Enzyme and Microbial Technology 38 (2006) 975–980

Improvement of the functional properties of a thermostable lipase fromalcaligenes sp. via strong adsorption on hydrophobic supports

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

a Departamento de Biocatalisis, Instituto de Catalisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spainb Escuela de Ingenierıa Bioquımica, Universidad Catolica de Valparaıso, Chile

Received 25 May 2005; received in revised form 22 August 2005; accepted 26 August 2005

Abstract

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at55 ◦C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to differentactivated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the mostioefeehat©

K

1

Emodist(ai

r

0d

nteresting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135%f catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilizednzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 ◦C, pH 8.5 (a 25-fold stabilizingactor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 ◦C (soluble enzyme) up to 70 ◦C (hydrophobic supportnzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on thexperimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highlyydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacialctivation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols usedo immobilize the enzyme hardly improved the properties of the lipase.

2005 Elsevier Inc. All rights reserved.

eywords: Lipase QL from Alcaligenes sp.; Protein immobilization; Stabilization; Enantioselectivity; Lipase modulation

. Introduction

The natural function of lipases (glycerol ester hydrolases.C. 3.1.1.3) is the hydrolysis of triglycerides. However, theyay be used in vitro to catalyze many different reactions,

ften very different from the natural ones (regarding con-itions, substrates, etc.). Thus, lipases may be used in thendustry not only to modify oils and fats [1–3], but also toynthesize fatty esters as cosmetic or surfactants [4–7] ando produce many different intermediates for organic synthesise.g. resolution of racemic mixtures) [8–12]. In fact, lipasesre very likely the most used enzymes in organic chem-stry because these enzymes combine a broad range of sub-

∗ Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760.E-mail addresses: jmguisan@icp.csic.es (J.M. Guisan),

fl@icp.csic.es (R. Fernandez-Lafuente).

strate with a high regio- and enantioselectivity in many cases[13].

In most cases, lipases need to be immobilized before beingused as industrial biocatalysts [14]. It has been recently shownthat different immobilization techniques could alter the exactshape of the open form of lipases, and this technique has beensuccessfully used in the modulation of enantioselectivity andregioselectivity of lipases [15–21].

Lipase QL is an extracellular enzyme produced by Alcali-genes sp. with a molecular mass of 31 kDa. It is inhibited bycationic detergents and activated by non-ionic detergents (spec-ifications of the supplier Meito Sangyo Co.). It has been usedto catalyze the acylation of primary and secondary alcohols[22–24] and the production of different key intermediates forthe preparation of several pharmaceutical products [25,26]. Oneof the main advantages of the enzyme is its very high thermosta-bility [27]. However, there are not reports on the immobilizationof this enzyme.

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.08.032

976 L. Wilson et al. / Enzyme and Microbial Technology 38 (2006) 975–980

In this study, we have prepared different immobilized prepa-rations of the lipase described above and their properties havebeen investigated.

2. Experimental

2.1. Materials

Lipase from Alcaligenes sp. (lipase QL) was obtained from Meito SangyoCo. Ltd. (Tokyo, Japan). Glyoxyl 6BCL and 10BCL were kindly donated byHispanagar SA (Burgos, Spain). Octadecyl-sepabeads was a kind gift fromResindion S.R.L. (Milan, Italy). p-Nitrophenyl propionate (pNPP), R- andS-glycidyl butyrate, Triton X-100, �-hydroxy-phenylacetic acid methyl esterand polyethyleneimine (Mw 25,000) were purchased from Sigma ChemicalCo. (St. Louis, USA). 2-O-Butyryl-2-phenylacetic acid was prepared as previ-ously described [28]. Other reagents and solvent used were of analytical grade.Glutaraldehyde-agarose [29] and polyethyleneimine-agarose [30,31] supportswere prepared as previously described.

2.2. Enzymatic activity assays

2.2.1. Standard assayThis assay was performed by continuously measuring the increase in the

absorbance at 348 nm produced by the released p-nitrophenol in the hydrolysisof 0.4 mM pNPP in 25 mM sodium phosphate buffer at pH 7 and 25 ◦C. Toinitialize the reaction, 0.05 mL of lipase solution or suspension was added to2.5 mL of substrate solution. One international unit of pNPP activity was definedas the amount of enzyme necessary to hydrolyze 1 �mol of pNPP per minute(IU) under the conditions described above.

2

1tMiSCua

tw

2o

omsuwtPafvm

2

2

sm

was removed by filtration and the supported lipase washed several times withdistilled water.

2.3.2. Immobilization on glutaraldehyde-agaroseFive grams of activated agarose gel modified with glutaraldehyde was added

to 50 mL of 25 mM sodium phosphate buffer lipase solution (0.285 mg pro-tein/mL) at pH 7 in the presence of 0.1% Triton X-100 (to prevent the formationof lipase dimmers) [32,33]. The mixture was then stirred at 25 ◦C and 250 rpmfor 1 h. After that, the supernatant was removed by filtration. Fifty milliliterssodium bicarbonate buffer 100 mM pH 10 containing 1 mg/mL of sodium boro-hydride was added to the immobilized preparation. The mixture was then stirredat 25 ◦C for 30 min. After that, the supernatant was removed by filtration and thesupported lipase washed properly with distilled water to remove the reductionagent excess and keep it at 4 ◦C.

2.3.3. Immobilization on polyethyleneimine-agaroseFive grams of polyethyleneimine-agarose support was added to 50 mL of

25 mM sodium phosphate buffer lipase solution (0.285 mg protein/mL) with0.1% Triton X-100 at pH 8. The mixture was then shaken at 4 ◦C and 250 rpmfor 4 h. After that, the solution was removed by filtration and the supported lipasewashed several times with distilled water.

2.4. Temperature–enzyme activity profile of different lipasespreparations

The temperature effect on the enzyme activity of the lipases preparationswas checked in the hydrolysis of butyl butyrate 30 mM in 25 mM sodium phos-phate at pH 7. The buffer was pre-incubated to reach the desired temperaturebefore adding the butyl butyrate and the enzyme. In the case of experimentsthat considered saturated substrate concentration, 150 mM of butyl butyrate wasu

2

B

3

3

abipttl

bewti

paiwba

.2.2. Enzymatic hydrolysis of R- and S-glycidyl butyrateTwo hundred and fifty milligrams of immobilized preparation was added to

0 mL of substrate 10 mM in 25 mM sodium phosphate buffer at pH 7, acetoni-rile 5% (v/v). The mixture was then stirred at 25 ◦C and 250 rpm. A pH-stat

ettler Toledo DL50 graphic was used to maintain the pH value constant dur-ng the reactions. The conversion was analyzed by RP-HPLC (Spectra PhysicP 100 coupled with an UV detector Spectra Physic SP 8450) using a Kromasil

18 (25 cm × 0.4 cm) column. Products were eluted at flow rate of 1.5 mL min−1

sing acetonitrile-10 mM ammonium phosphate buffer at pH 2.95 (35:65, v/v)nd UV detection performed at 225 nm.

The enantiomeric ratio (E) was calculated directly from the ratio betweenhe reaction rates of both isomers (using hydrolysis degrees between 10 and 20%here the enzyme kinetics is in the first order region).

.2.3. Enzymatic hydrolysis of α-hydroxy-phenylacetic acid methyl esterr 2-O-butyryl-2-phenylacetic acid

Five hundred milligrams of immobilized preparation was added to 3 mLf substrate solution. 10 mM pure enantiomers of �-hydroxy-phenylacetic acidethyl ester or 0.5 mM 2-O-butyryl-2-phenylacetic acid, at 25 ◦C in 25 mM

odium phosphate buffer, pH 7. A pH-stat Mettler Toledo DL50 graphic wassed to maintain the pH value constant during the reactions. The conversionas analyzed 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.roducts were eluted at flow rate of 1.5 mL min−1 using acetonitrile–10 mMmmonium phosphate buffer at pH 2.95 (35:65, v/v) and UV detection per-ormed at 225 nm in the case of 2-O-butyryl-2-phenylacetic acid, and (25:75,/v) and UV detection performed at 254 nm to �-hydroxy-phenylacetic acidethyl ester.

.3. Immobilization of lipase QL

.3.1. Immobilization on octadecyl-sepabeads supportFive grams of octadecyl-sepabeads support was added to 50 mL of 25 mM

odium phosphate buffer lipase solution (0.285 mg protein/mL) at pH 7. Theixture was then stirred at 25 ◦C and 250 rpm for 3 h. After that, the solution

sed.

.5. Protein concentration

The protein concentration of the soluble enzyme was determined by theradford method [34].

. Results and discussion

.1. Immobilization of enzyme on different supports

Offering octadecyl-sepabeads to the lipase, 100% of enzymectivity was immobilized after 60 min of incubation. The immo-ilization promoted a certain degree of hyperactivation (35%),n agreement with previous supports using this hydrophobic sup-orts to immobilize lipases [35,36] (Fig. 1). The SDS-PAGE ofhis enzyme preparation revels only one band, confirming thathe only protein having esterase activity in the extract was theipase QL.

The enzyme could be fully desorbed from the support byoiling in the presence of detergent, incubation on guanidine,tc. This support could be reused to immobilize fresh enzymeithout any difference compared to the use of initial support. In

his way, it was possible to reuse the support after the biocatalystsnactivation and enzyme desorption.

On the other hand, the ionic adsorption of lipase QL onolyethyleneimine-agarose was also quite rapid. All enzymectivity become immobilized on the supports after 2 h. Themmobilization have no effect on the enzyme activity (measuredith pNPP). Moreover, the enzyme could be desorbed by incu-ation in 3 M NaCl/10 mM HCl, and the support could be reusedfter the enzyme inactivation.

L. Wilson et al. / Enzyme and Microbial Technology 38 (2006) 975–980 977

Fig. 1. Immobilization of lipase QL on octadecyl-sepabeads. Adsorption wasperformed as described in Methods. Soluble enzyme kept unaltered its activityunder the immobilization conditions: (�) suspension; (�) supernatants.

Immobilization on glutaraldehyde activated supports wasvery slow and only 20% of the activity was immobilized onthis support. Glyoxyl-agarose was unable to immobilize thisenzyme even after 24 h of incubation. Considering the mecha-nism of immobilization using this support (a first simultaneousmultipoint attachment) [37], this result suggests that the enzymehas very few Lys available to react with the support surface (thatis, fully exposed to the medium). This could be due to the gli-cosylated nature of this enzyme.

3.2. Effect of immobilization on the enzyme stability

Fig. 2 shows the thermal inactivation courses of the dif-ferent QL immobilized preparations compared to the solublelipase, at 80 ◦C and pH 8.5. It was possible to observe impor-

Ft(r

tant differences between the thermal stability of the differentimmobilized preparations. The enzyme immobilized on glu-taraldehyde support presented lower stability than the solubleenzyme, perhaps because soluble enzyme could be as dimmerswhile glutaraldehyde presented monomers (it has been preparedin the presence of detergent to ensure the enzyme desegregation)[32,33]. The polyethyleneimine-agarose preparation presented aslightly higher stability than that of the soluble enzyme, whereasthe octadecyl-sepabeads immobilized preparation was the moststable one. The half-lives of the different immobilized prepa-rations were: 11.7 (the octadecyl-sepabeads preparation), 1.58(the polyethyleneimine-agarose preparation) and 0.5 (solubleenzyme).

These results suggest that the immobilization by interfacialactivation on hydrophobic supports is a very suitable techniqueto stabilize this enzyme. This result is consistent with otherresults reported in the immobilization of lipases on these kindof support [35].

3.3. Temperature/activity profile of different QLpreparations

Fig. 3 shows the effects of temperature on the activity of sol-uble and immobilized lipase QL. Using fully soluble substrate(to prevent the interfacial adsorption of the lipase by the sub-s ◦letfdtrl[

Fpde

ig. 2. Thermostability of different immobilized preparations of lipase QL. Inac-ivations were carried out at 80 ◦C in 25 mM sodium phosphate buffer, pH 8.5.�) Glutaraldehyde-QL preparation; (�) polyethyleneimine-agarose-QL prepa-ation; (�) octadecyl-sepabeads-QL preparation; (♦) soluble enzyme.

trate drops), the optimal temperatures were 70 and 50 C, foripase QL immobilized on octadecyl-sepabeads and the solublenzyme, respectively. However, when we utilized substrate overhe saturation concentration where the enzyme may be inter-acially activated by the hydrophobic surface of the substraterops, the soluble enzyme showed an increase in the optimalemperature, now comparable to the octadecyl-sepabeads prepa-ation. This suggested again that the open and adsorbed form ofipases is much more stable than the standard form of the lipase35].

ig. 3. Effect of temperature on enzyme activity of different QL immobilizedreparations. Experiments were carried out using butyl butyrate as substrate asescribed in Section 2. (�) Octadecyl-sepabeads-QL preparation; (♦) solublenzyme; (�) soluble enzyme in the presence of the substrate drop.

978 L. Wilson et al. / Enzyme and Microbial Technology 38 (2006) 975–980

Table 1Enzyme activity of different immobilized preparations of lipase QL

Preparation Enzyme activity (IU/gpreparation)

pNPP �-hydroxy-phenylaceticacid methyl ester (×10−4)

2-O-butyryl-2-phenylaceticacid (×10−5)

Glycidyl butyrate

Glutaraldehyde-QL 7.1 48 16 15Polyethyleneimine-agarose-QL 39.1 49 16 2.2Octadecyl-sepabeads-QL 41.0 43 18 10

The reactions were carried out at 25 ◦C in 25 mM sodium phosphate buffer pH 7. Ten millimolar �-hydroxy-phenylacetic acid methyl ester; 0.5 mM 2-O-butyryl-2-phenylacetic acid; 10 mM glycidyl butyrate.

3.4. Activity of different QL immobilized preparationsagainst different substrates

Table 1 shows the activity of different lipase QL preparationsagainst different substrates: pNPP, �-hydroxy-phenylacetic acidmethyl ester, 2-O-butyryl-2-phenylacetic acid and glycidylbutyrate.

In the case of �-hydroxy-phenylacetic acid methyl ester and2-O-butyryl-2-phenylacetic acid, all immobilized preparationspresented similar activity. However, the activity was pretty dif-ferent using pNPP and glycidyl butyrate.

Using pNPP, the lipase immobilized on glutaraldehyde sup-port was almost 6 times less active than that immobilized onoctadecyl-sepabeads or polyethyleneimine-agarose. However,against glycidyl butyrate, the glutaraldehyde preparation pre-sented the higher activity. This immobilized preparation wasseven times more active than the polyethyleneimine-agarose oneusing this substrate. Thus, the immobilization of the lipase QLon different supports greatly alters their specificity against dif-ferent substrates.

3.5. Enantioselectivity of different lipase QL immobilizedpreparations in the kinetic resolution of glycidyl butyrate.

Table 2 shows the results obtained in the resolution of gly-co

daMsd

TEl

P

GPO

T

Fig. 4. Effect of the co-solvent concentration on the enantioselectivity of the dif-ferent immobilized preparations of lipase QL in the hydrolysis of (R,S) glycidylbutyrate. The reactions were carried out at 25 ◦C in 25 mM sodium phosphatebuffer, pH 7, glycidyl butyrate 10 mM. (�) Glutaraldehyde-QL preparation;(�) polyethyleneimine-agarose-QL preparation; (�) octadecyl-sepabeads-QLpreparation.

on the E-value of the octadecyl-sepabeads and glutaraldehydeimmobilized preparations.

The effect of temperature also varied depending on theenzyme preparation. Preparations on glutaraldehyde and octade-cyl decreased the E-value when the temperature decrease, whilethe enzyme adsorbed on PEI increased the E-value.

Fig. 4 shows the effect of the presence of acetonitrile onthe enantioselectivity of immobilized lipase QL. Lipase QLimmobilized on polyethyleneimine-agarose support presentedan increase in the enantioselectivity value in the presence ofacetonitrile from E = 4.1 (without solvent) to E = 7.2 in the pres-ence of 15% solvent (v/v). The glutaraldehyde preparation alsopresented an improvement in the enantioselectivity value in thepresence of co-solvent from 4.5 to 7.6. However, the highestenantioselectivity value, was obtained using the QL immobilizedon octadecyl-sepabeads in presence of 15% co-solvent (E = 9).

4. Conclusion

We can conclude that the lipase QL is a very stable enzymeand the stability could be further increased when the enzyme was

idyl butyrate catalyzed by different immobilized preparationsf lipase QL under different reaction conditions (pH and T).

At 25 ◦C and pH 7, the immobilized preparations presentedifferent enantioselectivity values, and the polyethyleneimine-garose preparation displayed the lowest E-value (E = 3.8).oreover, this lipase preparation did not seem to be very sen-

ible to the reaction conditions (pH and T). If the pH wasecreased from 7 to 5, significant decrement were observed

able 2ffect of pH and temperature on the enantioselectivity of the different immobi-

ized preparations of lipase QL in the hydrolysis of (R,S)-glycidyl butyrate

reparation pH 7 pH 5

25 ◦C 4 ◦C 25 ◦C 4 ◦C

lutaraldehyde-QL 5.0 3.2 4.6 4.0olyethyleneimine-agarose-QL 3.8 4.4 3.7 4.8ctadecyl-sepabeads-QL 5.2 3.0 4.0 2.8

he reactions were carried out with glycidyl butyrate 10 mM, acetonitrile 5%.

L. Wilson et al. / Enzyme and Microbial Technology 38 (2006) 975–980 979

immobilized via interfacial adsorption on a very hydrophobicsupport (octadecyl-sepabeads). Furthermore, the enzyme afterthe immobilization on this support exhibited an important shiftin its optimal temperature respect to the soluble one. Anotheradvantage for this immobilization protocol is that it is an easy-to-perform procedure and the support can be recovered and reused[35].

Very interestingly, the lipase properties could be stronglymodulated by immobilizing it on different supports, alteringactivity, specificity and enantioselectivity towards several com-pounds.

Acknowledgments

The authors gratefully recognize the support from the Span-ish CICYT with the project BIO2000-0747-C05-02. We thankCONICYT-BID (Chile) for a fellowship for L. Wilson. We grate-fully recognize the support given by the Program of InternationalCooperation CSIC (Spain)–CONICYT (Chile). We thank Dr.Angel Berenguer for his help during the writing of this article.

References

[1] Undurraga D, Markovits A, Erazo S. Cocoa butter equivalent throughenzymatic interesterification of palm oil midfraction. Process Biochem

[

[

[

[

[

[

of (±)-�-hydroxy-phenylacetic acid derivatives. Tetrahedron: Asymmetr2002;13:1337–45.

[16] 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.

[17] Palomo JM, Fernandez-Lorente G, Mateo C, Fernandez-Lafuente R,Guisan JM. Enzymatic resolution of (±)-trans-4-(4′-fluorophenyl)-6-oxo-piperidin-3-ethyl carboxylate, an intermediate in the synthesis of(−)-paroxetine. Tetrahedron: Asymmetr 2002;13:2375–81.

[18] Fernandez-Lorente G, Fernandez-Lafuente R, Palomo JM, Mateo C,Bastida A, Coca J, et al. Biocatalyst engineering exerts a dra-matic effect on selectivity of hydrolysis catalyzed by immobilizedlipases in aqueous medium. J Mol Catal B Enzym 2001;11:649–56.

[19] Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, GuisanJM, Fernandez-Lafuente R. Enzymatic production of (3S, 4R)-(−)-4-(4′-fluorophenyl)-6-oxo-piperidin-3-carboxylic acid using a commercialpreparation from Candida antarctica A: the role of a contaminantesterase. Tetrahedron: Asymmetr 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 hetero-functional epoxy resins.Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid. J MolCatal B: Enzym 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: Asymmetr2003;14:3679–87.

[22] Naemura K, Murata M, Tanaka R, Yano M, Hirose K, Tobe Y. Enantios-

[

[

[

[

[

[

[

[

[

2001;36:933–9.[2] Plou JF, Barandiaran M, Calvo MV, Ballesteros A, Pastor E. High yield

production of mono and dioleylglycerol by lipase catalyzed hydrolysisof triolein. Enzyme Microb Technol 1996;18:66–71.

[3] Dandik L, Aksoy HA. Applications of Nigella sativa seed lipase inoleo-chemical reactions. Enzyme Microb Technol 1996;19:277–81.

[4] Yan Y, Bornscheuer UT, Cao L, Schmid RD. Lipase catalyzed solid-phase synthesis of sugar fatty acid esters: removal of byproducts byazeotropic distillation. Enzyme Microb Technol 1999;25:725–8.

[5] Watanabe Y, Miyawaki Y, Adachi S, Nakanishi K, Matsuno R. Continu-ous production of acyl mannoses by immobilized lipase using a packed-bed reactor and their surfactant properties. Biochem Eng J 2001;8:213–6.

[6] Jaeger KE, Reetz TM. Microbial lipases from versatile tools for biotech-nology. Trends Biotechnol 1998;16:396–403.

[7] Maugard T, Legoy MD. Enzymatic synthesis of derivatives of VitaminA in organic media. J Mol Catal B Enzym 2000;8:275–80.

[8] Kazlauskas RJ, Bornscheuer UT. Biotransformations with lipases. In:Rhem HJ, Philer G, Stadler A, Kelly PJW, editors. Biotechnology, vol.8. New York: VCH; 1998. p. 37–192.

[9] DeZeote MC, Kock-van Dalen AC, van Rantwijk F, Sheldon RA. Lipasecatalysed ammoniolysis of lipids: a facile synthesis of fatty acid amide.J Mol Catal B Enzym 1996;1:109–13.

10] van Dyck SMO, Lemiere GLF, Jonckers THM, Dommisse R, PietersL, Buss V. Kinetic resolution of a dihydrobenzofuran-type neolignan bylipase-catalysed acetylation. Tetrahedron: Asymmetr 2001;12:785–9.

11] Jaeger KE, Reetz MT. Directed evolution of enantioselective enzymesfor organic chemistry. Curr Opin Chem Biol 2000;4:68–73.

12] Koeller KM, Wong CH. Enzymes for chemical synthesis. Nature2001;409:232–40.

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

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

15] Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, Fernandez-Lafuente R, Guisan JM. Modulation of the enantioselectivity of Candidaantarctica B lipase via conformational engineering: kinetic resolution

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-metr 1996;7:1581–4.

23] 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: Asymmetr 1996;7:3285–94.

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

25] Tanaka K, Yasuda M. A practical synthesis of (R)-3-chlorostyreneoxide starting from 3-chloroethylbenzene. Tetrahedron: Asymmetr1998;9:3275–82.

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

27] 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.

28] Palomo JM, Segura R, Mateo C, Fernandez-Lafuente R, GuisanJM. Improving the activity of lipases from thermophilic organismsat mesophilic temperatures for biotechnology applications. Biomacro-molecules 2004;1:249–54.

29] Betancor, L., Lopez-Gallego, F., Hidalgo, A., Alonso-Morales, N.,Dellamora-Ortiz, G., Mateo, C., Fernandez-Lafuente, R., Guisan, JM.Different mechanisms of protein immobilization on glutaraldehyde acti-vated supports: effect of support activation and immobilization condi-tions. Enzyme Microb Technol, submitted for publication.

30] Mateo C, Abian O, Fernandez-Lafuente R, Guisan JM. Reversibleenzyme immobilization via a very strong and non-distorting ionicadsorption on support-polyethylenimine composites. Biotechnol Bioeng2000;68:98–105.

31] Torres R, Mateo C, Fuentes M, Palomo JM, Ortiz C, Fernandez-Lafuente R, et al. Reversible immobilization of invertase on Sepabeads

980 L. Wilson et al. / Enzyme and Microbial Technology 38 (2006) 975–980

coated with polyethyleneimine: optimization of the biocatalyst’s stability.Biotechnol Progr 2002;18:1221–6.

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

[33] 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.Biomacromolecules 2003;4:1–6.

[34] 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.

[35] Palomo JM, Munoz G, Fernandez-Lorente G, Mateo C, Fernandez-Lafuente R, Guisan JM. Interfacial adsorption of lipases on veryhydrophobic support (octadecyl-sepabeads): Immobilization, hyperacti-vation and stabilization of the open form of lipases. J Mol Catal BEnzym 2002;19–20:279–86.

[36] Segura R, Palomo JM, Mateo C, Cortes A, Terreni M, Fernandez-Lafuente R, et al. Different properties of the lipases contained in porcinepancreatic lipase extracts as enantioselective biocatalysts. BiotechnolProgr 2004;20:825–9.

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