Enzyme and Microbial Technology 36 (2005) 432–438

Esterification of phenylacetic and 2-phenylpropionic acids bymycelium-bound carboxylesterases

Andrea Romanoa, Raffaella Gandolfia, Francesco Molinaria,∗, Attilio Convertib,Mario Zilli b, Marco Del Borghib

a Department of Food and Microbiological Science and Technology, University of Milan, via Celoria 2, 20133 Milan, Italyb Department of Chemical and Process Engineering, University of Genoa, via Opera Pia 15, I-16145 Genoa, Italy

Received 9 December 2003; accepted 1 August 2004

Abstract

Lyophilized mycelia ofAspergillus oryzaeandRhizopus oryzaewere used in phosphate buffer to study the hydrolysis of ethyl esters ofphenylacetic and 2-phenylpropionic acids and in organic solvents to synthesize, by direct acylation, several esters of these acids, namely ethyl,p peraturesa ined at 50i cityi alcoholmt ineticso rofurane,p ith eitherb©

K

1

ihomaod(psb

g li-s suit-esisraseshesisuchs-ted to

prod-any

mar-op-

therityrom

0d

ropyl, butyl, pentyl, hexyl and isoamyl esters. Ethanol acylations with 2-phenylpropionic acid were also performed at different temnd in different solvents. In all cases, the conversion yield progressively increased with temperature and the best results were obta◦C

n n-heptane. The use ofA. oryzaeled to the preferential formation of the (S)-enantiomer of ethyl 2-phenylpropionate with enantiospecifincreasing with temperature, whereasR. oryzaebehaved oppositely. The molar conversions of these reactions also increased with the

olecular weight (MW), likely because of the higher hydrophobicity and chemical affinity of the products for the solvent.A. oryzaeprovedo be more effective and quicker thanR. oryzaein all the esterifications, probably due to a more favorable microenvironment. The kf direct ethanol acylations were also studied in various solvents, specifically dimethylsulfoxide, dioxane, acetonitrile, tetrahydyridine, diisopropyl ether, benzene, toluene,n-heptane, isooctane, and pentadecane. The starting formation rates of ethyl esters wiocatalyst were shown to progressively increase with the ratio of logP to the solvent molecular weight.2004 Elsevier Inc. All rights reserved.

eywords: Esterification; Phenylacetic acid; 2-Phenylpropionic acid; Organic solvent; Carboxylesterase; Kinetics

. Introduction

Selective conversions of natural or synthetic substratesnto useful products using whole cells or isolated enzymesave been gaining an increasing importance among the meth-ds for the production of organic substances[1]. The enzy-atic activity is often characterized by high chemo-, regio-,nd stereoselectivity, which are very useful for the synthesisf fine chemicals. The success of a biotransformation at in-ustrial scale depends upon a series of factors, among whicha) availability and low cost of the biocatalyst, (b) efficientroduct recovery processes, (c) competitive costs with re-pect to conventional methods, (d) environmental compati-ility, and (e) non-pathogenicity of the biological systems.

∗ Corresponding author. Tel.: +39 02 50316695; fax: +39 02 50316694.E-mail address:Francesco.Molinari@unimi.it (F. Molinari).

Esterifications can be performed enzymatically usinpases or esterases in organic solvents, under conditionable to address their catalytic activity towards the synth[2]. The production of esters catalyzed by lipases/estein organic solvent is nowadays a rather common synttechnique, because of the possibility of immobilizing senzymes to improve their stability[3]. Interesterifications uing more or less activated esters as acylants are exploishift the reaction equilibrium towards the ester formation[4].However, the use of synthetic substrates prevents theuct to be regarded as “natural”; therefore, the use of mnatural carboxylic acids and alcohols available on theket makes the direct esterification the most interestingtion.

As far as the choice of the solvent is concerned,esterification capacity is usually favored in low-polasolvents[5–8], although some enzymes, i.e., lipases f

141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2004.08.042

A. Romano et al. / Enzyme and Microbial Technology 36 (2005) 432–438 433

Candida antarctica, exhibited good activity even in po-lar solvents, among which acetonitrile ortert-butanol[9].

The specific acylations catalyzed by hydrolases in organicsolvent are numerous and some of them are also used inthe pharmaceutical industry, such as the synthesis of the 7-butanoyl-castanospermine (effective inhibitor of HIV virus)and the kinetic resolution of the arylglycidic ester precur-sor of Diltiazem[10,11]. The 2-arylpropionic acids, an im-portant class of anti-inflammatory non-steroidic drugs, showtheir pharmacological activity mainly in the (S)-enantiomer.Although most of them are still used in the form of racemicmixture[12], various methods are employed to obtain selec-tively the target enantiomer, among which asymmetric syn-thesis[13,14], chromatographic separation, diastereomericcrystallization[15,16]and chemical or enzymatic kinetic res-olution [17].

A direct enzymatic method to obtain the resolution ofacid racemic mixtures is the enantioselective esterification.Enzymatic esterifications of arylpropionic acids are aboveall obtained by interesterification as for 2-phenylpropionicacid [9], Naxopren and Ibuprofen[18], Flurbiprofen[19],and Suprofen[20]. Conversely, since the commercial en-zymes are unsuitable to carry out the direct esterificationowing to thermodynamic constraints, only few examples arer ylica ields[ inb hib-i wm on isv rop-e extra-c tablem byu

ellsca Fur-t ablet n ofe ertedb Li-p in thecm -t aci-d res iumo ylb le

R ifi-c or-g get

information about the mechanisms responsible for chemose-lectivity and stereoselectivity of the lipase action.

2. Materials and methods

2.1. Biocatalyst preparation and biotransformationconditions

A. oryzaeMIM (Microbiologia Industriale, Milano) andR. oryzaeCBS 112.07 (Centraal Bureau voor Schimmelcul-tures, Baarn, The Netherlands) were used throughout thisstudy and routinely maintained on malt extract (8 g l−1, agar15 g l−1, pH 5.5). The microorganisms were cultured in500 ml-Erlenmeyer flasks containing 100 ml of the medium[Difco yeast extract 1 g l−1, (NH4)2SO4 5 g l−1, K2HPO41 g l−1, MgSO4·7H2O 0.2 g l−1, pH 5.8] supplemented withTween 80 (0.5%) and incubated for 48 h at 28◦C on a recipro-cal shaker (100 rpm). Suspensions of spores (1.6× 104) wereused as inoculum. The microorganisms were also culturedin a 10 l-stirred tank reactor containing 2 l of the mediumat 28◦C, 200 rpm and aeration of 1 vvm. Mycelia grownfor 48 h in submerged cultures were harvested by filtrationat 4◦C, washed with phosphate buffer (pH 7.0, 0.1 M) andlyophilized. Ester synthesis was carried out in 10 ml-screwc n or-g acid.A iumc -t s.

2

0o of aC nyl-1 singa ilan,I tor;t( 0%1 ly).T tersw tiono erifi-c dw ro-c elyd stersu 086( ml 2-p 90f s(e

eported of lipase-catalyzed acylations with free carboxcids (especially acetic acid) able to furnish good ester y

21,22]. Direct acetylation of alcohols is difficult to obtay enzymatic catalysis, since lipase activity is often in

ted by the free acid[23,24]. Therefore, the discovery of neicrobial lipases and esterases suited to this applicati

ery attractive. Fungal lipases often show interesting prties as biocatalysts; they are generally secreted asellular enzymes, although evidences exist regarding noycelium-bound activity, which can be directly exploitedsing lyophilized mycelium[25–30].

In the absence of permeability problems, lyophilized can also be used in organic solvents[28–32], which wouldllow exploiting directly cell-bound lipases/esterases.

hermore, the cell structure may act as natural matrixo protect the enzymes from the possible negative actioxternal agents, providing an effect analogous to that exy common matrixes used for enzyme immobilization.ases with broad substrate specificity have been foundulture filtrates of several species ofAspergillus[33], but alsoycelium-bound lipase from a strain ofA. flavusshowed in

eresting substrate specificity, being able to catalyze theolysis of several vegetable oils[34,35]. Several esters weynthesized in organic solvent using lyophilized mycelf Rhizopussp. andAspergillussp., among which geranutyrate[27], ethyl and geranyl acetates[30–32]and severasters of 2-phenylpropionic acid[36].

The capability of lyophilizedAspergillus oryzaeMIM andhizopus oryzaeCBS 112.07 mycelia of catalyzing esterations of phenylacetic and 2-phenylpropionic acids inanic solvent was investigated in this work, in order to

apped test tubes by suspending lyophilized mycelia ianic solvent (5 ml) and then adding the alcohol and thell the tests were performed using a lyophilized myceloncentration of 30 g l−1 by dry weight. The reaction mixures were magnetically stirred at different temperature

.2. Analytical methods

Samples were periodically drawn and centrifuged; 20�lf the supernatant were added to an equal volumeHCl3 solution containing an internal standard (2-phe-propanol). Molar conversions were determined uFractovap G1 gas chromatograph (Carlo Erba, M

taly) equipped with a hydrogen flame ionization deteche column temperature was kept at 180◦C. The column3 mm× 2000 mm) was packed with Carbowax 20 M, 100/120 mesh, Supelcoport (Sigma-Aldrich, Milan, Itahe absolute configuration of the optically active esas determined by comparison with the specific rotaf samples of the pure enantiomers obtained by estation of enantiomerically pure (S)-2-phenylpropionic aciith different alcohols using conventional esterification pedures[37]. The enantiomeric composition was routinetermined by gas chromatographic analysis of the esing a chiral capillary column DMePeBeta-CDX-PSMEGA, Legnano, Italy), with 0.25 mm diameter, 25ength, and 0.25�m thickness. For ethyl, propyl and butylhenylpropionates, the column temperature was kept at◦C

or 15 min and then increased by l◦C/min; retention timemin): (R)-ethyl ester 26.8; (S)-ethyl ester 27.5; (R)-propylster 35.5; (S)-propyl ester 36.2; (R)-butyl ester 35.8; (S)-

434 A. Romano et al. / Enzyme and Microbial Technology 36 (2005) 432–438

butyl ester 36.5. For pentyl and hexyl 2-phenylpropionates,the column temperature was kept at 90◦C for 10 min andthen increased by l◦C/min; retention times (min): (R)-pentylester 30.7; (S)-pentyl ester 31.4; (R)-hexyl ester 34.9; (S)-hexyl ester 35.6. For isoamyl 2-phenylpropionate, the col-umn temperature was kept at 80◦C for 15 min and then in-creased by 1◦C/min; retention times (min): (R)-isoamyl ester44.1; (S)-isoamyl ester 27.5. The stereochemical outcome ofthe transformations was initially evaluated as enantiomericexcess (e.e.) of the major enantiomer and then as the enan-tiomeric ratio for the product (E) [2].

3. Results and discussion

3.1. Mycelia hydrolytic activity

The hydrolytic activities of lyophilized mycelia ofA.oryzaeMIM and R. oryzaeCBS 112.07 were evaluated bypreliminary hydrolysis tests performed on ethyl phenylac-etate (EPA) and racemic ethyl 2-phenylpropionate (EPP) so-lutions, with starting substrate concentration (S0) of 23 mM,in 0.1 M phosphate buffer at pH 7.0 andT= 50◦C (Table 1).

The lyophilized mycelium ofA. oryzaewas by far thequicker and more efficient catalyst, showing a starting spe-c ato y.B mo-l PP,l enceo

thee te (re-s enant

3

et ny-l

TY yl 2-p izedm

A

R

S

According to the endoergonic character of esterifications,the formation rates of both esters resulted to be lower thanthose observed for the hydrolysis of the corresponding esters.Moreover, the esterification of PPA was more than 100%slower than that of PAA with both mycelia. As far as theactivities of the selected catalysts are concerned, likewise thehydrolysis, the mycelium ofA. oryzaeproved to be moreeffective than that ofR. oryzae.

Finally, since PPA was used as racemic mixture, the stere-ochemical outcome of the EPP synthesis was also evaluated.The lyophilized mycelium ofA. oryzaewas found to acylateethanol with very highS-stereospecificity (EPP e.e. = 96S),while R. oryzaecatalyzed preferably the formation of the es-ter with opposite configuration with lower enantioselectivity(EPP e.e. = 79R).

3.3. Effect of substrate concentration on ethanolacylation

Considering the reversible character of the reactions cat-alyzed by carboxylesterases, the microbial esterifications inorganic solvent were previously described by a typical ping-pong mechanism[31,32], which is opposite to that proposedby Jencks for esters hydrolysis in water[38]. According tothis approach, it was demonstrated that, at the start of the re-a nten-t

ν

ws alco-h alMb

tu-a ationo testswe atao ulatet -

TE c and2

M e

P

2YeE

S

ific rate of ester hydrolysis (v0) 3–4 times higher than thf R. oryzaeand conversion yields (Y) > 90% after one daesides, either biocatalyst exhibited with EPA higher

ar conversion and starting rate of hydrolysis than with Eikely due to the steric hindrance associated to the presf the� methyl group in the latter ester.

Finally, both mycelia showed no valuable variation innantiomeric excess with respect to the starting racemaults not shown), thus demonstrating the absence of anyiospecificity of mycelium-bound hydrolytic activities.

.2. Acylation reactions

Mycelia ofA. oryzaeMIM andR. oryzaeCBS 112.07 werhen tested inn-heptane for ethanol acylations with pheacetic (PAA) and 2-phenylpropionic (PPA) acids (Table 2).

able 1ields and starting specific rates of ethyl phenylacetate and ethhenylpropionate hydrolysis in 0.1 M phosphate buffer by lyophilycelium ofA. oryzaeandR. oryzae

Y1a (%) Y2

b (%) v0c (mmolSgX

−1 h−1)

. oryzaeEPA hydrolysis 51 96 0.39EPP hydrolysis 45 94 0.34

. oryzaeEPA hydrolysis 15 56 0.11EPP hydrolysis 12 51 0.092

0 = 23 mM;T= 50◦C; pH = 7.0.a Y1: conversion yield estimated after 1 h.b Y2: conversion yield estimated after 24 h.c v0: specific rate of ester hydrolysis.

-

ction, the system can be described by a Michaelis–Meype equation:

0 = kcatS0

Km + S0

herev0 is the starting rate of the ester formation,S0 thetarting equimolar concentration of both substrates (ol and acid),Km a composite term involving the individuichaelis constants for both substrates andkcat= k3Et 0 is theiocatalyst kinetic constant.

To verify whether the acylation of ethanol with PAA aclly follows the mechanism described by the above equr not and to calculate the related kinetic parameters,ere performed inn-heptane at 50◦C, varyingS0 of boththanol and PAA from 35 to 86 mM. The experimental df product concentration versus time were used to calc

he initial rates of EPA formation at differentS0 values. Plot

able 2xperimental results after 72 h of ethanol acylations with phenylaceti-phenylpropionic acids using lyophilized mycelia ofA. oryzaeandR. oryzae

icroorganism A. oryzae R. oryza

henylacetic acidY (%)a 50 22

-Phenylpropionic acid(%)a 20 12

.e. (%)b 96S 79R(–)c 61 9.5

0 = 86 mM;T= 50◦C.a Y: conversion yield after 24 h.b e.e.: enantiomeric excess of the major product enantiomer.c E: enantiomeric ratio for the product.

A. Romano et al. / Enzyme and Microbial Technology 36 (2005) 432–438 435

Fig. 1. Lineweaver–Burk plot of ethanol acylation with phenylacetic acidby mycelium-bound carboxylesterase ofA. oryzaein n-heptane.T= 50◦C.

ting these results inFig. 1according to Lineweaver–Burk, anapparent Michaelis constant,Km, of 389 mM and akcat valueof 0.53 mmolP gX

−1 h−1 where estimated with excellent cor-relation (r2 = 0.994).

Although an apparent Michaelis–Menten model is toosimple to obtain certain information about the mechanismof this complex biocatalytic system, the very highKm valueestimated in this work could be ascribed to the presence ofdiffusion limitations of substrates and products through thedifferent phases. Supposing that the same carboxylesterasemight be responsible for the acylations of various alcoholswith different acids, the above value, which is about three andfive times higher than those estimated for ethanol acetylationat the same temperature[31] and for geraniol acetylation at80◦C[32], suggests a decreased enzyme affinity with increas-ing either the steric hindrance or the acidity of the carboxylicacid. On the other hand, comparing the abovekcat value withthose obtained for ethanol (1.41 mmolP gX

−1 h−1) and geran-iol (0.88 mmolP gX

−1 h−1) acetylations, it is evident that thesteric hindrance associated either to the alcohol or the acidcould have significantly affected this kinetic parameter.

The starting rate of the ester formation increased from0.045 to 0.10 mmolP gX

−1 h−1 when the equimolar concen-tration of both substrates was increased from 35 to 86 mM,whereas a further test performed by doubling the ethanolc ofP(

acy-l A.oam thes osite(t tings velyi rates(

with A. oryzae and v0 = 0.014 mmolP gX−1 h−1, respec-

tively). These results on the whole seem to confirm the oc-currence of an inhibition phenomenon likely exerted by thefree acid on the enzymatic activity, similar to that evidencedby other authors for acetic acid[21,22].

3.4. Effect of temperature on ethanol acylation

Ethanol acylation with PAA and PPA were also evaluatedat different temperatures. Following the suggestions of pre-liminary work[36],n-heptane was selected as a reference sol-vent, while additional tests were performed in toluene withA.oryzaeand in pentadecane withR. oryzaeto put in evidencepossible effects of the solvent properties on the esterificationreaction. The highest molar conversions were obtained withboth biocatalysts at 50◦C inn-heptane (Fig. 2a), thereby con-firming the positive effect of temperature on these systemsabove an inhibiting threshold[31,32]as well as the validityof selecting this compound as a general solvent for bioester-ifications by lyophilized mycelia of fungi.

The results ofFig. 2b also show the influence of tempera-ture on the enantiomeric excess of the major enantiomer of theesteric product.A. oryzaeexhibited the preferential formationof the (S)-enantiomer of EPP, the enantioselectivity being fa-vored by temperature and thermodynamically controlled. Ont ef-f d

F d (b)the enantiomeric excess of EPP from ethanol (B0 = 70 mM) acylations with2-phenylpropionic acid (A0 = 35 mM) performed at different temperaturesand in different solvents.A. oryzae: (�) in n-heptane; (�) in toluene.R.oryzae: (©) in n-heptane; (�) in pentadecane.

oncentration (B0 = 70 mM) and keeping constant thatAA (A0 = 35 mM) exhibited av0 increase by only 33%v0 = 0.060 mmolP gX

−1 h−1).On the basis of the results collected using PAA as an

ating agent, analogous tests were carried out with PPA.ryzaealways behaved better (v0 = 0.025 mmolP gX

−1 h−1

t A0 = 35 mM, B0 = 70 mM) than R. oryzae (v0 = 0.016molP gX

−1 h−1 under the same conditions); moreover,tereoselectivity of the reaction was found to keep oppe.e. = 90Swith the former biocatalyst and e.e. = 70R withhe latter, respectively). It is worthy to note that the starpecific rates of EPP formation were similarly and negatinfluenced by increasing the equimolar amount of substA0 = B0) from 35 to 86 mM (v0 = 0.023 mmolP gX

−1 h−1

he contrary,R. oryzaeexhibited an opposite temperatureect on the preferential (R)-EPP formation, which resulte

ig. 2. Experimental data after 144 h of (a) the molar conversion an

436 A. Romano et al. / Enzyme and Microbial Technology 36 (2005) 432–438

to be kinetically controlled. These results demonstrate thatthe selected biocatalysts could usefully be exploited for theseparate synthesis of both enantiomers, simply acting on thesolvent, the microbial source of biocatalyst and temperature.

Comparison with the results of EPP hydrolysis early dis-cussed put in evidence the apparent contradiction between thelack of stereoselectivity of the hydrolysis and the stringentstereospecificity of either catalyst in the bioconversion. Ac-cording to the state-of-the-art development of bio-technologyand bio-information, it is likely that the enzymes in these twostrains could catalyze the reaction of acylation with differentstereospecificity, due to different conformational constraintsand solvation effects which occur in water and organic media.

3.5. Effect of the alcohol structure on the acylationreactions

Table 3shows the effect of an increased alcohol chainlength on the molar conversion of acylations performed withboth mycelia. Because of the slowness of the esterificationwith PPA, this parameter was calculated after 96 h of reactionrather than 24 h as for PAA.

The evidentY increase with the alcohol molecular weight(MW) can be ascribed to the increased hydrophobicity andchemical affinity of the product for the organic solvent, ifc l hy-d thant for-m uctsa singt sulto stratea olarc ves-t aredt be-c

3

uslya -

TE ) ofa acid(

O

AE11113

A

tions of ethanol with PAA and PPA were studied atT= 50◦C, A0 = 35 mM andB0 = 70 mM, in 11 different sol-vents with progressively increasing hydrophobicity (logP),namely: dimethylsulfoxide (−1.3), dioxane (−1.1), acetoni-trile (−0.33), tetrahydrofurane (0.49), pyridine (0.71), diiso-propyl ether (1.9), benzene (2.0), toluene (2.5),n-heptane(4.0), isooctane (4.5), and pentadecane (8.1) (Fig. 3).

Both biocatalysts showed a linear increase in the start-ing formation rate either of EPA (r2 = 0.977 and 0.951 forA.oryzaeandR. oryzae, respectively) or EPP (r2 = 0.730 and0.735 forA. oryzaeandR. oryzae, respectively) with increas-ing the ratio of logP to the solvent molecular weight. Theseresults are consistent with the general strong positive influ-ence of an increased apolarity of the organic solvent on lipaseactivity and the less marked negative effect of solvent molec-ular complexity[5–8].

At least three different causes could be recognized as beingresponsible for such a behavior. As previously proposed[31],the progressive increase in carboxylesterase activity with thesolvent hydrophobicity could be primarily the result of an in-creased thermostability. This situation, already observed byVolkin et al.[39] for other systems, would be consistent withthe supposed hydrophobic nature of this biocatalyst and itslink to the membrane[30]. According to a second thermo-dynamic hypothesis, the use of a solvent with increased hy-d ffec-t singt r,w dro-c col-l ed by

F ient( tate,aT

ompared with the reactants. Therefore, a longer alcohorophobic chain could have stabilized the ester more

he alcohol, thereby shifting the equilibrium towards theation of products. The enantiomeric excess of the prodppreciably decreased with both biocatalysts with increa

he alcohol chain length (results not shown), likely as a ref the improved rates. Moreover, these results demonnegligible influence of the steric hindrance on the m

onversion, at least within the molecular weight range inigated throughout this study. In contrast, this factor appeo become predominant using 3-methyl-1-butanol, likelyause of the bulky presence of a lateral methyl group.

.6. Effect of the solvent on the acylation reactions

Since the nature of the reaction medium notorioffects the activity of a biocatalyst[6,8], the acyla

able 3ffect of the alcohol structural complexity on the molar conversion (%cylations with phenylacetic acid (after 24 h) and 2-phenylpropionicafter 96 h) by lyophilized mycelium ofA. oryzaeandR. oryzae

rganic acid A. oryzae R. oryzae

PAA PPA PAA PPA

lcoholthanol 60 15 20 20-Propanol 65 20 26 24-Butanol 85 31 30 26-Pentanol 91 24 45 27-Hexanol 100 30 50 29-Methyl-1-butanol 86 29 30 17

0 = 35 mM;B0 = 70 mM;T= 50◦C; solvent:n-heptane.

rophobicity could stabilize the transition states more eively than the alcoholic and acid reactants, thereby cauhe activation energy to decrease andv0 to increase. Howevehen the solvent is a highly hydrophobic long-chain hyarbon (i.e., pentadecane), the frequency of the effectiveisions between substrates and molecules can be inferr

ig. 3. Influence of the ratio of the logarithm of solvent partition coefficlogP) to molecular weight on the starting rates of (a) ethyl phenylacend (b) ethyl 2-phenylpropionate formation. (�) A. oryzae; () R. oryzae.= 50◦C; A0 = 35 mM;B0 = 70 mM.

A. Romano et al. / Enzyme and Microbial Technology 36 (2005) 432–438 437

Fig. 4. Simultaneous dependence of the enantiomeric ratio (E) of EPP fromethanol (B0 = 70 mM) acylation with 2-phenylpropionic acid (A0 = 35 mM)on the solvent partition coefficient (P) and the molecular weight. Esterifica-tions catalyzed by dry mycelia of (�) A. oryzaeand () R. oryzae. T= 50◦C.

the interaction with those of solvent, which can act as a latex[40].

Fig. 3 also shows thatv0 was even negligible below aminimum logP/MW threshold (≤0.01), regardless of the na-ture of the catalyst and the acid. It is likely that, in thesepolar solvents, both substrates were stabilized by solvationto such an extent that the activation energy dramatically in-creased. These results as well are consistent with the already-suggested localization of the carboxylesterase activity on thecytoplasmic membrane of either catalyst[30]. A comparisonamong the values ofv0 obtained in the tested solvents showsthat A. oryzaewas twice and six times more effective thanR. oryzaein the acylations of ethanol with PPA and PAA,respectively. In addition, both mycelia catalyzed the reactionof the latter acid much faster than that of the former, therebyconfirming the significance of the steric hindrance on thisreaction.

Fig. 4shows that the stereospecificity of either biocatalystkept qualitatively the same in the different solvents, i.e.,A.oryzaeandR. oryzaeled to the preferential formations of (S)-and (R)-EPP, respectively.

As expected, the increase inv0 with solvent apolar-ity (log P) caused the enantioselectivity ofR. oryzaecar-boxylesterase to decrease, whereas the mycelium ofA. oryzaeshowed unexpectedly the opposite behavior. This can be ex-p ymec re-o site.F n al-m tt eighta sterf

4

ofP s in

various solvents using lyophilized mycelia ofA. oryzaeandR. oryzae. The results of these tests evidenced that:

• the mycelium-bound carboxylesterase ofA. oryzaewasmore efficient than that ofR. oryzaeto perform the hy-drolysis of both EPA and EPP, showing conversion yields>90% after one day, without any enantiospecificity.

• The conversion yield of ethanol acylation with PPA pro-gressively increased with temperature using either biocat-alyst or exhibited maximum values at 50◦C in n-heptane.

• The use ofA. oryzaeled to the preferential formation of the(S)-enantiomer of EPP with enantiospecificity increasingwith temperature, whereasR. oryzaebehaved oppositely.

• The molar conversion of this reaction also increased withthe alcohol molecular weight, likely because of the in-creased hydrophobicity and chemical affinity of the prod-uct for the solvent.

• Using either biocatalyst, the enantiomeric excess of EPPappreciably decreased with increasing the alcohol chainlength, likely because of the improved rates.

• Rhizopus oryzaeproved to be the more effective biocatalystin all the esterifications, probably due to a more favorablemicroenvironment.

• In the presence of different solvents, the starting formationrates of EPA and EPP with either biocatalyst were shown to

t

A

t on“ theM onso ts”(

R

H;

ns ofkinendia.

in8–50.ger

atal-ors.–84.

of–7.sys-eng

lained with an additional effect of the solvent on the enzonformation, which likely influenced oppositely the stespecific entry of one or both substrates into the activeinally, the product enantiomeric ratio appeared to be aost logarithmic function of logP/MW2, which means tha

he dependence of this parameter on the molecular wnd solvent polarity is quite different from that of the e

ormation rate.

. Conclusions

Ethyl, propyl, butyl, pentyl, hexyl and isoamyl estersAA and PPA were synthesized by direct esterification

progressively increase with the ratio of logP to the solvenmolecular weight.

cknowledgements

This work was supported by the C.N.R. Target ProjecBiotechnology” (no. 97.01019. PF 115.08601) and by.I.U.R. Program of Relevant National Interest “applicatif whole lyophilized cells in monophasic organic solvenno. 2002095553003).

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