High-yield purification of an organic solvent-tolerant ... - INBIOSIS

ANALYTICALBIOCHEMISTRYAnalytical Biochemistry 341 (2005) 267–274www.elsevier.com/locate/yabioHigh-yield puriWcation of an organic solvent-tolerantlipase from Pseudomonas sp. strain S5Raja Noor Zaliha R.A. Rahman ¤ , Syarul Nataqain Baharum,Mahiran Basri, Abu Bakar SallehEnzyme and Microbial Technology Research, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MalaysiaReceived 2 December 2004Available online 22 March 2005AbstractAn organic solvent-tolerant S5 lipase was puriWed by aYnity chromatography and anion exchange chromatography. The molecularmass of the lipase was estimated to be 60 kDa with 387 puriWcation fold. The optimal temperature and pH were 45 °C and 9.0,respectively. The puriWed lipase was stable at 45 °C and pH 6–9. It exhibited the highest stability in the presence of various organicsolvents such as n-dodecane, 1-pentanol, and toluene. Ca 2+ and Mg 2+ stimulated lipase activity, whereas EDTA had no eVect on itsactivity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride.It showed random positional speciWcity on the thin-layer chromatography.© 2005 Elsevier Inc. All rights reserved.Keywords: Organic solvent-tolerant lipase; Pseudomonas sp. strain S5; PuriWcation; CharacterizationLipases (E.C. 3.1.1.3) are glycerol ester hydrolases thatcatalyze the hydrolysis of triaclyglycerols into fatty acid,partial acylglycerols, and glycerol. The world market forindustrial enzymes has been estimated at approximatelyUS $600 million, with lipases comprising approximatelyUS $20 million [1]. The interest of lipases stems primarilyfrom their ability to preferentially hydrolyze long/short or saturated/unsaturated fatty acyl residues, butthey also exhibit a positional speciWcity for either the1(3) or 1,2(2,3) positions of a triacylglycerol molecule.In addition, the ability of enzyme being active in thepresence of organic solvents has received much attentionduring the past two decades [2]. In an organic solventenvironment, some enzymes have enhanced thermostabilityand there is a possibility to perform reactions thatare suppressed in water [3].Substrates and products of lipase-catalyzed mechanismsare often insoluble in aqueous solution, and the* Corresponding author. Fax: +60 3 8943 0913.E-mail address: rnzaliha@putra.upm.edu.my (R.N.Z.R.A. Rahman).enzyme is usually insoluble in organic solvents. Reactionscatalyzed by lipases were usually carried out in organicaqueoustwo-phase media that are favorable because theseparation of enzyme from substrates or products is easy.However, in general, enzymes are easily denatured andtheir catalytic activities disappear in the presence oforganic solvents, even if they are nearly water insoluble [4].The bacterial genus Pseudomonas secretes a number ofextracellular enzymes, which include lipases, in response toXuctuating external nutrients. Interest in Pseudomonas lipasesstems either from their potential usefulness in a varietyof biotechnological applications or from their detrimentaleVect on stored food products such as refrigerated milk [5].We previously isolated from local source a benzene, toluene,ethyl-benzene, and xylene (BTEX) 1 degrader identi-Wed as Pseudomonas sp. strain S5, which secreted an organicsolvent-tolerant lipase [6]. The lipase was very stable in the1 Abbreviations used: BTEX, benzene, toluene, ethyl-benzene, andxylene; FFA, free fatty acid; BSA, bovine serum albumin; Con A, concanavalinA.0003-2697/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.03.006

268 Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274presence of various organic solvents such as n-hexane,cyclohexane, toluene, and 1-octanol. In this article, wereport the puriWcation and characterization of organic solvent-tolerantlipase secreted by Pseudomonas sp. strain S5.Materials and methodsCulture conditionPseudomonas sp. strain S5, which secreted organic solvent-tolerantlipase, was used in this study [6].CultivationThe culture was grown at 37 °C for 48 h in themedium prepared by optimizing the previously usedmedium for lipase production [6]. In the cultivation procedure,6% of inoculum was inoculated into a 250 mlblue cap bottle containing 50 ml of the medium.Preparation of culture supernatantThe culture supernatant was prepared by removingthe cells in the culture by centrifugation at 12,000g for20 min at 4 °C.Measurement of lipase activityDetermination of liberated free fatty acid (FFA) wasmeasured by the modiWed method of Kwon and Rhee [7].Olive oil was used as a substrate. The reaction mixture,consisting of 1.0 ml of crude enzyme, 2.5 ml olive oil emulsion,and 0.02 ml of 20 mmol CaCl 2·2H 2 O, was incubatedfor 30 min with shaking at 200 rpm at 37 °C. Then thereaction was stopped by adding 1.0 ml of 6 N HCl and5.0 ml isooctane. The upper layer (4 ml) was pipetted outinto a test tube, and 1.0 ml cupric acetate pyridine wasadded. The FFA dissolved in isooctane was determinedby measuring the absorbency of isooctane solution at715 nm. Lipase activity was determined by measuring theamount of FFA from the standard curves of oleic acid.One unit of lipase activity is deWned as the amount ofenzyme that liberated 1 μmol FFA in 1 min at 37 °C.Determination of protein concentrationsThe protein concentrations in this study were determinedby the method of Bradford [8] using bovine serumalbumin (BSA, Sigma Chemical, St. Louis, MO, USA) asa standard.Con A–Sepharose column chromatographyThe enzyme solution was loaded onto the column(1.6 cm diameter, 20.0 cm height) of concanavalin A(Con A)–Sepharose (Amersham–Pharmacia Biotech,Uppsala, Sweden) preequilibrated with 25 mM Tris–HClbuVer (pH 7.5). The column was washed with 5£ of columnbed volume of the equilibration buVer. The proteinsbound to the aYnity gel were eluted by increasing theconcentration of methyl α-D-glucoside from 0.1 to 0.5 Mwith a step gradient in 100 ml of 25 mM Tris–HCl buVer(pH 7.5) and 100 ml of the same buVer containing methylα-D-glucoside. Fractions (approximately 5 ml of each)that exhibited lipase activity in the gradient region of themethyl α-D-glucoside concentration were collected. Theenzyme solution was then dialyzed overnight againstbuVer containing methyl α-D-glucoside.DEAE–Sephacel column chromatographyThe enzyme solution from the previous column wasloaded onto a column (1.6 cm diameter, 10.0 cm height)of DEAE–Sephacel (Amersham–Pharmacia Biotech)preequilibrated with 25 mM Tris–HCl buVer (pH 7.5).The column was washed with 100 ml of the equilibrationbuVer. The proteins bound to the anion exchange gelwere eluted by increasing the concentration of NaCl in25 mM Tris–HCl buVer (pH 7.5) from 0.1 to 0.5 M with alinear gradient using 50 ml of 25 mM Tris–HCl buVer(pH 7.5) and 50 ml of the same buVer containing NaCl.Fractions (approximately 2 ml of each) that exhibitedlipase activity in the gradient region of the NaCl concentrationwere collected. The enzyme solution was dialyzedovernight against buVer containing NaCl.Determination of the purity and molecular mass of puriWedS5 lipase by SDS–PAGEThe solution containing puriWed S5 lipase andsample buVer solution was heated in a boiling waterbath for 3 min. The sample was run in a stacking gelcontaining 4.35%(w/v) polyacrylamide and 0.3%(w/v)N,N-methylene-bis(acrylamide) and in a separating gelcontaining 14.8%(w/v) polyacrylamide and 0.2%(w/v)N, N-methylene-bis(acrylamide) under the conditionsdeveloped by Laemmli [9]. The protein was stained bysilver staining.Organic solvent stability of the enzymeThe stability of the puriWed lipase in the organic solventwas investigated by the method as described byOgino et al. [10] with slight modiWcation. The puriWed S5lipase-containing 25 mM Tris–HCl (pH 7.5) was Wltersterilizedwith a cellulose acetate membrane Wlter (0.2 μmpore size). Then 1 ml of an organic solvent was added to3ml of the Wltrate in a vial bottle, and the mixture wasincubated at 37 °C with shaking at 150 rpm for 2 h. Theremaining lipase activity was measured by the methodby Kwon and Rhee [7] as described above.

Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274 269EVect of pH and pH stability of the S5 lipaseThe eVect of pH on the S5 lipase activity was investigatedby the method using olive oil as a substrate. VariousbuVer systems (pH 4.0–12.0) at 25 mM were added toolive oil to perform olive oil emulsion. Then the enzymewas assayed at diVerent pH values at 37 °C for 30 minwith shaking at 200 rpm. For pH stability studies, 200 μlof puriWed enzyme solution was mixed with 800 μl of25 mM buVer at speciWc pH. The mixture was incubatedat 37 °C for 30 min, and the residual activity was determined.BuVer systems were used at a concentration of25 mM: glycine–HCl buVer (pH 4.0–5.0), sodium hydrogenphosphate–NaOH buVer (pH 5.0–7.0), Tris–HClbuVer (pH 7.0–9.0), disodium hydrogen orthophosphate–NaOHbuVer (pH 9.0–11.0), and glycine–NaOHbuVer (pH 10.0–12.0).EVect of temperature on activity and thermostability of S5lipaseThe eVect of temperature on lipase activity wasstudied by carrying out the enzyme reaction at diVerenttemperatures in the range of 30–55 °C at pH 9.0using Tris–HCl buVer (25 mM). The reaction was carriedout for 0 and 30 min. The thermostability oflipase was tested at various temperatures, rangingfrom 37 to 45 °C, for diVerent time intervals, rangingfrom 0 to 6 h. At each time interval, 500 μl sampleswere pippeted out and immediately frozen prior tobeing assayed.Substrate speciWcityTo determine the eVect of substrates on puriWedlipase, the olive oil emulsion was substituted with thevarious substrates and was assayed with 1.0 ml enzymesolution at 37 °C for 30 min at pH 9.0.EVect of metal ions and oxidizing, reducing, and chelatingagentsThe eVect of metal ions and oxidizing, reducing, andchelating agents was investigated by incubating theenzyme in a 1:1 ratio at pH 9 for 15 and 30 min at 37 °C.The solution of metal ions and oxidizing, reducing, andchelating agents was prepared at a concentration of1mM.Positional speciWcityMixtures containing 100 mM of triolein, 9 ml phosphatebuVer (pH 7.0), and 2 ml enzyme solution wereshaken at 200 rpm at 37 °C. At 0, 3, 6, 9, and 12 h, thereaction products were extracted with 5 ml n-hexane andanalyzed by thin-layer chromatography. A silica gelplate (Silica gel 60, Merck, Darmstadt, Germany) wasdeveloped in a mixture of chloroform and acetone (96:4).Spots of the hydrolysis products from triolein werevisualized by iodine vapor. As standards, 1(3)-monoolein,2-monoolein, 1,2(2,3)-diolein, triolein, and oleic acidpurchased from Sigma were used. The commercialenzymes Lipozyme and Novozyme were included in thisexperiment as a control because the positional speciWcityhas already been veriWed.Results and discussionPuriWcation of S5 lipaseThe objective of enzyme puriWcation is to get rid of asmuch unwanted protein as possible while retaining theenzyme activity. Lipase was puriWed with Con A–Sepharose as the initial steps, followed by DEAE–Sephacel. When the enzyme was loaded onto Con A–Sepharose column chromatography, the absorbedenzyme was eluted out at a 250 mM concentration ofmethyl α-D-glucoside. At this stage, a high percentage ofrecovery of 89% protein with 30 puriWcation fold wasobtained. The enzyme was further puriWed with anionexchange column chromatography and eluted out byNaCl treatment at a concentration of 0.3 M. A singleband was obtained for S5 lipase, which was puriWed toapproximately 387.5-fold with a speciWc activity of1240 U/mg and an overall yield of 52%.Homogeneity of the eluted peak was conWrmed bySDS–PAGE. The silver staining revealed a single proteinband with a molecular weight of approximately 60kDa(Fig. 1). The puriWcation results are summarized in Table 1.So far, S5 lipase is the Wrst organic solvent-tolerantPseudomonas lipase to be puriWed using combinationof aYnity chromatography and anion exchange chromatography.An organic solvent-stable lipase fromPseudomonas aeruginosa LST-03 was puriWed by ionexchange and hydrophobic interaction chromatography[10]. Other Pseudomonas lipases were puriWed byusing (NH 4 ) 2 SO 4 saturation as a step in partial puriWcation[11,12]. Lin et al. [13] puriWed an alkaline lipasefrom Pseudomonas pseudoalcaligenes F-111 by acetoneprecipitation, gel Wltration, and hydrophobic interactionfollowed by another gel Wltration. Compared withother puriWed Pseudomonas lipases, S5 lipase showed ahigher fold and recovery. According to Ogino et al.[10], LST-03 organic solvent-tolerant lipase was puri-Wed to 34.7-fold with an overall yield of 12.6%. Pseudomonaslipase KWI-56 was puriWed to 13.9-fold byacetone precipitation and gel Wltration by HPLC with2.9% recovery [14]. Other Pseudomonas lipasesobtained 31-fold with 18% recovery and 140-fold with15% recovery [13,15]. Meanwhile, Pseudomonas mendocinaPK-12CS lipase was puriWed to 240-fold with

270 Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274Table 2Stability of puriWed lipase in presence of various organic solventsOrganic solvent Log P Relative activity30 min incubation 2 h incubationNone — 1.00 1.001-Pentanol 1.3 5.40 36.70Benzene 2.0 30.60 0.00Chloroform 2.0 23.30 5.33Toluene 2.5 1.66 0.001-Octanol 2.9 0.29 44.50Cyclohexane 3.2 31.80 23.20n-Hexane 3.6 18.40 0.001-Decanol 4.0 3.48 0.00n-Decane 5.6 4.51 0.19n-Dodecane 6.6 1.17 4.95n-Tetradecane 7.6 2.84 0.68n-Hexadecane 8.8 1.32 0.15Note. Data are means of triplicate determinations. The enzyme andorganic solvent were mixed in a 3:1 ratio, and the mixture was incubatedat 37 °C with shaking at 150 rpm for 2 h and assayed for thelipase activity. Activities of S5 lipase in the presence of various organicsolvents are shown as values relative to those in the absence of anorganic solvent.Fig. 1. SDS–PAGE of puriWed S5 lipase. The puriWed protein was electrophoresedon 12%(w/v) of SDS–PAGE and stained with silver stain.Lane 1, marker; lane 2, crude enzyme; lane 3, aYnity chromatography;and lane 4, ion exchange (puriWed S5 lipase).14.8% recovery using acetone precipitation and anionexchange chromatography [16].Organic solvent stability of the enzymeExposure of the puriWed organic solvent-tolerant S5lipase to various organic solvents for 30 min elucidatedthat this enzyme was stable to all organic solvents tested(Table 2). The highest activity was achieved by cyclohexane,benzene, and n-hexane, with increases in relativeactivity of 31.8, 30.6, and 18.9%, respectively, after30 min. The S5 lipase was activated to 6 and 153 times by1-pentanol and 1-octanol after 2 h exposure to both solvents.Similarly, Sharma et al. [12] reported that theirAG-8 lipase was activated in the presence of ethanol andmethanol. In contrast, the stability of LST-03 lipase waslower in the presence of alcohol such as tert-butanol,ethanol, and 1,4-butanediol [10]. In this study, when25%(v/v) of benzene, toluene, n-hexane, and 1-decanolwas added to the puriWed lipase solution and incubatedfor 2 h, the enzyme was drastically inactivated comparedwith 30 min incubation. This phenomenon is due to thehigh toxicity of the organic solvent to the enzyme [17].Similarly, Sugihara et al. [18] reported that Pseudomonascepacia lipase was inactivated at all concentrations ofbenzene and hexane after 30 min incubation under assayconditions. In contrast, S5 lipase was very stable after30 min incubation with benzene and n-hexane but wasreduced drastically after 2 h incubation. Water-misciblesolvents generally cause more striking enzyme denaturationthan do water-immiscible solvents.The stability of the S5 lipase in organic solvents didnot follow the log P trends. According to Klibanov [19],the less hydrophobic the solvent, the higher its aYnity towater and hence the more likely it is to strip the essentialwater from the enzyme molecules. It is well known thatwater acts as a lubricant that aVords a high conformationalXexibility to enzyme molecules. If one follows thetrends of log P, the lower the log P values, the less hydrophobicthe solvent, so the enzyme is less stable and theremay be change in the conformation of the enzyme molecules.However, diVerent organic solvents showed diVerenttolerance proWles to the S5 lipase. It is well-known thatthe eVect of organic solvents on enzyme activity diVersfrom lipase to lipase [18]. There was no clear correlationbetween the solubility of an organic solvent in water andstability of lipase in its presence [10].The stability and activation eVects of the organic solvent-tolerantS5 lipase in aqueous–organic mixtures suggestedthe ability of this enzyme to resist denaturationby organic solvents and to form multiple hydrogenTable 1Summary of puriWcation of S5 lipase in culture supernatantPuriWcation step Total activity (U) Total protein (mg) SpeciWc activity (U/mg) PuriWcation (fold) Yield (%)Culture supernatant 54.1 16.9 3.2 1.0 100Con A–Sepharose 48.0 0.5 96.0 30.0 89DEAE–Sephacel 24.8 0.02 1240 387.5 52

272 Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274S5 lipase was found to have the highest activityagainst triolein, which possessed longer carbon chainlengths. This result proved, in principle, that the lipase ispreferred to hydrolyze longer carbon chains of thesubstrates. In contrast, Ogino et al. [10] reported thatLST-03 lipase had the highest hydrolytic activity againsttricaproin (C6).EVect of metal ions and oxidizing, reducing, and chelatingagentsFig. 5. Thermal stability of puriWed lipase. The enzyme solution wasincubated at 30 °C (), 37 °C (), 45 °C (), 50 °C (), and 55 °C ()for 6 h. The incubated enzyme was assayed at diVerent time intervalsto check for the residual activity.against substrate are shown relative to the olive oil inTable 3. S5 lipase was found to have the highest activityagainst palm oil, followed by coconut oil (125%).According to Ogino et al. [10], enzyme had the highestactivity against coconut oil because the principal fattyacid component was lauric acid (12:0), which was morehydrolyzable by the lipase than was oleic acid (18:1), theprincipal fatty acid component of olive oil. Similarly,the puriWed lipases eYciently hydrolyzed various oilsand fats such as palm oil, soybean oil, and coconut oil[14,16].In the oleochemical industry, enzymatic hydrolysis ofoils and fats is an energy-saving process compared withconventional high-temperature and high-pressure processes,and unsaturated fatty acids can be produced withoutoxidation [14]. Therefore, S5 lipase with the aboveproperties presents a suitable candidate for this purpose.Table 3Hydrolytic activities of S5 lipase against various natural oils and triacylglycerolsNatural oil/triglycerol Relative activity a (%)Olive oil (18:1) 100Coconut oil (12:0) 125Palm oil (16:0) 322Groundnut oil (18:1) 42Sesame oil (18:2) 50Soybean oil (18:3) 56Triacetin (C2) 50Tributyrin (C4) 58Tricaproin (C6) 65Tricaprylin (C8) 66Tricaprin (C10) 96Trilaurin (C12) 122Trimyristin (C14) 186Tripalmitin (C16) 217Triolein (C18) 247Note. Data are means of triplicate determinations. The olive oil emulsionwas substituted with the various substrates and assayed with1.0 ml enzyme solution at 37 °C for 30 min at pH 9.0.a The activities of the enzyme against substrates are shown as valuesrelative to those with olive oil.According to Voet et al. [22], nearly one-third of allknown enzymes require the presence of metal ions forcatalytic activity. This group of enzymes includes themetal enzymes, which contain tightly bound metal ioncofactors, most commonly transition metal ions such asFe 2+ , Fe 3+ , Cu 2+ , Mn 2+ , and Zn 2+ . Metal-activatedenzymes, in contrast, loosely bind metal ion from solution,usually the alkali and alkaline earth metal ionsNa + , K + Mg 2+ , and Ca 2+ .The eVect of metal ions and oxidizing, reducing, andchelating agents was tested at 1 mM in 25 mM Tris–HClbuVer at pH 9.0 (Table 4). Lipase was completely inhibitedby Fe 3+ , Ba 2+ , K + , Zn 2+ , and Cu 2+ , known as transitionmetal ions, after 30 min incubation. Alkaline earthmetal ions, Mg 2+ and Ca 2+ , were found to stimulate thelipase activity at 15 min incubation. However, prolongedincubation (30 min) led to decreases in enzyme activityof 52 and 95% for Mg 2+ and Ca 2+ , respectively. This suggestedthat S5 lipase is a metal-activated enzyme. In thisgroup of enzymes, the ions often play a structural rolerather than a catalytic one. The ions bind to the enzymeand change the conformation of the protein to countergreater stability to the enzyme. But transition metal ionschange the conWrmation of the protein to less stable dueto ion toxicity.Table 4EVect of various reagents on lipase activityReagent (1 mM) Relative activity (%)15 min incubation 30 min incubationNo addition 100 100NaCl 48 53MgCl 2 161 52CaCl 2 114 95KCl 52 36BaCl 2 45 25FeCl 3 23 20CuCl 2 32 25ZnSO 4 23 20Ammonium persulfate 50 48KI 18 12Sodium citrate 19 15EDTA 100 992-Mercaptoethanol 25 15Note. Data are means of triplicate determinations. S5 lipase was incubatedwith diVerent reagents in a 1:1 ratio at pH 9.0 for 15 and 30 minat 37 °C before the residual activity was measured.

Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274 273Fig. 6. Thin-layer chromatogram of the hydrolysis products of triolein catalyzed by S5 lipase. Mixtures containing 100 mM of triolein, 9 ml phosphatebuVer (pH 7.0), and 2 ml enzyme solution were shaken at 200 rpm at 37 °C. At 0, 3, 6, 9, and 12 h, the reaction products were extracted with 5 mlof n-hexane and analyzed by thin-layer chromatography. Lane 1, triolein; lane 2, 1(3) monoolein; lane 3, 1(2) diolein; lane 4, 1(3) diolein; lane 5, oleicacid; lane 6, 0 h; lane 7, 3 h; lane 8, 6 h; lane 9, 9 h; lane 10, 12 h; lane 11, Lipozyme; lane 12, Novozyme; and lane 13, marker.Similar results were reported by Sharma et al. [12]with their lipase from Pseudomonas sp. AG-8. Theydescribed that Ca 2+ ions activated the enzyme, whereasFe 3+ and Zn 2+ strongly inhibited its activity. A possibleexplanation of this phenomenon is that Ca 2+ has a specialenzyme-activating eVect that it exerts by concentratingat the fat–water interface. Therefore, calcium ionsmay carry out three distinct roles in lipase action:removal of fatty acids as insoluble Ca 2+ salts in certaincases, direct enzyme activation resulting from concentrationat the fat–water interface, and stabilizing eVect onthe enzyme. Fe 3+ and Zn 2+ , and especially Cu 2+ , werehighly toxic to lipase even at diluted solution [23].The activity of the lipase from Pseudomonas sp. S5decreased with both oxidizing and reducing agents.Among the reducing agents, the enzyme retained 48%activity after 30 min exposure with ammonium persulfate,whereas it retained 12% of the maximal activityafter 30 min exposure with potassium iodide. When thereducing agent 2-mercaptoethanol was added at a concentrationof 1 mM, enzyme activity dropped by 25 and15% after 15 and 30 min, respectively.The enzyme activity was not aVected by 1 mM EDTA,indicating that the lipase from Pseudomonas sp. S5 wasnot a metalloenzyme. Lin et al. [13] also showed thatactivity of lipase produced from P. pseudoalcaligenesF-111 was not aVected by 1 mM EDTA.Positional speciWcity against triacylglycerolsAccording to Farias et al. [24], lipase can be classiWedin terms of speciWcity toward the position of the acylgroup of triglycerides into two groups: 1,3-speciWc andnonspeciWc or random. The results of the thin-layerchromatography analysis of the hydrolysis products oftriolein are shown in Fig. 6. The enzyme cleaved not onlythe 1,3-positioned ester bonds but also the 2-positionedester bond of triolein. Thus, S5 lipase could hydrolyzethe ester bonds of triolein nonspeciWcally. Similarly,LST-03 organic solvent-tolerant lipase also hydrolyzedester bonds nonspeciWcally [10]. Similar Wndings werereported by Sigiura et al. [25] with their PseudomonasXuorescens lipase that cleaved ester bond nonspeciWcally.Lipozyme and Novozyme were included in this experimentas a control because the 1,3-positional speciWcityand nonspeciWc position, respectively, have already beenveriWed.According to Gao et al. [26], Pseudomonas sp. strain2106 lipase shows 1,3-positional speciWcity. Sugihara etal. [18] reported that P. cepacia lipase cleaved all of theester bonds of triolein, with some preference for the 1,3-ester bonds. The same Wndings were reported by Mencherand Alford [27] with their Pseudomonas fragi lipasethat hydrolyzed 1,3-ester bonds.References[1] M.V. Arbridge, W.H. Pitcher, Industrial enzymology: a looktowards the future, Trends Biotechnol. 7 (1989) 330–335.[2] G.A. Sellek, J.B. Chaudhuri, Biocatalysis in organic media usingenzymes from extramophiles, Enzyme Microbiol. Technol. 25(1999) 471–482.[3] B.C. Koops, H.M. Verheij, A.J. Slotboom, M.R. Egmond, EVect ofchemical modiWcation on the activity of lipases in organic solvents,Enzyme Microbiol. Technol. 25 (1999) 622–631.[4] H. Ogino, K. Miyamoto, M. Yasuda, K. Ishimi, H. Ishikawa, Growthof organic solvent-tolerant Pseudomonas aeruginosa LST-03 in thepresence of various organic solvents and the production of lipolyticenzyme in the presence of cyclohexane, Biochem. Eng. 4 (1999) 1–6.[5] R. Stead, Microbial lipases: their characteristics, role in food spoilage,and industrial uses, J. Dairy Res. 53 (1986) 481–505.

274 Organic solvent-tolerant lipase from Pseudomonas / R.N.Z.R.A. Rahman et al. / Anal. Biochem. 341 (2005) 267–274[6] S.N. Baharum, A.B. Salleh, C.N.A. Razak, M. Basri, M.B.A. Rahman,R.N.Z.R.A. Rahman, Organic solvent tolerant lipase byPseudomonas sp. strain S5: stability of enzyme in organic solventand physical factors aVecting its production, Ann. Microbiol. 53(2003) 75–83.[7] D.Y. Kwon, J.S. Rhee, A simple and rapid colorimetric methodfor determination of free fatty acid for lipase assay, J. Am. OilChem. Soc. 63 (1986) 69–92.[8] M.M. Bradford, A rapid and sensitive method for the quantitationof microgram quantities of protein using the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.[9] U.K. Laemmli, Cleavage of structure proteins during assembly ofthe head of bacteriophage T 4 , Nature 277 (1970) 680–685.[10] H. Ogino, S. Nakagawa, K. Shinya, T. Muto, N. Fujimura, M.Yasudo, H. Ishikawa, PuriWcation and characterization of organicsolvent tolerant lipase from organic solvent tolerant Pseudomonasaeruginosa LST-03, J. Biosci. Bioeng. 89 (2000) 451–457.[11] C. Schuepp, S. Kermasha, M.C. Michalski, A. Marin, Production,partial puriWcation, and characterization of lipase from Pseudomonasfragi CRDA 037, Proc. Biochem. 32 (1997) 225–232.[12] A.K. Sharma, R.P. Tiwari, G.S. Hoondal, Properties of a thermostableand solvent stable extracellular lipase from a Pseudomonassp. AG-8, J. Basic Microbiol. 41 (2001) 363–366.[13] S.F. Lin, C.M. Chiou, C.M. Yeh, Y.C. Tsai, PuriWcation and partialcharacterization of an alkaline lipase from Pseudomonaspseudoalcaligenes F-111, Appl. Environ. Microbiol. 62 (1996)1093–1095.[14] T. Iizumi, K. Nakamura, T. Fukase, PuriWcation and characterizationof a thermostable lipase from newly isolated Pseudomonas sp.KWI-56, Agric. Biol. Chem. 54 (1990) 1253–1258.[15] E.J. Gilbert, A. Cornish, C.W. Jones, PuriWcation and properties ofextracellular lipase from Pseudomonas aeruginosa EF2, J. Gen.Microbiol. 137 (1991) 2223–2229.[16] U.K. Jinwal, U. Roy, A.R. Chowdhury, A.P. Bhaduri, P.K. Roy,PuriWcation and characterization of an alkaline lipase from anewly isolated Pseudomonas mendocina PK-12CS and chemoselectivehydrolysis of fatty acid ester, Bioorg. Med. Chem. 11 (2003)1041–1046.[17] J. Sikkema, D.J.A.M. Bont, B. Poolman, Intercalations of cyclichydrocarbons with biological membranes, J. Biol. Chem. 269(1994) 8022–8028.[18] A. Sugihara, M. Ueshima, Y. Shimada, S. Tsunasawa, Y. Tominaga,PuriWcation and characterization of a novel thermostablelipase from Pseudomonas cepacia, J. Biochem. 112 (1992) 598–603.[19] A.M. Klibanov, Enzymes that work in organic solvents, Chem-Tech. (1986) 354–359.[20] A.M. Klibanov, Improving enzymes by using them in organic solvents,Nature 409 (2001) 241–246.[21] R.P. Yadav, P. Agarwal, S.N. Upadhay, Microbial lipases: toolsfor drug discovery, J. Sci. Ind. Res. 59 (2000) 977–987.[22] D. Voet, J.G. Voet, C.W. Pratt, Fundamentals of Biochemistry,John Wiley, New York, 1999.[23] E.D. Wills, The relation of metals and –SH groups to the activityof pancreatic lipase, Biochim. Biophys. Acta 40 (1960) 481–490.[24] R.N. Farias, M. Torres, R. Canela, Spectrophotometric determinationof the positional speciWcity of nonspeciWc and 1,3-speciWclipases, Anal. Biochem. 252 (1997) 186–189.[25] M. Sigiura, M. Isobe, F. Hirata, T. Inigawa, Application of lipoproteinlipase for the assay of serum triglyceride, Chem. Pharm.Bull. (Tokyo) 23 (1975) 1417–1420.[26] X.G. Gao, S.G. Cao, K.C. Zhang, Production, properties andapplication to nonaqueous enzymatic catalysis of lipase fromnewly isolated Pseudomonas strain, Enzyme Microbiol. Technol.27 (2000) 74–82.[27] J.R. Mencher, J.A. Alford, PuriWcation and characterization of thelipase of Pseudomonas fragi, J. Gen. Microbiol. 48 (1967) 317–328.