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Enzyme and Microbial Technology 39 (2006) 750–755

CLEAs of lipases and poly-ionic polymers: A simple way ofpreparing stable biocatalysts with improved properties

Lorena Wilson 1, Gloria Fernandez-Lorente, Roberto Fernandez-Lafuente ∗,Andres Illanes 1, Jose M. Guisan ∗∗, Jose M. Palomo ∗∗

Departamento de Biocatalisis, Instituto de Catalisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain

Received 2 November 2005; received in revised form 9 December 2005; accepted 20 December 2005

bstract

Standard CLEAs preparation using commercial preparations of lipases from Alcaligenes sp. (QL) and Candida antarctica (fraction B) (CAL-B)s not fully effective, some leakage of enzyme from the CLEA can be observed, and the SDS-PAGE of that preparations reveals that many enzyme

olecules have not cross-linked properly. The co-precipitation of the lipases with poly-ethyleneimine (PEI) or PEI-sulfate dextran (DS) mixturesermitted to get fully physically stable CLEAs, with higher stability in the presence of organic solvents. Very interestingly, the conditions ofrecipitation and the nature of the polymers permitted to significantly alter the lipases activity, enantio-selectivity and specificity. For example,

he QL showed changes in activity and enantio-selectivity in the hydrolysis of (±)-glycidyl butyrate when the derivative was prepared in presencer absence of Triton X-100. Results were further improved if the enzyme was co-precipitated with DS (from around 4 to more than 14). Similarhanges in the lipase properties were found using CAL-B.

2005 Elsevier Inc. All rights reserved.

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eywords: CLEAs; Artificial environments; Polymers; Enzyme stabilization; M

. Introduction

Cross-linked enzyme aggregates (CLEAs) have been pro-osed as an alternative to conventional immobilization onre-existing solid supports or cross-linked crystals of proteins1–10]. The preparation of CLEAs involves the precipitationf the enzyme (that may be not pure) and the further chemicalross-link of the protein aggregate. The cross-linking preventsnzyme re-dissolution when the precipitating agent is retired,herefore, this kind of biocatalysts may be used in any kindf reaction media. Since no pre-existing support is required,

roduction costs are reduced and a high activity per gram ofiocatalyst can be obtained since most of the biocatalyst massill be protein.

∗ Corresponding author. Tel.: +34 91 585 4809; fax: +34 91 585 4760.∗∗ Co-corresponding authors.

E-mail addresses: rfl@icp.csic.es (R. Fernandez-Lafuente),mguisan@icp.csic.es (J.M. Guisan), jose.palomo@mpi-dortmund.mpg.deJ.M. Palomo).

1 Permanent address: School of Biochemical Engineering, Universidadatolica de Valparaıso, Chile.

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141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.12.011

ation of lipases properties

Lipases are among the most relevant enzymes in organichemistry because they combine a broad range of substratepecificity with a high regio- and enantio-selectivity [11–13].hese enzymes suffer drastic changes in their structure duringatalysis, and it has been proposed that enzyme properties cane strongly modulated by designing strategies that permit tolter those changes [14–18]. In fact, it has been shown thatifferent immobilization techniques can strongly modulate thenantio- and regio-selectivity of lipases [19–23]. Furthermore,he preparation of lipase CLEAs using different precipitationonditions have been already used to alter enzyme properties [7].

When the proteins are very poor in external Lys groups,he preparation of CLEAs may become more complicated,ince these Lys residues are the main residues involved in theross-linking step (using glutaraldehyde or aldehyde-dextrans cross-linking agent) [1–10]. Recently, it has been shown thathis problem may be solved by co-precipitating the enzyme andome polymers containing a large number of primary amino

roups: e.g. polyethyleneimine [24].

Moreover, this co-precipitation of enzymes and ionicolymers has been proposed to alter the microenvironment ofnzymes, generating a “polymer-salt environment” surrounding

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he enzyme that may, for example, reduce the concentration ofrganic solvent in the enzyme environment [6].

The presence of ionic polymers near to the proteins couldave two effects on the lipases: (i) the change in the micro-nvironment due to the high saline effect of the polymers andii) the reduced mobility of the enzyme structure caused byultipoint polymer-enzyme interaction and cross-linking.Herein, we have studied the effect of the presence of dif-

erent polymers in the preparation of CLEAs of lipases fromlcaligenes sp. [25–30] and from Candida antarctica B [31–33].oreover, we have evaluated the activity, enantio-selectivity and

tability of the CLEAs.

. Materials and methods

.1. Materials

Lipase from Alcaligenes sp. (QL) was from Meito Sangyo Co., Ltd., Tokyo,apan and lipase from C. antarctica fraction B, CAL-B (Novozym 525L) wasurchased from Novo Nordisk, Denmark. Polyethyleneimine (PEI) of 25,000 Dand (R) and (S)-glycidyl butyrate was purchased from Aldrich (Milwaukee,I, USA); p-nitrophenyl propionate (pNPP), Triton X-100, (R) and (S)-�-

ydroxyphenylacetic acid methyl ester and dextran sulfate (DS) of 25,000 Daere purchased from Sigma Chemical Co. (St. Louis, USA). Polyethylenegly-

ol (PEG) of 600 Da was from Merck (Darmstadt, Germany). Ethylene glycolimethyl ether and glutaraldehyde solution were from Fluka. 2-O-butyryl-2-henylacetic acid was prepared as previously described [34] (R). Cyanogenromide activated Sepharose 4 B support (CNBr) was obtained from Pharmaciaine Chemicals (Uppsala, Sweden). Other reagents and solvents used were ofnalytical grade.

.2. Enzyme activity assay

This was the standard assay used for both enzymes. This assay was performedy measuring the increase in the absorbance at 348 nm produced by the released-nitrophenol in the hydrolysis of 0.4 mM p-nitrophenyl propionate in 25 mModium phosphate buffer at pH 7.0 and 25 ◦C. To start the reaction, 0.05 mL ofipase solution or suspension was added to 2.5 mL of substrate solution. Onenternational unit of activity (IU) was defined as the amount of enzyme thatydrolyzes 1 �mol of pNPP per minute under the conditions described above.

.3. Solvent stability of different lipase preparations

Different lipase preparations were incubated at 25 ◦C in 90% (v/v) dioxanend 10% (v/v) 5 mM sodium phosphate buffer at pH 7.0. Periodically, the resid-al activity was determined as described in Section 2.2. The experiments werearried out in triplicate and error was always below 5%.

.4. Enzymatic hydrolysis of (+) or (−)-glycidyl butyrate

0.03 mL of CLEA preparation (between 30 and 100 mg/mL) was added to0 mL of 10 mM glycidyl butyrate (pure isomer R or S) in 25 mM sodium phos-hate buffer at pH 7.0 and 5% (v/v) acetonitrile. The mixture was then stirred at5 ◦C and 250 rpm. A pH-stat Mettler Toledo DL50 graphic was used to keep theH value constant during the reaction. Substrates and products of reaction wereetermined by RP-HPLC (Spectra Physic SP 100 coupled with an UV detec-or Spectra Physic SP 8450) using a Kromasil C18 (25 cm × 0.4 cm) column.lution was performed at a flow rate of 1.5 mL min−1 using acetonitrile–10 mM

mmonium phosphate buffer at pH 2.95 (35:65, v/v), and UV detection per-ormed at 225 nm. Standard curves (using commercial products) were used toalculate the concentrations of both substrates and products using the area ofach peak in the calculations. E was defined as the ratio between reaction ratesith each isomer.

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l Technology 39 (2006) 750–755 751

.5. Enzymatic hydrolysis of mandelic acid derivatives

0.1 or 0.5 mL of CLEA preparation (100 mg/mL) were added to 10 mL of0 mM pure “R” or “S” �-hydroxyphenylacetic acid methyl ester in 25 mModium phosphate buffer pH 7.0 or 0.5 mM of pure “R” or “S” 2-O-butyryl-2-henylacetic acid in 25 mM sodium acetate buffer pH 5.0 at 25 ◦C. A pH-statettler Toledo DL50 graphic was used to maintain the pH value constant during

he reactions. Substrates and products of reaction were determined by RP-HPLCSpectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450)sing a Kromasil C18 (25 cm × 0.4 cm) column. Elution was performed at a flowate of 1.5 mL min−1 using acetonitrile–10 mM ammonium phosphate buffer35:65, v/v) at pH 2.95 and UV detection performed at 225 nm in the case of-O-butyryl-2-phenylacetic acid (25:75, v/v) and UV detection performed at54 nm in the case of �-hydroxyphenylacetic acid methyl ester. Conversion wasetermined using the area of peak and standard curve as described above.

.6. Preparation of CLEAs

PEG 600 Da and ethylene glycol dimethyl ether were selected as the bestrecipitants for QL and CAL-B, respectively. Cross-linked aggregates of QLere prepared by adding 100 mL of PEG under agitation to 50 mL of QL solu-

ion (250 mg/mL, pH 7.0) to precipitate the enzyme. After 30 min, 2 mL oflutaraldehyde solution (25%, v/v) were added to cross-link the enzyme pre-ipitate, and the mixture was kept under stirring for 1 h. Then, the volume wasuplicated by adding 100 mM sodium bicarbonate buffer at pH 10 and 100 mgf sodium borohydride were added. After 15 min, another 100 mg of sodiumorohydride were added and allowed to react for 15 min. Finally, the resultingLEA was washed five times with 10 volumes of 100 mM sodium phosphateuffer at pH 7.0 and centrifuged at 12,000 rpm for 15 min. All the steps wereerformed in an ice bath at approximately 2 ◦C, and the pH was controlled at aalue of 7.0.

In the case of CLEA of CAL-B, the procedure was the same as above, butsing ethylene glycol dimethyl ether as precipitant.

.7. Preparation of CLEA with polymers

Before adding the precipitant (PEG or ethylene glycol dimethyl ether) tohe lipase solution, 6.25 mL of DS solution (100 mg/mL) was added to thenzyme solution, under stirring for 15 min; and then, 6.25 mL of the PEI solu-ion (100 mg/mL) were added to the previous solution, submitting the solutiono stirring for 10 min. DS and PEI solutions were adjusted to pH 7.0 prior to use.ll operations were carried out in an ice bath at approximately 2 ◦C, and the pHas controlled at a value of 7.0.

CLEAs with enzymes and polyethyleneimine was denominated CLEA-P,LEAs with polyethylenimine and dextrans sulfate were called CLEA-DP.

.8. SDS-PAGE analysis

Soluble enzyme and CLEAs preparations were analyzed by sodium dode-yl sulfate (SDS)-PAGE [35]. SDS-PAGE was performed according to theaemmli’s method [36] in a SE 250-Mighty Small II electrophoretic unit (Hoefero.) using gels of 12% polyacrylamide in a separation zone of 9 cm × 6 cm andconcentration zone of 5% polyacrylamide. The gels were stained following theoomassie method. Molecular weight markers (LMW kit (14,400–94,000 Da))ere from Pharmacia.

The protein concentration of the different soluble enzyme preparations wasetermined by the Bradford’s method [37].

. Results and discussion

.1. Preparation of CLEAs

High immobilization yields (around 90%) and recoveredctivities (over 65%) were obtained with CLEAs produced usingirectly the commercial preparations of both enzymes (CAL-B

752 L. Wilson et al. / Enzyme and Microbia

Table 1Immobilization yield and expressed activity of the different CLEAs

Type of biocatalyst Immobilization yield (%) Expressed activity (%)

QLCLEA 92 ± 3 82 ± 3CLEA with PEI 95 ± 3 73 ± 2

CAL-BCLEA 90 ± 2 75 ± 2CLEA with PEI 93 ± 2 65 ± 2

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mmobilization yield was defined as 100 − (100 × activity in supernatant/initialctivity). Expressed activity is defined as observed activity/offered enzyme.

nd QL) (Table 1). However, release of some lipase activity to theupernatant was produced when the CLEAs were incubated inuffers, suggesting that the glutaraldehyde cross-linking was notully effective. In fact, the boiling of the CLEA in the presencef SDS (to desorb from the aggregate any enzyme molecule notovalently cross-linked) produced the release of a large amountf protein from the CLEA to the supernatant (Fig. 1). CAL-Bhows only some few Lys (6) distributed around the enzyme

urface, this can explain the low efficiency on the cross-linking31]. In the case of QL, the structure is not available, but resultsf slow immobilization on glyoxyl support suggested that there

ig. 1. SDS-PAGE analysis of different CLEAs of lipases. Lane 1: Standardolecular weight (kDa). Lane 2: Soluble lipase QL. Lane 3: CLEA of lipaseL. Lane 4: CLEA with polymers of lipase QL.

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l Technology 39 (2006) 750–755

re not Lys rich areas in the external surface of this enzyme38,39].

To overcome this problem, the commercial preparations ofoth lipases were mixed with PEI before adding the precipitant.mmobilization yields were over 90% in both cases, and recov-red activities were slightly higher than those obtained withoutEI (Table 1). More significant, no enzyme was released justy incubation on buffers, even the drastic treatment of boil-ng the CLEAs in the presence SDS for 1 h did not resultedn the release of <ny enzyme from the CLEA to the mediumFig. 1), suggesting that a proper cross-linking of the pro-ein aggregate had been achieved. Thus, the addition of PEIas used as a standard preparation of CLEAs from both

nzymes.

.2. Effect of the preparation of the CLEA on the functionalroperties of lipases

.2.1. QL

.2.1.1. Effect of Triton X-100. Table 2 shows the activitiesnd enantiomeric ratios (E) obtained in the hydrolysis of gly-idyl butyrate catalyzed by CLEAs of QL co-precipitatedith PEI (CLEA-P) prepared in the presence or absence ofriton X-100. The immobilized preparations that had beenroduced in the presence of Triton yielded two-fold lowerctivity than the CLEA-P prepared in the absence of deter-ent. However, the enantio-selectivity ratio was lower (E = 4)sing this preparation than the one prepared in the pres-nce Triton X-100 (E = 6.2). Considering that QL presentsstrong tendency to form bimolecular aggregates [40], the

ddition of detergents (before the precipitation) should pro-ote the breaking of the dimer, producing the monomeric

orm of the enzyme. Thus, in one case the enzyme wasrecipitated as a dimmer while in the other it was precipi-ated as a monomer. Obviously, a certain direct effect of theriton on the enzyme conformation may be not fully dis-arded.

The CLEA-P prepared in the absence of detergent was incu-ated in the presence of Triton for 24 h and washed as the formererivative top eliminate any Triton molecule from the CLEA.he properties of the CLEA were not affected by this treatment,

uggesting that the Triton X-100 could be eliminated followinghis protocol and that the differences between CLEAs-P pre-ared in the presence or absence of Triton X-100 were due toeal differences in the enzyme structure.

able 2pecific activity and enantio-selectivity ratio of different CLEAs with polymersf lipase QL, in the hydrolysis of 10 mM glycidyl butyrate at pH 7, 5% (v/v)cetonitrile and 25 ◦C

iocatalyst Activity(UI/gCLEA)S-isomer

Activity(UI/gCLEA)R-isomer

Enantiomericratio (E)

LEA-P without Triton 237 59 4.0 ± 0.2LEA-P with Triton 154 25 6.2 ± 0.3LEA-DP with Triton 496 35 14.2 ± 0.5

L. Wilson et al. / Enzyme and Microbial Technology 39 (2006) 750–755 753

Table 3Specific activity and enantio-selectivity ratio of different CLEAs with polymers of CAL-B in the hydrolysis of different substrates at 25 ◦C at pH 7

Biocatalyst �-Hydroxyphenylacetic acid methyl ester 2-O-butyryl-2-phenylacetic acid Glycidyl butyrate

Activity (isomer R) (IU/g) E (R) Activity (isomer S) (IU/g) E (S) Activity (isomer R) (IU/g) E (R)

CLEA-P 16 18 ± 2 1.60 × 10−3

CLEA-DP 34 29 ± 2 1.87 × 10−3

Fig. 2. Stability in organic media of different CLEAs prepared with polymers.Ibd

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nactivation was carried out at 90% (v/v) dioxane in 5 mM sodium phosphateuffer pH 7 at 25 ◦C. The residual activity was determined using pNPP assay asescribed in Section 2. CNBr-QL (�), CLEA-DP (�).

.2.1.2. Effect of polymer composition. Next, the effect of theo-precipitation of the enzyme with a mixture of DS and PEICLEA-DP) was assessed. Since the CLEA is now formed with aigher mass of polymer (DS), the expected activity of this prepa-ation per gram of biocatalyst should be lower than when it wasrepared just with PEI. However, the CLEA-DP-QL derivativead an increased activity: 40% higher regarding the R-isomernd more than three times higher regarding the S-isomer. Thisuggested that the DS promoted a significant increment in thectivity of each individual enzyme molecule. Moreover, theissimilar increment in the activity regarding both isomers pro-uced an increase in the E ratio (Table 2).

.2.1.3. Stability in the presence of organic solvents. The sta-ility of the CLEA-DP of QL was compared to that of thenzyme covalently attached to a pre-existing support: (CNBr-garose) (Fig. 2). The CLEA-DP was much more stable thanhe covalently immobilized derivative, very likely because ofhe partition of the organic solvent away from the polymericaline environment surrounding the enzyme molecules [6,41].

.2.2. CAL-BThe presence of Triton X-100 during the preparation of the

LEAs presented no significant effect on the final propertiesf the biocatalyst, perhaps because this enzyme does not tendo form dimers [42–44]. However, the enzyme properties couldgain be modulated by co-precipitation with different polymers.

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Table 3 shows the activity and enantiomeric ratio of CAL--CLEAs prepared using only PEI or PEI and DS, expressed

n the kinetic resolution of three different substrates: glycidylutyrate (a small and relatively simple molecule), and two man-elic acid derivatives: �-hydroxyphenylacetic acid methyl esterthe chiral center is in the acyl donor side) and 2-O-butyryl-2-henylacetic acid (the chiral center is in the nucleophile side, aecondary alcohol). Using both mandelic acid derivatives, theo-aggregation with PEI and DS increased the activity. More-ver, in both cases, the highest E ratios were achieved usingLEAs with both PEI and DS.

However, using glycidyl butyrate, the situation was the oppo-ite; the highest activity and enantio-selectivity were achievedsing the CLEA prepared using only PEI.

Considering only the activity, the isomer R of �-ydroxyphenylacetic acid methyl ester was hydrolyzed eight-old slower than glycidyl butyrate, using lipase co-aggregatedith PEI, while difference was only a 40% using the lipase coag-regated with PEI and Ds.

All results suggest that the co-precipitation of lipase withifferent polymers may be a very simple way to alter all its func-ional properties: activity, specificity and enantio-preference.

. Conclusions

The preparation of CLEAs is a very simple technology tommobilize proteins, but when the proteins have low Lys con-ent, the final cross-linking step may be not efficient enougho prevent some enzyme leakage. This may be solved by co-ggregating the enzyme with a polymer rich in amino groups thatill permit that the glutaraldehyde cross-links the enzyme and

he polymer. This was the case for both lipases used in this studyand other enzymes, e.g. glutarayl acylase [24]) suggesting thatay be a more general problem. Moreover, in this paper we have

hown that the addition of polymers during the aggregation ofipases to yield CLEAs may be suitable to modulate the enzymeroperties.

The activity and enantio-selectivity properties of the enzymeL – with a strong tendency to form aggregates [40] – could beodulated if the precipitation step was performed in the presence

f Triton X-100. This effect could be related to a change in thetructure of the enzyme or to the precipitation of the enzyme ints monomeric or dimeric forms.

The nature of the polymers co-aggregated with the lipases

ould also alter their properties, strongly affecting activity, speci-city and enantio-specificity.

Thus, the co-aggregation of QL with DS and PEI permittedo improve the volumetric activity (by a three-fold factor) and

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nantio-selectivity (by a two-fold factor) of QL CLEAs preparednly with PEI in the hydrolysis of glycidyl butyrate. Consideringhat the total mass of CLEA was increased by the use of DS, these

eans that the improvement in activity per lipase molecule –lthough difficult to determine – should be around a six-foldactor.

On the contrary, with this substrate CAL-B CLEAs withnly PEI presented around two-fold more activity and enantio-electivity ratios than CLEAs with PEI and DS. However, againpposite results were observed using �-hydroxyphenylaceticcid methyl ester, where the co-immobilization with DS andEI permitted to doubling the activity and have a higher E ratio,hile differences in activity were no so clear using 2-O-butyryl--phenylacetic acid.

That is, the presence of different polymers, able to establishifferent interactions with the enzyme lipase, produces signif-cant changes in activity, specificity and enantio-selectivity ofhe lipases. The strategy is extremely simple and may be of gen-ral use to modulate the properties of any enzyme that sufferonformational changes during catalysis.

cknowledgments

The authors gratefully recognize the support from the Span-sh CICYT (Projects BIO2001-2259 and PPQ 2002-01231). Wehank CONICYT-BID (Chile) for a fellowship for L. Wilson.

e gratefully recognize the support given by the Program ofnternational Cooperation CSIC (Spain)—CONICYT (Chile).

e thank Dr. Angel Berenguer for his kind help during the writ-ng of this manuscript.

eferences

[1] Broun G. Chemically aggregated enzymes. In: Mosbach K, editor. Meth-ods in enzymology. New York: Academic Press; 1976. p. 263–9.

[2] Tyagi R, Batra R, Gupta M. Amorphous enzyme aggregates: stabilitytoward heat and aqueous-organic cosolvent mixtures. Enzyme MicrobTechnol 1999;24:348–54.

[3] Cao L, van Rantwijk F, Sheldon. Cross-linked enzyme aggregates: a sim-ple and effective method for the immobilization of penicillin acylaseR.Org Lett 2000;2:1361–4.

[4] Cao L, van Langen F, van Rantwijk F, Sheldon R. Cross-linked aggre-gates of penicillin acylase: robust catalysts for the synthesis of �-lactamantibiotics. J Mol Catal B: Enzymatic 2001;11:665–70.

[5] Cao L, van Langen L, Sheldon R. Immobilised enzymes: carrier-boundor carrier-free? Curr Opin Biotechnol 2003;14:1–8.

[6] Wilson L, Illanes A, Abian O, Pessela B, Fernandez-Lafuente R, GuisanJM. Co-aggregation of penicillin g acylase and polyionic polymers: asimple methodology to prepare enzyme biocatalysts stable in organicmedia. Biomacromolecules 2004;5(3):852–7.

[7] Lopez-Serrano P, Cao L, van Rantwijk F, Sheldon R. Cross-linkedenzyme aggregates with enhanced activity: application to lipases.Biotechnol Lett 2002;24:1379–83.

[8] Mateo C, Palomo JM, van Lancen LM, van Rantwijk F, Sheldon RA.A new, mild cross-linking methodology to prepare cross-linked enzyme

aggregates. Biotechnol Bioeng 2004;86(3):273–6.

[9] Wilson L, Betancor L, Fernandez-Lorente G, Fuentes M, Hidalgo A,Guisan JM, et al. Crosslinked aggregates of multimeric enzymes: a sim-ple and efficient methodology to stabilize their quaternary structure.Biomacromolecules 2004;5(3):814–7.

[

l Technology 39 (2006) 750–755

10] Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom AP,Van Rantwijk F, et al. Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 2004;87:754–62.

11] Reetz MT. Lipases as practical biocatalysts. Curr Opin Chem Biol2002;6:145–50.

12] Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH. Kineticsand mechanisms of reactions catalysed by immobilized lipases. EnzymeMicrob Technol 1992;14:426–46.

13] Varma IK, Albertsson AC, Rajkhowa R, Srivastava RK. Enzyme cat-alyzed synthesis of polyesters. Prog Polym Sci 2005;30:949–81.

14] Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, Fernandez-Lafuente R, Guisan JM. Modulation of the enantioselectivity of thelipase from Candida antarctica B via conformational engineering. Res-olution of �-hydroxy-phenylacetic acid derivatives. Tetrahedron: Asym-metry 2002;13:1337–45.

15] Palomo JM, Fernandez-Lorente G, Mateo C, Ortiz C, Fernandez-Lafuente R, Guisan JM. Modulation of the enantioselectivity of lipasesvia controlled immobilization and medium engineering: hydrolytic res-olution of Mandelic acid esters. Enzyme Microb Technol 2002;31:775–83.

16] Palomo JM, Fernandez-Lorente G, Mateo C, Fernandez-Lafuente R,Guisan JM. Enzymatic resolution of (±)-trans-4-(4′-fluorophenyl)-6-oxopiperidin-3-ethyl carboxylate, intermediate in the synthesis of (−)-Paroxetine. Tetrahedron: Asymmetry 2002;13:2375–81.

17] Fernandez-Lorente G, Fernandez-Lafuente R, Palomo JM, Mateo C,Bastida A, Coca J, et al. Biocatalyst engineering exerts a dramatic effecton selectivity of hydrolysis catalyzed by immobilized lipases in aqueousmedium. J Mol Catal B Enzymat 2001;11:649–56.

18] Queiroz N, Nascimento MG. Pseudomonas sp. lipase immobilized inpolymers versus the use of free enzyme in the resolution of (R,S)-methylmandelate. Tetrahedron Lett 2002;43:5225–7.

19] Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, GuisanJM, Fernandez-Lafuente R. Enzymatic production of (3S,4R)-4-(4′-fluorophenyl)-6-oxopiperidin-3-carboxylic acid by using acommercial preparation from Candida antarctica A: the roleof a contaminant esterase. Tetrahedron: Asymmetry 2002;13:2653–9.

20] Palomo JM, Munoz G, Fernandez-Lorente G, Mateo C, Fuentes M,Guisan JM, et al. Modulation of Mucor miehei lipase properties viadirected immobilization on different heterofunctional epoxy resins.Hydrolytic resolution of (R,S)-2-phenyl-2-butyroylacteic acid. J MolCatal B Enzymat 2003;21:201–10.

21] Palomo JM, Fernandez-Lorente G, Rua ML, Guisan JM, Fernandez-Lafuente R. Evaluation of the lipase from Bacillus thermocatenu-latus as an enantioselective biocatalyst. Tetrahedron: Asymmetry2003;14:3679–87.

22] Palomo JM, Segura RL, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R. Resolution of (±)-glycidyl butyrate catalyzed by a lipasefrom Rhizopus oryzae in aqueous media. Tetrahedron: Asymmetry2004;15:1157–61.

23] Palomo JM, Segura RL, Mateo C, Terreni M, Guisan JM, Fernandez-Lafuente R. Synthesis of enantiomerically pure glycidol via a fullyenantioselective lipase-catalyzed resolution. Tetrahedron: Asymmetry2005;16:869–74.

24] Lopez-Gallego F, Betancor L, Hidalgo A, Alonso N, Fernandez-LafuenteR, Guisan JM. Co-aggregation of enzymes and polyethyleneimine: asimple method to prepare stable immobilized derivatives of glutaryl acy-lase without support. Biomacromolecules 2005;6:1839–42.

25] Naemura K, Murata M, Tanaka R, Yano M, Hirose K, Tobe Y. Enantios-elective acylation of alcohols catalyzed by lipase QL from Alcaligenessp.: a predictive active site model for lipase QL to identify the fasterreacting enantiomer of an alcohol in this acylation. Tetrahedron: Asym-metry 1996;7:1581–4.

26] Naemura K, Murata M, Tanaka R, Yano M, Hirose K, Tobe Y. Enantios-elective acylation of primary and secondary alcohols catalyzed by lipaseQL from Alcaligenes sp.: a predictive active site model for lipase QL toidentify which enantiomer of an alcohol reacts faster in this acylation.Tetrahedron: Asymmetry 1996;7:3285–94.

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Biomacromolecules 2003;4:1–6.

L. Wilson et al. / Enzyme and Mi

27] Muralidhar RV, Chirumamilla RR, Marchant R, Ramachandran VN,Ward OP, Nigam P. Understanding lipase stereoselectivity. World JMicrobiol Biotechnol 2002;18:81–97.

28] Tanaka K, Yasuda M. A practical synthesis of (R)-3-chlorostyreneoxide starting from 3-chloroethylbenzene. Tetrahedron: Asymmetry1998;9:3275–82.

29] Sharfuddin M, Narumi A, Iwai Y, Miyazawa K, Yamada S, KakuchiT, et al. Lipase-catalyzed dynamic kinetic resolution of hemiaminals.Tetrahedron: Asymmetry 2003;14:1581–5.

30] Negishi S, Shirasawa S, Arai Y, Suzuki J, Mukataka S. Activationof powdered lipase by cluster water and the use of lipase powdersfor commercial esterification of food oils. Enzyme Microb Technol2003;32:66–70.

31] Uppenberg J, Hansen MT, Patkar S, Jones TA. The sequence, crystalstructure determination and refinement of two crystal forms of lipase Bfrom Candida Antarctica. Structure 1994;2:293–308.

32] Lutz S. Engineering lipase B from Candida Antarctica. Tetrahedron:Asymmetry 2004;15:2743–8.

33] Chang QL, Lee CH, Parkin KL. Comparative selectivities of immobi-lized lipases from Pseudomonas cepacia and Candida antarctica (frac-tion B) for esterification reactions with glycerol and glycerol analoguesin organic media. Enzyme Microb Technol 1999;25:290–7.

34] Palomo JM, Segura R, Mateo C, Fernandez-Lafuente R, Guisan JM.Improving the activity of lipases from thermophilic organisms at mod-erate temperatures for biotechnology applications. Biomacromolecules2004;1:249–54.

35] Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J,

Guisan JM. A single step purification, immobilization and hyperac-tivation of lipases via interfacial adsorption on strongly hydrophobicsupports. Biotechnol Bioeng 1998;58:486–93.

36] Laemmli UK. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 1970;277:680–5.

[

l Technology 39 (2006) 750–755 755

37] Bradford MM. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein dyebinding. Anal Biochem 1976;72:248–54.

38] Wilson L, Palomo JM, Fernandez-Lorente G, Illanes A, Guisan JM,Fernandez-Lafuente R. Improvement of functional properties of a ther-mostable lipase from Alcaligenes sp. via strong adsorption on hydropho-bic supports. Enzyme Microb Technol 2006;38:975–80.

39] Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, et al. Some special features of glyoxyl supports to immobilizeproteins. Enzyme Microb Technol 2005;37:456–62.

40] Wilson L, Palomo JM, Fernandez-Lorente G, Illanes A, GuisanJM, Fernandez-Lafuente R. Effect of aggregation in the proper-ties of a lipase from Alcaligenes sp. Enzyme Microb Technol.doi: 10.1016/j.enzmictec.2005.10.015.

41] Abian O, Mateo C, Fernandez-Lorente G, Palomo J, Fernandez-LafuenteR, Guisan JM. Stabilization of immobilized enzymes against organicsolvents: generation of hyper-hydrophilic micro-environments fullysurrounding enzyme molecule. Biocatal Biotransform 2001;19:489–503.

42] Fernandez-Lorente G, Palomo JM, Fuentes M, Mateo C, Guisan JM,Fernandez-Lafuente R. Self-assembly of Pseudomonas fluorescens lipaseinto bimolecular aggregates dramatically effects functional properties.Biotechnol Bioeng 2003;82:232–7.

43] Palomo JM, Fuentes M, Fernandez-Lorente G, Mateo C, Guisan JM,Fernandez-Lafuente R. General trend of lipase to self-assemble giv-ing bimolecular aggregates greatly modifies the enzyme functionality.

44] Palomo JM, Ortiz C, Fuentes M, Fernandez-Lorente G, GuisanJM, Fernandez-Lafuente R. Use of immobilized lipases for lipasepurification via specific lipase–lipase interactions. J Chromatogr A2004;1038:267–73.