Enzyme and Microbial Technology 39 (2006) 167–171 Rapid communication Improvement of the enantioselectivity of lipase (fraction B) from Candida antarctica via adsorpiton on polyethylenimine-agarose under different experimental conditions Rodrigo Torres 1 , Claudia Ortiz 2 , Benevides C.C. Pessela, Jose M Palomo, César Mateo, Jose M. Guisán ∗ , Roberto Fernández-Lafuente ∗ Departamento de Biocatálisis, Instituto de Catálisis-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-B preparations were evaluated as catalysts of the enantioselective hydrolysis of R,S-mandelic acid methyl ester under identical conditions. Interestingly, the best enantioselectivity was achieved with lipase adsorbed at pH 9.0 and 4 ◦ C, conditions where the enzyme exhibited the best enantioslectivity when the reaction was carried out under those conditions. Even more interestingly, these properties were even improved if this preparation was used 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 the same experimental conditions. That is, the same lipase (CAL-B) adsorbed on the same support (PEI-agarose) and used under the same conditions exhibits very different activity–selectivity properties just by using different adsorption conditions. It seems that different conformations of CAL-B can be fixed by intense multipoint anion-exchange involving very large regions of the enzyme surface interacting with these dense layers of polyethyleneimine. © 2006 Elsevier Inc. All rights reserved. Keywords: Lipase immobilization; CAL-B; Agarose-PEI; Kinetic resolution; Frozen of enzyme structures 1. Introduction Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are relevant enzymes used in organic chemistry because they couple a broad specificity with high regio-and enantiospecificity and selectivity. They have been employed as biocatalysts for kinetic resolution of racemates [1–5]. When using lipases, we must take into account the dramatic conformational changes of the lipase 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. Guisán), rfl@icp.csic.es (R. Fernández-Lafuente). 1 Present address: Escuela de Quı́mica, Facultad de Ciencias, Edificio “Camilo, Torres”, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia. 2 Present address: Escuela de Bacteriologia, Facultad de Salud, Universidad Industrial de Santander, Bucaramanga, Santander, Colombia. ∗ 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.03.025 According to this mechanism, lipases present two very dissimilar structural forms: a closed and an open form. In the closed form, the active site of the lipase is secluded from the reaction medium by an oligopeptide chain called “lid”, thus this form is considered inactive. In the open form of lipase, the lid is displaced and the active site is exposed to the reaction medium, allowing the enzyme activity. However, upon exposure to a hydrophobic substrate such as a lipid droplet, the equilibrium shifts towards the open form (interfacial activation) [6–9]. The open and closed forms of the lipases are the result of many different intra-protein interactions, easily modulated by the experimental conditions. In fact, this mechanism promotes that the lipases properties are extremely modulated by the reaction conditions [10–13]. We can expect that if we are able to, to some extent, “freeze” the enzyme structure on different conditions; it may be possible to keep the properties of the enzyme on other conditions. The use of polymeric polycationic supports, such as PEI-coated supports may be very useful to achieve this 168 R. Torres et al. / Enzyme and Microbial Technology 39 (2006) 167–171 goal. PEI-coated supports [14] is a support able to strongly immobilize proteins under a wide range of conditions. In the immobilization, the polymeric nature of this support permits a large percentage of the protein surface to interact with the polymeric bed. In this way, it is possible that enzymes immobilized under different conditions could behave fully different. Thus, this methodology has been used to immobilize different structures of invertase [15]. To check the feasibility of this strategy, we have used the lipase B from Candida antarctica (CAL-B). This is one of the most used lipases, because of its high activity and stability. CALB has a molecular weight of 33 kDa, with an isoelectric point of 6.0 [16,17]; the 3D structure and amino acid sequence has been resolved by Uppenberg [18,19], and due to the fact that CAL-B presents a limited available space in the pocket of the active-site, this enzyme exhibits a high degree of selectivity [16,20–23] Although this enzyme has not a proper lid, it presented a small one. Moreover, the enzyme present a tendency to become adsorbed on hydrophobic surfaces at low ionic strengths [24]. 2. Materials and methods ysis of 0.4 mM p-NPP in 25 mM phosphate buffer at pH 7 and 25 ◦ C. One international unit (U) was defined as the amount of enzyme that is necessary to hydrolyze 1 ␮mol p-NPP/min under the assay conditions described above. 2.2.4. Enzymatic hydrolysis of (R,S)-mandelic acid methyl ester The activities of different preparations of lipase from C. antarctica B on the hydrolysis of (R,S)-mandelic acid methyl ester were investigated by adding the enzyme preparations (0.2 g) to a 10 mL of 10 mM (R,S)-mandelic acid methyl ester under different conditions (pH, T) under mechanical stirring. During the reaction, the pH-value was kept constant by automatic titration and the enzymatic activity (␮mol of substrate hydrolyzed per min/g of biocatalyst) was evaluated from NaOH consumption using a pH-stat Mettler Toledo DL50 graphic. At different times, the degree of hydrolysis was confirmed by reverse-phase HPLC (Spectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450) on a Kromasil C18 (25 cm × 0.4 cm) column supplied by Analisis Vinicos (Spain) with a mobile phase of acetonitrile (30%) and 10 mM ammonium phosphate buffer (70%) at pH 2.95 and a flow rate of 1.5 mL/min. The elution was monitored by recording the absorbance at 254 nm. Triplicates (at least) of each assay were made 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 mobile phase 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 by recording the absorbance at 225 nm. We determined the mandelic acid produced. 2.1. Materials Lipase from C. antarctica B (Novozym 525 L) was kindly supplied by Novozymes (Bagsvaerd, Denmark). Polyethyleneimine (600–1000 kDa), Triton X-100, p-nitrophenyl propionate (p-NPP), and (R,S)-mandelic acid methyl ester were from Sigma Chem. Co. (St. Louis, USA). Octyl-agarose 4 BCL and cyanogen bromide (CNBr-activated sepharose 4 BCL) were purchased from Pharmacia Biotech (Uppsala, Sweden). Glyoxyl-agarose 4 BCL was prepared according to Guisan [25]. PEI-agarose was prepared as previously described [14]. Other reagents and solvents were of analytical grade. 2.2. Methods 2.2.1. Preparation of CAL-B To purify the lipase from any other proteins (e.g. esterases), the enzyme preparation was incubated in the presence of octyl-agarose at low ionic strength, following the previously described procedure [24]. Periodically, the activity of the suspensions and supernatants was assayed using the p-NPP assay as described below. After 1 h, most of the activity had been immobilized. The adsorbed lipase was washed thoroughly with distilled water. To desorbe the enzyme, the adsorbed lipase was washed with 1% (v/v) Triton X-100 in 5 mM sodium phosphate buffer at pH 7 and 25 ◦ C. 2.2.2. Enzyme immobilization of CAL-B 2.2.2.1. Immobilization on CNBr-activated supports. Covalent immobilization on CNBr-activated support was carried out at pH 7 and 25 ◦ C using the protocol recommended by the supplier. 2.2.2.2. Immobilization on PEI-agarose supports. Immobilization of the enzyme on PEI-agarose by ionic exchanger was performed at different pH and temperature. In the immobilization process of CAL-B, enzyme activity of suspensions and supernatants was assayed at different immobilization times by using the p-NPP assay as described below. The enzyme loading was 1 mg protein/mL of support (that is approx. 5% of the maximum loading) in order to prevent diffusion problems. Protein concentration was determined by the Bradford’s method [26]. 2.2.3. Standard determination of CAL-B activity This assay was performed by measuring the increase in the absorbance at 348 nm produced by the release of p-nitrophenol (p-NP) in the enzymatic hydrol- 2.2.6. Calculation of E (enantiomeric ratio)-values The E-value was calculated from the ratio between the enantiomers of both released acid. Conversion degree was always between 10 and 15%. 3. Results and discussion 3.1. Effect of reaction conditions on enantioselectivity of CAL-B immobilized on CNBr-agarose CNBr-agarose immobilized lipase was used as a reference to study the effect of the reaction conditions DURING REACTION because of the low possibilities to get a significant multipoint covalent attachment using this support, therefore, we can get a lipase with a stability and rigidity very similar to the soluble one [27] but without the possibility of suffer any protein–protein interaction that could alter its catalytic behavior [28–31]. The enantioselectivity of CAL-B against this substrate was not very high, therefore the changes of the enzyme properties could be easily detected. Table 1 shows that the enantioselectivity of the immobilized lipase decreased when the pH-value diminished. Thus, the R-isomer was always hydrolyzed quicker, with the higher enantiomeric ratio obtained at pH 9 [32,33]. On the other hand, at pH 7, the best E-values were obtained at low temperature: CNBr-agarose preparation displayed 2-fold Table 1 Effect of the reaction pH-value on activity and enantioselectivity of CNBragarose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acid methyl ester at 25 ◦ C Entry Reaction pH-value Activity (U/g) E-value (R/S) 1 2 3 5 7 9 0.15 0.25 0.20 8.5 9.0 12 169 R. Torres et al. / Enzyme and Microbial Technology 39 (2006) 167–171 Table 2 Effect of reaction temperature on activity and enantioselectivity of CNBragarose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acid methyl ester at pH 7 Table 4 Effect of immobilization temperature on activity and enantioselectivity of different PEI-agarose CAL-B preparations in the hydrolysis of 10 mM (R,S)-mandelic acid methyl ester Entry Reaction T-value (◦ C) Activity (U/g) E-value (R/S) Entry Immobilization T-value (◦ C) Activity (U/g) E-value (R/S) 1 2 3 4 25 37 0.06 0.25 0.36 13 9 6 1 2 3 4 25 37 0.21 0.21 0.39 25 16 10.5 higher enantioselectivity at 4 ◦ C than at 37 ◦ C (see Table 2). Obviously, the higher temperature values, the higher activity was observed. 3.2. Effect of the immobilization pH-value on the enantioselectivity of CAL-B immobilized on PEI-agarose Lipase from C. antarctica B was immobilized on PEI-agarose supports at different pH-values (5, 7 and 9), where the enantioselctivity of the enzyme immobilized on CNBr-agarose was significantly different (see above). Because of the strong adsorption strength of the support, in all cases around 90% of the activity could be readily immobilized on the support. This is one of the advantages of using this ionic exchanger: immobilization may be performed under a great range of conditions with very high yield [14]. When these three preparations, bearing identical lipase but adsorbed under different conditions, were assayed at identical reaction pH-value (pH 7) in the kinetic resolution of (R,S)mandelic acid methyl ester, CAL-B immobilized on PEI-agarose support at pH 9 (conditions where if the reaction was performed the enzyme reached a maximum E-value) was 4-fold more enantioselective than CAL-B immobilized at pH 5 (see Table 3). Moreover, the highest activity of the enzyme was obtained when it was immobilized at pH 9, in fact doubling the activity of the enzyme immobilized at pH 7 and eight times higher than that obtained with the enzyme immobilized at pH 5, although in all cases almost full immobilization had been achieved and the enzyme was fully stable under those conditions. We must remark out that these very different results were achieved using an identical enzyme immobilized under an identical support and measured under identical conditions, being the only difference the immobilization conditions. Table 3 Effect of immobilization pH on activity and enantioselectivity of different PEIagarose CAL-B derivatives in the hydrolysis of 10 mM (R,S)-mandelic acid methyl ester Entry 1 2 3 Immobilization pH-value 5 7 9 Activity (U/g) E-value (R/S) Reaction pH-value Reaction pH-value 5 7 5 7 0.015 0.045 0.21 0.035 0.11 0.27 3.5 6.5 16 2.5 6.0 11 Immobilization was performed at 25 ◦ C. Enzyme activity was determined at 25 ◦ C. Experiments were performed at pH 5 and 25 ◦ C. The immobilizations were performed at pH 9. When these three preparations were utilized at pH 5, we detected a significant increment in the E-value, in opposition to the results observed with the covalently immobilized preparation, the enzyme immobilized at pH 9 increased the E-value by a 50% (from 11 to 16). The activity of the enzyme preparations decreased when measured at pH 5 for all of them, mainly for the immobilized enzymes prepared at pH 5 and 7 (by more than 50%), whereas the effect on the activity of the enzyme immobilized at pH 9 was quite smaller (around 20%). That is, the same enzyme immobilized under different conditions in the same support yielded very different catalytic properties when assayed under the same conditions, and moreover the change of the reaction conditions affected in a very different way the different enzyme preparations. Moreover, it was clear that if the immobilization was performed under conditions where the enzyme was more enantioselective, the biocatalyst become more enantioselective. That is, the immobilization on PEI permitted to keep different conformations of the lipase, and a correlation between properties under the immobilization conditions and properties under other conditions of these enzymes could be found: in a certain way it was possible to retain the enzyme structure present under the immobilization conditions. 3.3. Effect of immobilization temperature on the enantioselectivity of CAL-B preparations CAL-B was immobilized at pH 9 on PEI-coated supports and at three different temperatures: 4, 25 or 37 ◦ C. Table 4 shows effects of the temperature of immobilization on the enantioselectivity of the PEI-immobilized CAL-B. Again, when the immobilization was performed under conditions where the Evalues (during reaction) obtained with the covalently immobilized enzyme were higher, the E-value of the enzyme was higher: the E-value increased from 10 when immobilized at 37 ◦ C to E = 24 if the enzyme was immobilized at 4 ◦ C. Moreover, the enzyme immobilized at 37 ◦ C (perhaps where the enzyme was in a larger percentage exhibiting the open form) presented 90% more activity than the enzyme immobilized under the other two T. 4. Discussion The results showed on this paper suggest that the conformations 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 enzyme on PEI-coated support may permit to keep, at least in some extension, these different enzyme conformations. Considering the results presented in this paper, optimal resolution using mildly covalently immobilized lipase was achieved at pH 9, but under those conditions the esters are not very stable and it is difficult to have a pure product. The enzyme immobilized on PEI-coated supports at pH 9 keeps these good properties, and even these good properties are more significant when the preparations was used at pH 5 (where the covalently enzyme presented the worse properties). It looks that, after having the best enzyme structure, we can benefic from other changes in the enzyme, like ionization of groups that 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 thanks kindly to Novozymes by the donation of CAL-B. We gratefully recognize the help from Dr. Martinez (Novo). We thank a PhD fellowship from Universidad Industrial de Santander, Colombia for R. Torres. The interesting suggestions and help of Dr. Angel Berenguer during the writing of this paper are gratefully recognized. References [1] Bornscheuer UT. Methods to increase enantioselectivity of lipases and esterases. 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