Enzyme and Microbial Technology 39 (2006) 167–171

Rapid communication

Improvement of the enantioselectivity of lipase (fraction B) from Candidaantarctica via adsorpiton on polyethylenimine-agarose

under different experimental conditions

Rodrigo Torres 1, Claudia Ortiz 2, Benevides C.C. Pessela, Jose M Palomo, Cesar Mateo,Jose M. Guisan ∗, Roberto Fernandez-Lafuente ∗

Departamento de Biocatalisis, Instituto de Catalisis-CSIC, Campus UAM, Cantoblanco, Madrid, Spain

Received 13 February 2006; received in revised form 3 March 2006; accepted 14 March 2006

Abstract

Agarose gels coated with a dense layer of polyethylenimine (PEI-agarose containing 1000 �mol of ionised groups per wet gram of support)are able to adsorb proteins under a very wide range of experimental conditions (different temperatures and pH ranging from pH 5.0 to 9.0).Candida antarctica lipase (fraction B) (CAL-B) was adsorbed on PEI-agarose under very different experimental conditions. The different CAL-Bptwusecp©

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reparations were evaluated as catalysts of the enantioselective hydrolysis of R,S-mandelic acid methyl ester under identical conditions. Interestingly,he best enantioselectivity was achieved with lipase adsorbed at pH 9.0 and 4 ◦C, conditions where the enzyme exhibited the best enantioslectivityhen the reaction was carried out under those conditions. Even more interestingly, these properties were even improved if this preparation wassed at pH 5 and 4 ◦C (E = 25). On the contrary, CAL-B adsorbed at pH 5.0 and 25 ◦C exhibits a much lower enantioselectivity (E = 3.5) under theame experimental conditions. That is, the same lipase (CAL-B) adsorbed on the same support (PEI-agarose) and used under the same conditionsxhibits very different activity–selectivity properties just by using different adsorption conditions. It seems that different conformations of CAL-Ban be fixed by intense multipoint anion-exchange involving very large regions of the enzyme surface interacting with these dense layers ofolyethyleneimine.

2006 Elsevier Inc. All rights reserved.

eywords: Lipase immobilization; CAL-B; Agarose-PEI; Kinetic resolution; Frozen of enzyme structures

. Introduction

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are rel-vant enzymes used in organic chemistry because they couplebroad specificity with high regio-and enantiospecificity and

electivity. They have been employed as biocatalysts for kineticesolution of racemates [1–5]. When using lipases, we mustake into account the dramatic conformational changes of theipase molecule during catalysis, due to its complex mechanism.

∗ Corresponding authors. Tel.: +34 91 5854809; fax: +34 91 5854760.E-mail addresses: jmguisan@icp.csic.es (J.M. Guisan),

fl@icp.csic.es (R. Fernandez-Lafuente).1 Present address: Escuela de Quımica, Facultad de Ciencias, Edificio

Camilo, Torres”, Universidad Industrial de Santander, Bucaramanga, San-ander, Colombia.

2 Present address: Escuela de Bacteriologia, Facultad de Salud, Universidadndustrial de Santander, Bucaramanga, Santander, Colombia.

According to this mechanism, lipases present two very dissimilarstructural forms: a closed and an open form. In the closed form,the active site of the lipase is secluded from the reaction mediumby an oligopeptide chain called “lid”, thus this form is consid-ered inactive. In the open form of lipase, the lid is displacedand the active site is exposed to the reaction medium, allowingthe enzyme activity. However, upon exposure to a hydrophobicsubstrate such as a lipid droplet, the equilibrium shifts towardsthe open form (interfacial activation) [6–9].

The open and closed forms of the lipases are the result ofmany different intra-protein interactions, easily modulated bythe experimental conditions. In fact, this mechanism promotesthat the lipases properties are extremely modulated by the reac-tion conditions [10–13]. We can expect that if we are able to, tosome extent, “freeze” the enzyme structure on different condi-tions; it may be possible to keep the properties of the enzyme onother conditions. The use of polymeric polycationic supports,such as PEI-coated supports may be very useful to achieve this

141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.03.025

168 R. Torres et al. / Enzyme and Microbial Technology 39 (2006) 167–171

goal. PEI-coated supports [14] is a support able to stronglyimmobilize proteins under a wide range of conditions. In theimmobilization, the polymeric nature of this support permits alarge percentage of the protein surface to interact with the poly-meric bed. In this way, it is possible that enzymes immobilizedunder different conditions could behave fully different. Thus,this methodology has been used to immobilize different struc-tures of invertase [15].

To check the feasibility of this strategy, we have used thelipase B from Candida antarctica (CAL-B). This is one of themost used lipases, because of its high activity and stability. CAL-B has a molecular weight of 33 kDa, with an isoelectric pointof 6.0 [16,17]; the 3D structure and amino acid sequence hasbeen resolved by Uppenberg [18,19], and due to the fact thatCAL-B presents a limited available space in the pocket of theactive-site, this enzyme exhibits a high degree of selectivity[16,20–23] Although this enzyme has not a proper lid, it pre-sented a small one. Moreover, the enzyme present a tendency tobecome adsorbed on hydrophobic surfaces at low ionic strengths[24].

2. Materials and methods

2.1. Materials

Lipase from C. antarctica B (Novozym 525 L) was kindly supplied byNtecPa[

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ysis of 0.4 mM p-NPP in 25 mM phosphate buffer at pH 7 and 25 ◦C. Oneinternational unit (U) was defined as the amount of enzyme that is necessary tohydrolyze 1 �mol p-NPP/min under the assay conditions described above.

2.2.4. Enzymatic hydrolysis of (R,S)-mandelic acid methyl esterThe activities of different preparations of lipase from C. antarctica B on the

hydrolysis of (R,S)-mandelic acid methyl ester were investigated by adding theenzyme preparations (0.2 g) to a 10 mL of 10 mM (R,S)-mandelic acid methylester under different conditions (pH, T) under mechanical stirring. During thereaction, the pH-value was kept constant by automatic titration and the enzymaticactivity (�mol of substrate hydrolyzed per min/g of biocatalyst) was evaluatedfrom NaOH consumption using a pH-stat Mettler Toledo DL50 graphic. Atdifferent times, the degree of hydrolysis was confirmed by reverse-phase HPLC(Spectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450) ona Kromasil C18 (25 cm × 0.4 cm) column supplied by Analisis Vinicos (Spain)with a mobile phase of acetonitrile (30%) and 10 mM ammonium phosphatebuffer (70%) at pH 2.95 and a flow rate of 1.5 mL/min. The elution was monitoredby recording the absorbance at 254 nm. Triplicates (at least) of each assay weremade and experimental error was never higher than 5%.

2.2.5. Determination of enantiomeric excess (ee)At different degrees of conversion, the ee of the produced acid was analyzed

by Chiral Reverse-Phase HPLC. The column was a Chiracel OD-R, the mobilephase was an isocratic mixture of 5% acetonitrile and 95% NaClO4/HClO4

0.5 M at pH 2.1 and the analyses were performed at a flow rate of 0.5 mL/min byrecording the absorbance at 225 nm. We determined the mandelic acid produced.

2.2.6. Calculation of E (enantiomeric ratio)-valuesThe E-value was calculated from the ratio between the enantiomers of both

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ovozymes (Bagsvaerd, Denmark). Polyethyleneimine (600–1000 kDa), Tri-on X-100, p-nitrophenyl propionate (p-NPP), and (R,S)-mandelic acid methylster were from Sigma Chem. Co. (St. Louis, USA). Octyl-agarose 4 BCL andyanogen bromide (CNBr-activated sepharose 4 BCL) were purchased fromharmacia Biotech (Uppsala, Sweden). Glyoxyl-agarose 4 BCL was preparedccording to Guisan [25]. PEI-agarose was prepared as previously described14]. Other reagents and solvents were of analytical grade.

.2. Methods

.2.1. Preparation of CAL-BTo purify the lipase from any other proteins (e.g. esterases), the enzyme

reparation was incubated in the presence of octyl-agarose at low ionic strength,ollowing the previously described procedure [24]. Periodically, the activityf the suspensions and supernatants was assayed using the p-NPP assay asescribed below. After 1 h, most of the activity had been immobilized. Thedsorbed lipase was washed thoroughly with distilled water. To desorbe thenzyme, the adsorbed lipase was washed with 1% (v/v) Triton X-100 in 5 mModium phosphate buffer at pH 7 and 25 ◦C.

.2.2. Enzyme immobilization of CAL-B

.2.2.1. Immobilization on CNBr-activated supports. Covalent immobilizationn CNBr-activated support was carried out at pH 7 and 25 ◦C using the protocolecommended by the supplier.

.2.2.2. Immobilization on PEI-agarose supports. Immobilization of thenzyme on PEI-agarose by ionic exchanger was performed at different pH andemperature. In the immobilization process of CAL-B, enzyme activity of sus-ensions and supernatants was assayed at different immobilization times bysing the p-NPP assay as described below. The enzyme loading was 1 mg pro-ein/mL of support (that is approx. 5% of the maximum loading) in order torevent diffusion problems. Protein concentration was determined by the Brad-ord’s method [26].

.2.3. Standard determination of CAL-B activityThis assay was performed by measuring the increase in the absorbance at

48 nm produced by the release of p-nitrophenol (p-NP) in the enzymatic hydrol-

eleased acid. Conversion degree was always between 10 and 15%.

. Results and discussion

.1. Effect of reaction conditions on enantioselectivity ofAL-B immobilized on CNBr-agarose

CNBr-agarose immobilized lipase was used as a reference totudy the effect of the reaction conditions DURING REACTIONecause of the low possibilities to get a significant multipointovalent attachment using this support, therefore, we can getlipase with a stability and rigidity very similar to the solublene [27] but without the possibility of suffer any protein–proteinnteraction that could alter its catalytic behavior [28–31].

The enantioselectivity of CAL-B against this substrate wasot very high, therefore the changes of the enzyme propertiesould be easily detected. Table 1 shows that the enantioselec-ivity of the immobilized lipase decreased when the pH-valueiminished. Thus, the R-isomer was always hydrolyzed quicker,ith the higher enantiomeric ratio obtained at pH 9 [32,33].On the other hand, at pH 7, the best E-values were obtained

t low temperature: CNBr-agarose preparation displayed 2-fold

able 1ffect of the reaction pH-value on activity and enantioselectivity of CNBr-garose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acidethyl ester at 25 ◦C

ntry Reaction pH-value Activity (U/g) E-value (R/S)

5 0.15 8.57 0.25 9.09 0.20 12

R. Torres et al. / Enzyme and Microbial Technology 39 (2006) 167–171 169

Table 2Effect of reaction temperature on activity and enantioselectivity of CNBr-agarose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acidmethyl ester at pH 7

Entry Reaction T-value (◦C) Activity (U/g) E-value (R/S)

1 4 0.06 132 25 0.25 93 37 0.36 6

higher enantioselectivity at 4 ◦C than at 37 ◦C (see Table 2).Obviously, the higher temperature values, the higher activitywas observed.

3.2. Effect of the immobilization pH-value on theenantioselectivity of CAL-B immobilized on PEI-agarose

Lipase from C. antarctica B was immobilized on PEI-agarosesupports at different pH-values (5, 7 and 9), where the enan-tioselctivity of the enzyme immobilized on CNBr-agarose wassignificantly different (see above). Because of the strong adsorp-tion strength of the support, in all cases around 90% of theactivity could be readily immobilized on the support. This isone of the advantages of using this ionic exchanger: immobi-lization may be performed under a great range of conditionswith very high yield [14].

When these three preparations, bearing identical lipase butadsorbed under different conditions, were assayed at identicalreaction pH-value (pH 7) in the kinetic resolution of (R,S)-mandelic acid methyl ester, CAL-B immobilized on PEI-agarosesupport at pH 9 (conditions where if the reaction was performedthe enzyme reached a maximum E-value) was 4-fold more enan-tioselective than CAL-B immobilized at pH 5 (see Table 3).Moreover, the highest activity of the enzyme was obtained whenit was immobilized at pH 9, in fact doubling the activity of theeoae

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Table 4Effect of immobilization temperature on activity and enantioselectivity of differ-ent PEI-agarose CAL-B preparations in the hydrolysis of 10 mM (R,S)-mandelicacid methyl ester

Entry Immobilization T-value (◦C) Activity (U/g) E-value (R/S)

1 4 0.21 252 25 0.21 163 37 0.39 10.5

Experiments were performed at pH 5 and 25 ◦C. The immobilizations wereperformed at pH 9.

When these three preparations were utilized at pH 5, wedetected a significant increment in the E-value, in oppositionto the results observed with the covalently immobilized prepa-ration, the enzyme immobilized at pH 9 increased the E-valueby a 50% (from 11 to 16). The activity of the enzyme prepa-rations decreased when measured at pH 5 for all of them,mainly for the immobilized enzymes prepared at pH 5 and7 (by more than 50%), whereas the effect on the activity ofthe enzyme immobilized at pH 9 was quite smaller (around20%).

That is, the same enzyme immobilized under different condi-tions in the same support yielded very different catalytic prop-erties when assayed under the same conditions, and moreoverthe change of the reaction conditions affected in a very differentway the different enzyme preparations.

Moreover, it was clear that if the immobilization was per-formed under conditions where the enzyme was more enantios-elective, the biocatalyst become more enantioselective. That is,the immobilization on PEI permitted to keep different confor-mations of the lipase, and a correlation between properties underthe immobilization conditions and properties under other con-ditions of these enzymes could be found: in a certain way itwas possible to retain the enzyme structure present under theimmobilization conditions.

3.3. Effect of immobilization temperature on thee

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nzyme immobilized at pH 7 and eight times higher than thatbtained with the enzyme immobilized at pH 5, although inll cases almost full immobilization had been achieved and thenzyme was fully stable under those conditions.

We must remark out that these very different results werechieved using an identical enzyme immobilized under an iden-ical support and measured under identical conditions, being thenly difference the immobilization conditions.

able 3ffect of immobilization pH on activity and enantioselectivity of different PEI-garose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acidethyl ester

ntry ImmobilizationpH-value

Activity (U/g) E-value (R/S)

ReactionpH-value

ReactionpH-value

5 7 5 7

5 0.015 0.035 3.5 2.57 0.045 0.11 6.5 6.09 0.21 0.27 16 11

mmobilization was performed at 25 ◦C. Enzyme activity was determined at5 ◦C.

nantioselectivity of CAL-B preparations

CAL-B was immobilized at pH 9 on PEI-coated supportsnd at three different temperatures: 4, 25 or 37 ◦C. Table 4hows effects of the temperature of immobilization on the enan-ioselectivity of the PEI-immobilized CAL-B. Again, when themmobilization was performed under conditions where the E-alues (during reaction) obtained with the covalently immobi-ized enzyme were higher, the E-value of the enzyme was higher:he E-value increased from 10 when immobilized at 37 ◦C to= 24 if the enzyme was immobilized at 4 ◦C. Moreover, the

nzyme immobilized at 37 ◦C (perhaps where the enzyme wasn a larger percentage exhibiting the open form) presented 90%

ore activity than the enzyme immobilized under the otherwo T.

. Discussion

The results showed on this paper suggest that the confor-ations of the lipases really are very different under different

170 R. Torres et al. / Enzyme and Microbial Technology 39 (2006) 167–171

reaction conditions and that the immobilization of the enzymeon PEI-coated support may permit to keep, at least in someextension, these different enzyme conformations.

Considering the results presented in this paper, optimal reso-lution using mildly covalently immobilized lipase was achievedat pH 9, but under those conditions the esters are not verystable and it is difficult to have a pure product. The enzymeimmobilized on PEI-coated supports at pH 9 keeps these goodproperties, and even these good properties are more signif-icant when the preparations was used at pH 5 (where thecovalently enzyme presented the worse properties). It looksthat, after having the best enzyme structure, we can beneficfrom other changes in the enzyme, like ionization of groupsthat could be important for the recognition of the substrates[34].

Acknowledgments

This work has been sponsored by the Spanish CICYT(projects BIO2001-2259 and PPQ 2002-01231). Authors thankskindly to Novozymes by the donation of CAL-B. We gratefullyrecognize the help from Dr. Martinez (Novo). We thank a PhDfellowship from Universidad Industrial de Santander, Colom-bia for R. Torres. The interesting suggestions and help of Dr.Angel Berenguer during the writing of this paper are gratefully

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