viscosity, no clear consensus has yet emerged on a singleparameter applied to quantitatively describe the solvent influenceon the enantioselectivity of enzymatic reactions [21,24,25]. Theinvestigation of the influence of reaction media on the Tr, whichplayed an important role in the change of the enzymatic enantios-electivity, may bring us some new insights into the contributionsfrom organic media to enzymatic enantioselectivity. Furthermore,the possible change of Tr caused by the different reaction mediacan also be considered as an effective approach to the control of theenzymatic enantioselectivity and the expansion of the applicationsof enzymatic reactions.

In this paper, the T of a lipase-catalyzed model reaction, the res-

r

olution of ketoprofen vinyl ester (KVE) in different reaction media,was investigated. Three lipases, Candida rugosa lipase, lipase fromAYS Amano® and Mucor javanicus lipase were used as catalysts inthe resolution, where both reaction media and temperature were

bial Technology 48 (2011) 454–457 455

cita

2

2

(Slam

2

Rvcttup(

2

stw(hd

2

tE−cTMca

3

epCa

F((

R COOCH=CH2

CH3

R = m-C6H5CO-C6H4

in aqueous

Tr1= 29 oC

in DIPE

Tr2= 56 oC

< 29 oC

R COOH

CH3

> 29 oC R COOH

CH3

< 56 oC

R COOH

CH3

> 56 oC R COOH

CH3

X. Jin et al. / Enzyme and Micro

hanged. The calculation of Tr showed that it can be remarkablynfluenced by the reaction media. The change of the Tr predictedhe different enantioselectivity increasing tendencies and offeredrational control of the enzymatic enantioselectivity.

. Materials and methods

.1. Materials

Ketoprofen was purchased from Zhejiang Jiuzhou Pharmaceutical Co., Ltd.China). Lipase from C. rugosa (CRL) and M. javanicus (MJL) was purchased fromigma–Aldrich. Lipase AYS Amano® (L-AYS) was donated by Amano Enzyme Inc. Allipases were used in the resolution without any pretreatment. All other chemicalsnd reagents were of analytical grade. All the organic solvents were dried over 4 Aolecular sieves for 24 h.

.2. The synthesis of ketoprofen vinyl ester

The synthesis of ketoprofen vinyl ester was mentioned in our previous work [26].acemic ketoprofen (3.2 g) and mercuric acetate (0.3 g) were dissolved in 30 ml ofinyl acetate. After stirring the mixture for 30 min at room temperature, 0.2 ml ofoncentrated sulfuric acid was added and the solution was refluxed for 3 h. Thenhe mixture was cooled to room temperature, and sodium acetate (1.0 g) was addedo quench the catalyst. The solution was filtered and concentrated. The crude prod-cts were purified by silica gel column chromatography with the mobile phase ofetroleum ether/ethyl acetate (30/1, v/v). The product was identified by IR, 1H NMRsee Supplementary Material).

.3. The general resolution of KVE catalyzed by lipase

The resolution of 10 mg KVE was catalyzed by 10 mg CRL, MJL, L-AYS in 1 mlolvent and shaken at 200 r/min. The reaction was terminated by 5 ml diethyl ethero extract the KVE and ketoprofen for 1 h. The enantiomers of KVE and ketoprofenere analyzed by HPLC (Shimadzu LC-20A series) with a chiral column Chiral AD-H

250 mm × 4.6 mm, Regis, USA) at 250 nm (UV detector). The mobile phase was n-exane/ethanol/acetic acid: 85/15/0.1 (v/v/v) with a flow rate of 0.5 ml/min. All theata of the resolution are shown in Supplementary Material.

.4. The calculation of the racemic temperature

The E value is related to the difference in the free energy of activation (��G‡) ofhe paths of the two enantiomers, according to the equation: ��G‡ = −RT ln E. Theyring activation parameters (��H‡ and ��S‡) were also calculated by plotting theRT ln E versus T, according to the equation: ��G‡ = ��H‡ − T��S‡ . The Tr could be

alculated from the equations, when ��G‡ = −RT ln E = 0 and Tr = ��H‡/��S‡ [6].he thermodynamic parameters were calculated and are shown in Supplementaryaterial. All the determinations were repeated for two or three times and the linear

orrelation coefficients are shown in Supplementary Material to reflect the randomnd systematic errors of the results.

. Results and discussions

We observed a temperature-dependent reversal of the enantios-lectivity of C. rugosa lipase (CRL) in the resolution of KVE in sodiumhosphate buffer (0.1 M, pH 7.0) (Fig. 1, Scheme 1). The Tr was 29 ◦C.RL showed R enantioselectivity at T < Tr under enthalpic controlnd S enantioselectivity at T > Tr under entropic control. Then three

330320310300290280

-7500

-5000

-2500

0

2500

-RTl

nE

T/K

Tr = 29

oC

Tr = 56

oC

Tr = 54

oC

Tr could not exist

Tr = 68

oC

Tr = 33

oC

S rich

R rich

ig. 1. The −RT ln E versus T for the hydrolytic resolution of KVE catalyzed by CRL in�) aqueous, (�) DIPE, (�) isooctane, (�) aqueous/dioxane, (©) aqueous/DIPE, and�) aqueous/isooctane.

Scheme 1. The hydrolytic resolution of KVE catalyzed by CRL and the racemictemperature-induced reversal of the enzymatic enantioselectivity from S- (in aque-ous) to R-enantiomers (in DIPE) at 29 < T < 56 ◦C.

organic solvents, dioxane, diisopropyl ether (DIPE) and isooctane,were considered to modulate the Tr. CRL could not show activity inthe strongly hydrophilic dioxane (log P = −1.1) [27] and the Tr couldnot be obtained (Fig. 1). In hydrophobic DIPE and isooctane, the Tr

was changed to 56 ◦C and 54 ◦C, respectively (Fig. 1). Because theTr was changed from 29 ◦C to 56 ◦C in DIPE, R-ketoprofen could beobtained predominantly at 40 ◦C, while S-ketoprofen could be pro-duced preferentially in aqueous at the same temperature (Fig. 1,Scheme 1). Similarly, some samples of the enzymatic enantiose-lectivity reversal by reaction media might also be ascribed to thechange of the Tr, which had not been realized in these studies[28–31].

In aqueous/dioxane (1/1, v/v), CRL exhibited no discriminationbetween enantiomers and the E values were 1.0 at each tempera-ture (Fig. 1). The Tr could also be not obtained in aqueous/dioxane(Fig. 1). Similar results were obtained by adding DMSO and DMFinto the aqueous as a co-solvent (data were not shown). The pres-ence of the strongly hydrophilic solvents could induce a changeof the enzyme conformation for the binding enantiomers [32]. Thesimilar phenomenon was observed in the hydrolysis of methyl alkyldimethylmalonates catalyzed by pig liver esterase [33]. When 25%DMSO was added, temperature also hardly influenced the enan-tioselectivity of the hydrolysis reaction.

For aqueous/DIPE (1/1, v/v) and aqueous/isooctane (1/1, v/v),the resolution was carried out in a two-phase medium. CRL wasdissolved partially in the aqueous phase and KVE was in the organicphase. However, the two hydrophobic solvent exhibited differentinfluences on the Tr of CRL. The Tr in aqueous/DIPE was changedobviously from 29 ◦C to 68 ◦C (Fig. 1). The less polar solvent couldinfluence the loop conformation and the hydration level of CRL[34–36]. Isooctane (log P = 4.5) was much more hydrophobic thanDIPE (log P = 1.9) and it could hardly influence CRL in the aqueousphase. The structure of CRL could retain its natural state in the aque-ous phase. Thus, the Tr of CRL was slightly changed from 29 ◦C to33 ◦C in aqueous/isooctane (Fig. 1). The above results demonstratedthat the Tr of CRL was varied easily by the hydrophilicity of organicsolvents.

In the literature [12], the difference of Tr was ascribed to thedifferent optimal temperatures (Top) and thermostability for thehyperthermophilic and mesophilic lipases. The hyperthermophilic

◦

lipase with high Top at 95 C could have a conformationally rigidstate, which could lead to an increase of the Tr higher than the reac-tion temperature, compared to the mesophilic lipase (CRL) with amoderate Top (40 ◦C) and low Tr (−46 ◦C) for the enantioselectivehydrolysis of naproxen methyl ester. In this study, an increase of

4 bial Technology 48 (2011) 454–457

TewoTflba

itebwociawnfl

mphmootraa

sjTnstotataa

(I

Fi

330320310300290280

-8000

-6000

-4000

-2000

0

- RTl

nE

T (K)

Tr = 4.2

oC

Tr = -179

oC

Tinv

= 26 oC

R rich

56 X. Jin et al. / Enzyme and Micro

r was also observed due to the change of reaction media. How-ver, for example, the thermostability and Top of CRL in isooctaneere similar to those in aqueous [12,37,38]. Thus, Tr might not

nly be influenced by the lipase thermostability and the change ofop. Probably, the absence of water and the lipase conformationalexibility in DIPE and isooctane could lead to a change of the neigh-orhood of the CRL catalytic triad from the natural conformation inqueous, and consequently induced the change of the Tr [20,39,40].

In addition, ��H‡ and ��S‡ (Tr = ��H‡/��S‡, results shownn Tables S2, S6, S10 in Supplementary Material) were often usedo show the change of the reaction thermodynamic states. Thenthalpic component was dominated by the static interactionsetween the enzyme and enantiomers; the entropic componentas related to the differential solvation and the restricted motion

f the enzyme–substrate complex [18,24]. The enthalpy–entropyompensation was also observed for the CRL-catalyzed resolutionsn different media (Fig. S1). The simultaneous decrease of the ��H‡

nd ��S‡ suggested that the enzyme, substrate and their complexere all influenced by the change of the reaction media, which wasot only caused by the enzyme thermostability and conformationalexibility with temperature, as mentioned in some previous work.

According to above results, the Tr of CRL in different reactionedia could offer a simple and effective method to investigate and

redict the tendency of the enantioselectivity improvement. Theigh E value for R-enantiomer could be obtained in aqueous/DIPEedia at 10 ◦C. Compared with other reaction media, the choice

f aqueous/DIPE could be the most effective for the improvementf enzymatic enantioselectivity by lowering temperature due tohe highest Tr in aqueous/DIPE. Similarly, for the enantioselectivityeversal, the S-enantiomer could be obtained most effectively inqueous/isooctane at a high temperature according to the low Tr

nd the Eyring plots.To further investigate the relationship between the Tr and

olvents, two other lipases, lipase AYS Amano® (L-AYS) and M.avanicus lipase (MJL), were used to catalyze the resolution. Ther of L-AYS in aqueous was 57 ◦C (Fig. 2). However, L-AYS couldot catalyze the hydrolysis in hydrophilic or hydrophobic organicolvents, which indicated that L-AYS might have a low flexibilityowards organic solvents. The Tr was 64 ◦C and 61 ◦C in aque-us/DIPE and aqueous/isooctane, respectively, which was similaro that in aqueous (Fig. 2). It indicated that L-AYS could hardly beffected by hydrophobic organic solvents due to its low flexibilityowards them. The Tr could not be obtained in aqueous/dioxane

nd L-AYS did not show enantioselectivity (Fig. 2), which was ingreement with the results from CRL.

In isooctane, MJL could show a high enantioselectivityeep% > 90%, E > 20) with a moderate conversion (10% < c% < 40%).t could be attributed to the flexibility of MJL which was higher

330320310300290280

-10000

-7500

-5000

-2500

0

Tr = 57

oC

Tr = 64

oC

Tr = 61

oC

Tr could not exist

-RTl

nE

T/K

R rich

ig. 2. The −RT ln E versus T for the hydrolytic resolution of KVE catalyzed by L-AYSn (�) aqueous, (�) aqueous/dioxane, (©) aqueous/DIPE, and (�) aqueous/isooctane.

Fig. 3. The −RT ln E versus T for the hydrolytic resolution of KVE catalyzed by MJLin (�) aqueous, (�) isooctane, and (�) aqueous/isooctane.

than that of CRL and L-AYS towards hydrophobic isooctane [41].Thus, as seen from Fig. 3, the Tr of MJL was changed remarkablyfrom 4.2 ◦C in aqueous to −179 ◦C in isooctane. It might implythat the thermodynamic state of MJL in aqueous and isooctanewas different with a potential change of the conformation [24].As a result, the −RT ln E versus T was extremely changed in aque-ous/isooctane (Fig. 3). MJL could catalyze the resolution in bothaqueous phase and organic phase. Neither the linear relationshipcould be obtained, nor was the Tr observed or calculated. It demon-strated that the relationship between the enantioselectivity and thetemperature in aqueous/isooctane was different from that in aque-ous and isooctane. Two linear regions intersected at 26 ◦C, whichwas defined as the inversion temperature (Tinv) [42–44], instead ofthe Tr which represented two thermodynamic states with differ-ent substrate-lipase-solvent supramolecules in aqueous/isooctane[45]. The same phenomena were also observed in DIPE and aque-ous/DIPE (see Table S5 and Fig. S2 in the Supplementary Material).In the researches on the Tinv by Cainelli et al. [5,46], the Tinvphenomenon was ascribed to two independent solute–solventclusters that were in equilibrium at Tinv and it was proposed thatthe solute–solvent clusters could be the real reacting species inthe enzymatic asymmetric reactions. Moreover, for the enzymatichydrolysis, there were two potential steps, including binding anddeacylation. Both of them could show enantiospecificity in prin-ciple. The non-linear results of MJL-catalyzed hydrolysis could beprobably ascribed to the influence of the solvent on the relativerates and enantiospecificity of binding and deacylation steps.

4. Conclusion

The investigation of Tr in the lipase-catalyzed resolution of KVEindicated that organic solvents showed significant influence on theTr. The change of Tr in different solvents, including organic sol-vents and mixed solvents with water, could lead to the change ofthe enzymatic enantioselectivity, even a potential reversal of theenantiopreference. Hydrophilic organic solvents could induce thechange more easily than hydrophobic organic solvents. The changeof Tr might also be related to the lipase conformational flexibil-ity and the substrate solvation in different reaction media, whichcould also change their interaction and the freedom degree of theircomplex. Although the reason for the change of Tr was much lessunderstood, the investigation of Tr could offer a simple and effec-

tive method to predict the increasing tendency of the enzymaticenantioselectivity and thus guide the choice of an optimal reactionsystem with the high enantioselectivity.

bial T

A

dZY

A

t

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

X. Jin et al. / Enzyme and Micro

cknowledgements

The financial support from the National Natural Science Foun-ation of China (Nos. 20704037, 20872130, 20874086) and thehejiang Provincial Natural Science Foundation (Project no. 2009-4080198, 2010-Z4090225) is gratefully acknowledged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.enzmictec.2011.01.009.

eferences

[1] Sakai T. Low-temperature method for a dramatic improvement in enan-tioselectivity in lipase-catalyzed reactions. Tetrahedron: Asymmetry2004;15:2749–56.

[2] Berglund P. Controlling lipase enantioselectivity for organic synthesis. BiomolEng 2001;18:13–22.

[3] Turner NJ. Controlling chirality. Curr Opin Biotechnol 2003;14:401–6.[4] Fitzpartrick PA, Klibanov AM. How can the solvent affect enzyme enantioselec-

tivity. J Am Chem Soc 1991;113:3166–71.[5] Cainelli G, Galletti P, Giacomini D. Solvent effects on stereoselectivity more

than just an environment. Chem Soc Rev 2009;38:990–1001.[6] Phillips RS. Temperature modulation of the stereochemistry of enzymatic catal-

ysis: prospects for exploitation. Trends Biotechnol 1996;141:13–6.[7] Guan G, Chen JY. Racemic temperature and stereochemistry. Trends Biotechnol

1997;15:333.[8] Phillips RS. Racemic temperature and stereochemistry (Response). Trends

Biotechnol 1997;15:333–4.[9] Pham VT, Phillips RS, Ljungdahl LG. Temperature-dependent enantiospecificity

of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. JAm Chem Soc 1989;111:1935–6.

10] Pham VT, Phillips RS. Effects of substrate structure and temperature on thestereospecificity of secondary alcohol dehydrogenase from Thermoanaerobac-ter ethanolicus. J Am Chem Soc 1990;112:3629–32.

11] Phillips RS. Temperature effects on stereochemistry of enzymatic reactions.Enzyme Microb Technol 1992;14:417–9.

12] Sehgal AC, Kelly RM. Enantiomeric resolution of 2-aryl propionic esterswith hyperthermophilic and mesophilic esterases: contrasting thermodynamicmechanisms. J Am Chem Soc 2002;124:8190–1.

13] Sakai T, Kishimoto T, Tanaka Y, Ema T, Utaka M. Low-temperature method forenhancement of enantioselectivity in the lipase-catalyzed kinetic resolutionsof solketal and some chiral alcohols. Tetrahedron Lett 1998;39:7881–4.

14] Miyazawa T, Yukawa T, Koshiba T, Sakamoto H, Ueji S, Yanagihara R, et al. Reso-lution of 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylationin organic media. Tetrahedron: Asymmetry 2001;12:1595–602.

15] Miyazawa T, Kaito E, Yukawa T, Murashima T, Yamada T. Enzymatic resolutionof 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylation usingacid anhydrides as acyl donors. J Mol Catal B: Enzym 2005;37:63–7.

16] Sakai T, Kawabata I, Kishimoto T, Ema T, Utaka M. Enhancement of theenantioselectivity in lipase-catalyzed kinetic resolutions of 3-phenyl-2H-azirine-2-methanol by lowering the temperature to −40 ◦C. J Org Chem1997;62:4906–7.

17] Magnusson AO, Takwa M, Hamberg A, Hult K. An S-selective lipase was createdby rational redesign and the enantioselectivity increased with temperature.Angew Chem Int Ed 2005;44:4582–5.

18] Vallin M, Syrén P, Hult K. Mutant lipase-catalyzed kinetic resolution of bulkyphenyl alkyl sec-alcohols: a thermodynamic analysis of enantioselectivity.ChemBioChem 2010;11:411–6.

19] Tripp AE, Burdette DS, Zeikus JG, Phillips RS. Mutation of serine-39 to threoninein thermostable secondary alcohol dehydrogenase from Thermoanaerobacterethanolicus changes enantiospecificity. J Am Chem Soc 1998;120:5137–41.

20] Carrea G, Riva S. Properties and synthetic applications of enzymes in organic

solvents. Angew Chem Int Ed 2000;39:2226–54.

21] Klibanov AM. Improving enzymes by using them in organic solvents. Nature2001;409:241–6.

22] Wang PY, Chen YJ, Wu AC, Lin YS, Kao MF, Chen JR, et al. (R,S)-Azolides as novelsubstrates for lipase-catalyzed hydrolytic resolution in organic solvents. AdvSynth Catal 2009;351:2333–41.

[

echnology 48 (2011) 454–457 457

23] Ammazzalorso A, Amoroso R, Bmoni G, de Filippis B, Fantacuzzi M, GiampietroL, et al. Candida rugosa lipase-catalysed kinetic resolution of 2-substituted-aryloxyacetic esters with dimethylsulfoxide and isopropanol as additives.Chirality 2008;20:115–8.

24] Overbeeke PLA, Jongejan JA, Heijnen JJ. Solvent effect on lipase enantioselectiv-ity. Evidence for the presence of two thermodynamic states. Biotechnol Bioeng2000;70:278–90.

25] Liu Y, Wang F, Tan TW. Effects of alcohol and solvent on the performance oflipase from Candida sp in enantioselective esterification of racemic ibuprofen.J Mol Catal B: Enzym 2009;56:126–30.

26] Cai XQ, Wang N, Lin XF. The preparation of polymerizable, optically activenon-steroidal anti-inflammatory drugs derivatives by irreversible enzymaticmethods. J Mol Catal B: Enzym 2006;40:51–7.

27] The log P values were all according to;Janssen AEM, van der Padt A, van Sonsbeek HM, van’t Riet K. The effect oforganic solvents on the equilibrium position of enzymatic acylglycerol synthe-sis. Biotechnol Bioeng 1993;41:95–103;Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis inorganic solvents. Biotechnol Bioeng 1987;30:81–7.

28] Hirose Y, Kariya K, Sasaki I, Kurono Y, Ebiikef H, Achiwa K. Drasticsolvent effect on lipase-catalyzed enantioselective hydrolysis of prochiral 1,4-dihydropyridines. Tetrahedron Lett 1992;33:7157–60.

29] Tawaki S, Klibanov AM. Inversion of enzyme enantioselectivity mediated bythe solvent. J Am Chem Soc 1992;114:1882–4.

30] Kumar SS, Arora N, Bhatnagar R, Gupta R. Kinetic modulation of Trichosporonasahii MSR 54 lipase in presence of organic solvents: altered fatty acid speci-ficity and reversal of enantio selectivity during hydrolytic reactions. J Mol CatalB: Enzym 2009;59:41–6.

31] de Gonzalo G, Ottolina G, Zambianchi F, Fraaije MW, Carrea G. Biocatalyticproperties of Baeyer–Villiger monooxygenases in aqueous-organic media. J MolCatal B: Enzym 2006;39:91–7.

32] Watanabe K, Yoshida T, Ueji S. The role of conformational flexibilityof enzymes in the discrimination between amino acid and ester sub-strates for the subtilisin-catalyzed reaction in organic solvents. Bioorg Chem2004;32:504–15.

33] Andrade FAC, Andrade MAC, Phillips RS. Temperature and DMSO increase theenantioselectivity of hydrolysis of methyl alkyl dimethylmalonates catalyzedby pig liver esterase. Bioorg Med Chem Lett 1991;1:373–6.

34] Cygler M, Schrag JD. Structure and conformational flexibility of Candida rugosalipase. Biochim Biophys Acta 1999;1441:205–14.

35] Grochulski P, Li Y, Schrag JD, Bouthillier F, Smith P, Harrison D, et al. Insightsinto interfacial activation from an open structure of Candida rugosa lipase. J BiolChem 1993;268:12843–7.

36] Grochulski P, Li Y, Schrag JD, Cygler M. Two conformational states of Candidarugosa lipase. Protein Sci 1994;3:82–91.

37] Bezbradica D, Mijin D, Siler-Marinkovic S, Knezevic Z. The Candida rugosa lipasecatalyzed synthesis of amyl isobutyrate in organic solvent and solvent-freesystem: a kinetic study. J Mol Catal B: Enzym 2006;38:11–6.

38] Yu HW, Chen H, Yang YY, Ching CB. Effect of salts on activity, stability andenantioselectivity of Candida rugosa lipase in isooctane. J Mol Catal B: Enzym2005;35:28–32.

39] de María PD, Sánchez-Montero JM, Sinisterra JV, Alcántara AR. UnderstandingCandida rugosa lipases: an overview. Biotechnol Adv 2006;24:180–96.

40] Tejo BA, Salleh AB, Pleiss J. Structure and dynamics of Candida rugosa lipase therole of organic solvent. J Mol Model 2004;10:358–66.

41] Broos J, Visser AJWG, Engbersen JFJ, Verboom W, van Hoek A, Reinhoudt DN.Flexibility of enzymes suspended in organic solvents probed by time-resolvedfluorescence anisotropy. Evidence that enzyme activity and enantioselectivityare directly related to enzyme flexibility. J Am Chem Soc 1995;117:12657–63.

42] Buschmann H, Scharf HD, Hoffmann N, Esser P. The isoinversion princi-ple – a general-model of chemical selectivity. Angew Chem Int Ed Engl1991;30:477–515.

43] Hale KJ, Ridd JH. A reassessment of the isoinversion relationship. J Chem SocPerkin Trans 1995;2:1601–5.

44] Cainelli G, de Matteis V, Galletti P, Giacomini D, Orioli P. Temperature andsolvent effects on enzyme stereoselectivity: inversion temperature in kineticresolutions with lipases. Chem Commun 2000:2351–2.

45] Cainelli G, Giacomini D, Galletti P. Temperature and solvent effects in facial

diastereoselectivity of nucleophilic addition: entropic and enthalpic contribu-tion. Chem Commun 1999;535:567–72.

46] Cainelli G, Galletti P, Giacomini D, Gualandi A, Quintavalla A. Chemo-and enzyme-catalyzed reactions revealing a common temperature-dependent dynamic solvent effect on enantioselectivity. Helv Chim Acta2003;86:3548–59.