Enzyme and Microbial Technology 39 (2006) 259–264

Effect of lipase–lipase interactions in the activity, stabilityand specificity of a lipase from Alcaligenes sp.

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, Spain

b School of Biochemical Engineering, Universidad Catolica de Valparaıso, Chile

Received 18 August 2005; accepted 24 October 2005

Abstract

It has been found that the lipase QL from Alcaligenes sp. presents a tendency to form very strong bimolecular aggregates (as shown by gelfiltration experiments). The addition of detergents (e.g., Triton X-100) is an easy way to break this aggregate. Soluble enzyme in absence of Triton(that is, forming a dimer) was more stable than the enzyme in the presence of Triton. The lack of Triton effect on the stability of immobilizedpatetaTi©

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reparations of monomeric enzyme suggests that its main effect is the breakage of the aggregate. The enzyme was immobilized on supportsctivated with glutaraldehyde in the presence and absence of Triton X-100, to immobilized monomer, or dimers, respectively, and we have foundhat the properties of the immobilized preparations were very different (after exhaustive washing to eliminate the remaining Triton). When thenzyme was immobilized under conditions where the enzyme tended to form aggregates, stability was much higher, activity was also higher, andhe specificity and enantioselectivity of the enzyme were quite different than when the enzyme was immobilized in the presence of Triton. Theddition of detergent to the enzyme preparation produced in absence of Triton promoted a release of around 50% of the protein to the supernatant.his suggested that we can immobilize the dimer or the monomer depending on the immobilization conditions. Thus, the control of the lipase–lipase

nteraction during immobilization dramatically alters the final biocatalyst properties.2005 Elsevier Inc. All rights reserved.

eywords: Lipase QL from Alcaligenes sp.; Interfacial activation; Lipase–lipase interaction; Protein immobilization

. Introduction

Lipases are very relevant enzymes from both a physiologicalnd a biotechnological point of view. In addition to their naturalunction (hydrolysis of fats and oils), lipases are also able toatalyze the regio- and enantioselective hydrolysis or synthesisf many esters, which are very different from their natural sub-trates [1–3]. Thus, lipases are able to recognize very differentubstrates but, at the same time, are able to catalyze very highlyelective reactions.

Lipases display a peculiar mechanism of action, “interfa-ial activation” [4–9]. Lipases may exist in two different forms.ne of them, where the active centre of the lipase is secluded

rom the reaction medium by a polypeptide chain called “lid”,s considered to be inactive (closed form). The other one, pre-

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

ernandez-Lafuente).

senting the lid displaced and the active centre exposed to thereaction medium, is considered to be active (open form). In aque-ous, homogeneous media, lipase molecules exist in equilibriumbetween these two forms, with this equilibrium shifted towardsthe closed form. This interchange between open and closedforms is accompanied by complex conformational changes ofthe lipase [4–9].

In the presence of any hydrophobic interface, the open form ofthe lipase becomes adsorbed on it and the equilibrium is shiftedtowards the open form of the lipase [10–18].

Recently, it has been shown that most lipases have a naturaltrend to form biomolecular aggregates, by adsorption of openlipases on open lipases via the large hydrophobic pocket formedaround the active centre. These aggregates present completelydifferent catalytic properties when compared to the individuallipase molecule [19,20]. In fact, this interaction may be used topurify lipases by specific adsorption [21] and even to immobilizethem [22]. Thus, this possibility should be considered during anylipase study.

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.

oi:10.1016/j.enzmictec.2005.10.015

260 L. Wilson et al. / Enzyme and Microbial Technology 39 (2006) 259–264

Lipase QL is an extracellular enzyme produced by the strainAlcaligenes sp. with a molecular weight of 31,000 Da. It is inhib-ited by cationic detergents and activated by non-ionic detergents(specifications of the supplier Meito Sangyo Co. Ltd.). LipaseQL is an interesting but not very well characterized lipase. It hasbeen used to catalyze the acylation of primary and secondaryalcohols [23–25] and the production of different key interme-diates for the preparation of several pharmaceutical products[26,27]. The main feature of this enzyme is its very high stabil-ity [28]; therefore, it may be interesting to know the reasons forthis extremely high thermostability.

2. Materials and methods

2.1. Materials

Lipase from Alcaligenes sp. (QL) was from Meito Sangyo Co. Ltd. (Tokyo,Japan). Glyoxyl-agarose 6BCL and 10BCL were kindly donated by the com-pany Hispanagar SA (Burgos, Spain). Octyl-agarose 4BCL was purchased fromPharmacia Biotech (Uppsala, Sweden). p-Nitrophenyl propionate, R- and S-glycidyl butyrate, Triton X-100, �-hydroxyphenylacetic acid methyl ester andpolyethyleneimine (MW 25,000) were purchased from Sigma Chemical Co.(St. Louis, USA). 2-O-Butyryl-2-phenylacetic acid was prepared as describedpreviously [29]. Other reagents and solvent used were of analytical grade.

2.2. Gel filtration of Alcaligenes sp. lipase

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then stirred at 25 ◦C and 250 rpm for 4 h. After that, the solution was removedby filtration and the supported lipase washed several times with distilled water.

2.6. Immobilization of lipase QL on glutaraldehyde-agarose(Glut-M and Glut-BA QL preparations)

Five grams of activated agarose gel modified with glutaraldehydewas added to 50 mL of 25 mM sodium phosphate buffer lipase solution(0.285 mg protein/mL) at pH 7 in the absence or in the presence of 0.1% TritonX-100. The mixture was then stirred at 25 ◦C and 250 rpm for 1 h. After that, theliquid phase was removed by filtration. Fifty millilitres of sodium bicarbonatebuffer 100 mM, pH 10, containing 1 mg/mL of sodium borohydride was addedto the immobilized preparation. The mixture was then stirred at 25 ◦C for 30 min.After that, the liquid phase was removed by filtration and the supported lipasewashed properly with distilled water to remove the reduction agent excess andkeep it at 4 ◦C.

2.7. Enzymatic hydrolysis of R- and S-glycidyl butyrate

Two hundred and fifty milligrams of immobilized preparation was added to10 mL of substrate 10 mM in 25 mM sodium phosphate buffer at pH 7, acetoni-trile 5% (v/v). The mixture was then stirred at 25 ◦C and 250 rpm. A pH-statMettler Toledo DL50 graphic was used to maintain the pH value constant duringthe reactions. The conversion was analyzed by RP-HPLC (Spectra Physic SP100 coupled with an UV detector Spectra Physic SP 8450) using a KromasilC18 (25 cm × 0.4 cm) column. Products were eluted at flow rate of 1.5 mL/minusing acetonitrile–10 mM ammonium phosphate buffer at pH 2.95 (35:65, v/v)and UV detection performed at 225 nm.

The enantiomeric ratio (E) was calculated directly from the ratio betweenthe reaction rates of both isomers (using hydrolysis degrees between 10 and 20%w

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Gel filtration analyses were performed using a glass column packed witheaded Agarose 10BCL (column size: 10 mm × 509 mm; column bed volume:0 mL). The eluting buffer used was 100 mM sodium phosphate, pH 7; all sep-rations were carried out at 25 ◦C with a flow rate of 1.23 mL/min, and lipaseL concentration was 0.285 mg protein/mL.

Where indicated, 0.1% (v/v) Triton X-100 was added to the elution buffer.he column was equilibrated by passing 400 mL of the appropriate buffer. Theluted solution was collected in 1 mL aliquots and the enzymatic activity deter-ined as described below.

The molecular weight of lipase QL preparations was estimated from a cali-ration curve plotted using standard proteins: penicillin G acilase (PGA) 90 kDa,ovine serum albumin (BSA) 67 kDa and lipase of Candida antarctica B (CAL-) 33 kDa.

.3. Enzymatic activity assay

This assay was performed by measuring the increase in the absorbance at48 nm produced by the released p-nitrophenol in the hydrolysis of 0.4 mMNPP in 25 mM sodium phosphate buffer at pH 7 and 25 ◦C. To initialize theeaction, 0.05 mL of lipase solution or suspension was added to 2.5 mL of sub-trate solution. One international unit of pNPP activity was defined as the amountf enzyme necessary to hydrolyze 1 �mol pNPP/min (IU) under the conditionsescribed above.

.4. Thermal inactivation of soluble enzyme

To check their thermal stability, different lipase preparations were incubatedt pH 7.0 or 8.5 at 70 ◦C. Where indicated, 0.05% (v/v) Triton X-100 was addedo the solution buffer. Samples were withdrawn periodically and the activity was

easured using pNPP assay. The experiments were carried out by triplicate andrror was never over 5%.

.5. Immobilization of lipase QL on octyl-agarose support

One gram of octyl-agarose support was added to 16 mL of 25 mM sodiumhosphate buffer lipase solution (0.05 mg protein/mL) at pH 7. The mixture was

here the enzyme kinetics is in the first-order region).

.8. Enzymatic hydrolysis of α-hydroxyphenylacetic acid methylster or 2-O-butyryl-2-phenylacetic acid

Five hundred milligrams of immobilized preparation was added to 3 mLf substrate 10 mM �-hydroxyphenylacetic acid methyl ester or 0.5 mM 2-O-utyryl-2-phenylacetic acid, at 25 ◦C in 25 mM sodium phosphate buffer, pH 7,nder continuous stirring. A pH-stat Mettler Toledo DL50 graphic was used toaintain the pH value constant during the reactions. The conversion was ana-

yzed by RP-HPLC (Spectra Physic SP 100 coupled with an UV detector Spectrahysic SP 8450) using a Kromasil C18 (25 cm × 0.4 cm) column. Products wereluted at flow rate of 1.5 mL/min using acetonitrile–10 mM ammonium phos-hate buffer at pH 2.95 (35:65, v/v) and UV detection performed at 225 nm inhe case of 2-O-butyryl-2-phenylacetic acid, and (25:75, v/v) and UV detectionerformed at 254 nm to �-hydroxyphenylacetic acid methyl ester.

At different conversion degrees, the enantiomeric excess of the releasedcid was analyzed by Chiral Reverse Phase HPLC. The column was a ChiracelD-R, the mobile phase was an isocratic mixture of 5% acetonitrile and 95%aClO4/HClO4 0.5 M at pH 2.3 and the analyses were performed at a flow of.5 mL/min by recording the absorbance 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).

.9. Effect of the presence of Triton X-100 on the activity of theifferent soluble preparations

A standard enzyme solution was prepared (1 g/L of lipase QL) in 5 mModium phosphate buffer at pH 7 and Triton X-100 (concentration range betweenand 2%, v/v). After 15 min at 25 ◦C and stirring, the activity was measured.

.10. SDS-PAGE experiments

Soluble enzyme and the octyl-agarose immobilized preparation were boiledn 1 vol of 2% sodium dodecylsulfate (SDS) [13]. Then, SDS-PAGE analysis

L. Wilson et al. / Enzyme and Microbial Technology 39 (2006) 259–264 261

Fig. 1. SDS-PAGE analysis of different preparations of lipase QL. Lane 1: stan-dard molecular weight (kDa); Lane 2: soluble lipase QL; Lane 3: lipase QLimmobilized on octyl-agarose.

[30] of both samples was performed and the gel was stained with silver staining.As reference, high molecular weight markers from Ammersan were used.

The protein concentration of the different soluble enzyme preparations wasdetermined by the Bradford’s method [31].

3. Results and discussion

3.1. Characterization of the soluble lipase QL

The soluble enzyme was analyzed by SDS-PAGE. Fig. 1shows that the preparation of lipase QL presented a main bandwith a molecular weight of 31 kDa that corresponds to thereported MW for lipase QL. By incubation with octyl-agarose,the esterase activity is incorporated to the support and a hyper-activation may be observed (Fig. 2), as it has been reported formany other lipases [13,32,33]. The SDS-PAGE of the adsorbedprotein confirms that the main band corresponds to a molecular

Fig. 2. Immobilization course of lipase QL on octyl-agarose. Adsorption wasperformed as described in Section 2. Soluble enzyme kept unaltered its activityunder all the immobilization conditions: (�) suspension and (�) supernatants.

weight of 31 kDa. When we tried to release the enzyme from thesupport, we found a very strong adsorption of the protein on thesupport. Thus, using 1% (v/v) of Triton X-100 only 50% of theactivity could be released, and the enzyme become inactivatedwhen using higher detergent concentrations.

Fig. 3 shows that the molecular weight determined using gelfiltration of the lipase QL in the presence of Triton X-100 isagain 31 kDa. However, in the absence of detergent, the enzymepresented a higher molecular weight, approximately doublingit. This experiment suggested that lipase QL has a tendency toauto-assemble into a bimolecular structure as shown by manyother lipases [19–22,34,35]. Nevertheless, this aggregate can beeasily dissociated by detergent addition.

The effect that the Triton X-100 causes on the activity ofthe enzyme is presented in Fig. 4. Low detergent concentrationprovoked an increase in the activity higher than 40% comparedto the initial activity. However, concentrations higher than 0.6%(v/v) promoted a decrease in the enzyme activity.

The enzyme (1 mg/mL of commercial powder lipase QL)stability was studied in the presence and absence of detergent.No inactivation was detected in one day at 60 ◦C for any ofthem. Fig. 5 shows the inactivation at 70 ◦C, in absence ofdetergent (half life 3.28 h) the enzyme (that will be in dimericform) is much more stable than the enzyme in the presenceof detergent (monomeric form). In soluble state, it is difficult

F rofile3 f mol

ig. 3. Effect of detergent in the gel filtration of QL preparations. (A) Elution p) and (©) soluble lipase QL in presence of Triton X-100. (B) Determination o

s determined by lipase activity: (�) soluble lipase QL (values were divided byecular weight. Experiments were performed as described in Section 2.

262 L. Wilson et al. / Enzyme and Microbial Technology 39 (2006) 259–264

Fig. 4. Effect of the Triton X-100 in the QL activity. The experiment was carriedout at 25 ◦C in 5 mM sodium phosphate buffer, pH 7.

Fig. 5. Thermal inactivation of different QL preparations. Inactivation was car-ried out at 70 ◦C in 25 mM sodium phosphate buffer, pH 8.5. The activity wasdetermined using pNPP assay as described in Section 2. (�) Soluble enzymeand (�) soluble enzyme in presence of Triton X-100 (0.05%, v/v).

to compare the monomeric and dimeric forms of this lipase,under the same conditions, and it was not possible to discardsome effect on the enzyme stability promoted by the presence ofdetergent.

3.2. Effect of the immobilization of lipase QL onglutaraldehyde activated supports in the presence andabsence of Triton X-100 on its stability

Lipase QL was immobilized on glutaraldehyde support(GLU) in the presence and absence of Triton X-100, and afterthat the derivatives were washed with an excess of distilled waterto ensure the elimination of the detergent. Almost quantitativeimmobilization was achieved in both cases. Then, the stabil-ity of both enzymes (in monomeric or in dimeric forms) wascompared. Fig. 6A shows that the enzyme immobilized in thepresence of detergent (GLU-M QL) – monomeric form – wasrapidly inactivated at 70 ◦C, presenting similar stability thanthe soluble enzyme in the presence of 0.05% Triton X-100,while the enzyme immobilized in absence of Triton was muchmore stable. The enzyme immobilized in the presence of Triton(monomeric form) and thoroughly washed with water was inac-tivated in the presence of Triton and in the absence of Triton.Stability was found to be identical, that is, Triton has no effect onthe immobilized derivative during inactivation at the employedconcentrations. This suggests that the differences in the stabilityoftta

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Fig. 6. Study of thermal stability of different lipase QL preparations. Inactivations werdetermined using pNPP assay as described in Section 2. (A) Enzyme in the presence(B) Enzyme in absence of Triton: (�) soluble enzyme and (�) enzyme immobilized

f the soluble enzyme in presence or absence of Triton X-100,ound in the previous point, were not caused by the presence ofhe detergent but that these differences in stabilities could reflecthe different state of the enzyme (as monomer or as bimolecularggregate).

On the other hand, the enzyme immobilized in absencef Triton X-100 (GLU-BA QL immobilized preparation) –imolecular aggregate – was much more stable than the otherreparation at 70 ◦C, with values similar to those of the solu-le enzyme in absence of detergent (Fig. 6B). The addition ofetergent to this immobilized preparation promoted the releasef about 50% of the activity to the supernatant, suggestinghat in this case we have the dimer of the lipase with onlyne subunit involved in the covalent attachment [23,24], andhe other enzyme molecule may be desorbed by breaking thisggregate.

e carried out at 70 ◦C in 25 mM sodium phosphate buffer, pH 7. The activity wasof Triton: (�) soluble enzyme and (�) enzyme immobilized on GLU-agarose.on GLU-supports.

L. Wilson et al. / Enzyme and Microbial Technology 39 (2006) 259–264 263

Table 1Enzymatic activity of different immobilized preparations of lipase QL against different substrates

Immobilized preparation Enzyme activity (IU/mg)

pNPP �-Hydroxyphenylacetic acid methyl ester 2-O-Butyryl-2-phenylacetic acid Glycidyl butyrate

GLU-BA QL 31.8 3.7 × 10−3 8.4 × 10−5 17.8GLU-M QL 19.8 2.5 × 10−3 6.3 × 10−5 7.9

Table 2Enantioselective hydrolysis of different substrates catalyzed by immobilized preparations of lipase QL at pH 7 and 25 ◦C

Immobilized preparation �-Hydroxyphenylacetic acid methyl ester 2-O-Butyryl-2-phenylacetic acid Glycidyl butyrate

GLU-BA QL 1.4 (R) 1.1 (R) 8.0 (S)GLU-M QL 1.4 (R) 1.3 (R) 5.1 (S)

These results suggested two facts:

- We have been able to immobilize the dimeric or the monomericform of the lipase.

- The stability of the lipase in a monomeric or a bimolecularaggregate form is very different. These results are in agreementwith the behaviour of other lipases [21,22].

Table 1 presents the catalytic activity of both immobilizedpreparations of lipase QL, utilizing four different substrates:pNPP, �-hydroxyphenylacetic acid methyl ester, 2-O-butyryl-2-phenylacetic and glycidyl butyrate. GLU-BA QL immobilizedpreparation shows always higher activity than GLU-M QL,although the difference between the activities of GLU-BA QL orGLU-M is different when using different substrates. The activityof both immobilized preparations is very similar using the 2-O-butyryl-2-phenylacetic acid, but the activity of GLU-BA QL ismore than twice greater than the one of the GLU-M QL usingthe glycidyl butyrate substrate. This shows that the specificityof both preparations was also different.

Table 2 shows that the both GLU-QL immobilized prepa-rations were not very enantiospecific towards 2-O-butyryl-2-phenylacetic acid or �-hydroxyphenylacetic acid methyl ester.However, in the resolution of glycidyl butyrate, the enzymehydrolyzed more rapidly the S enantiomer. In the latter case, itwpft(

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this dimer. The specificity of the aggregates and monomers ofthe enzyme was also different when changing the preparation.

Acknowledgments

The authors gratefully recognize the support from the SpanishCICYT with the project BIO 2005-08576. We thank CONICYT-BID (Chile) for a fellowship for L. Wilson. We gratefullyrecognize the support given by the Program of InternationalCooperation CSIC (Spain)—CONICYT (Chile). The kind helpand suggestions by Angel Berenguer (Universidad de Alicante)are gratefully acknowledged.

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