h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Industrial
Microbiology
Process
optimization
for
production
and
purification
of
a
thermostable,
organic
solvent
tolerant
lipase
from
Acinetobacter
sp.
AU07
P.
Gururaj,
Subramanian
Ramalingam,
Ganesan
Nandhini
Devi
∗,
Pennathur
Gautam
∗CentreforFoodTechnology,AnnaUniversity,Chennai,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received25October2014 Accepted16April2015 Availableonline26April2016 AssociateEditor:JorgeGonzalo FariasAvendano
Keywords: Acinetobactersp.
Responsesurfacemethodology MALDI-TOF
Organicsolventtolerantlipase Thermostablelipase
a
b
s
t
r
a
c
t
Thepurposeofthisstudywastoisolate,purifyandoptimizetheproductionconditions ofanorganicsolventtolerantandthermostablelipasefromAcinetobactersp.AU07isolated fromdistillerywaste.Thelipaseproductionwasoptimizedbyresponsesurface methodol-ogy,andamaximumproductionof14.5U/mLwasobservedat30◦CandpH7,usinga0.5% (v/v)inoculum,2%(v/v)castoroil(inducer),andagitation150rpm.Theoptimized condi-tionsfromtheshakeflaskexperimentswerevalidatedina3Llabscalebioreactor,andthe lipaseproductionincreasedto48U/mL.Theenzymewaspurifiedbyammoniumsulfate precipitationandionexchangechromatographyandtheoverallyieldwas36%.SDS-PAGE indicatedamolecularweightof45kDaforthepurifiedprotein,andMatrixassistedlaser desorption/ionizationtimeofflightanalysisofthepurifiedlipaseshowedsequence sim-ilaritywithGDSLfamilyoflipases.TheoptimumtemperatureandpHforactivityofthe enzymewasfoundtobe50◦Cand8.0,respectively.Thelipasewascompletelyinhibitedby phenylmethylsulfonylfluoridebutminimalinhibitionwasobservedwhenincubatedwith ethylenediaminetetraaceticacidanddithiothreitol.Theenzymewasstableinthepresence ofnon-polarhydrophobicsolvents.DetergentslikeSDSinhibitedenzymeactivity;however, therewasminimallossofenzymeactivitywhenincubatedwithhydrogenperoxide,Tween 80andTritonX-100.Thekineticconstants(KmandVmax)revealedthatthehydrolytic activ-ityofthelipasewasspecifictomoderatechainfattyacidesters.TheVmax,KmandVmax/Km ratiooftheenzymewere16.98U/mg,0.51mM,and33.29,respectivelywhen4-nitrophenyl palmitatewasusedasasubstrate.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Lipases(EC 3.1.1.3)are enzymes thatcleave ester bondsin lipidicsubstrates.Inthepresenceofwater,theycatalyzethe
∗ Correspondingauthorsat:DepartmentofBiotechnology,AnnaUniversity,Chennai,India. E-mails:[email protected](G.NandhiniDevi),[email protected](P.Gautam).
hydrolysis of triglycerides to form monoglycerides, diglyc-erides, glycerol and free fatty acids. Lipases are serine hydrolasesandareactiveatthelipid-waterinterface.1,2They
areubiquitousinnatureandarefoundinavarietyofplants, animals and microorganisms.3 Most bacterial lipases are
http://dx.doi.org/10.1016/j.bjm.2015.04.002
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
secreted extracellularly and are versatile biocatalysts that carry out a variety of reactions viz. hydrolysis, esterifica-tion, transesterification,inter esterification, acidolysis, and aminolysis.4,5 Lipasesutilizeawidespectrumofsubstrates,
andsomeofthemarestableatextremetemperature,andpH conditionsandinorganicsolvents.Theyareusedascatalysts forreactionsinreducedwaterenvironments.6Frequently,the
substratesoflipasesareinsolubleinaqueoussolution.Hence, conductingthereactionsinorganicsolventscanimprovethe dissolutionofsubstrates andincreasesubstrateavailability, inadditiontoaidingintheeasyseparationofenzymesfrom substratesorproducts.7,8
Lipolytic strains isolated from industrial effluentsshow potentialutility inbiodegradation andbioremediation. The biofilmformedbylipasesecretingorganismscanbeusedto degradefatsandoils.9Therefore,wehaveproduced,purified
andbiochemicallycharacterizedalipaseisolatedfrom Acine-tobactersp.AU07.Wealsooptimizedthephysicalconditions byemployingresponsesurfacemethodology(RSM)toimprove lipaseproduction.
Materials
and
methods
ChemicalsEnzyme substrates (4-nitrophenyl esters) and inhibitors were procured from Sigma (St. Louis, USA). Chemicals for mediapreparationwerepurchasedfromHi-Media(Mumbai, India). The ion-exchange chromatography sorbent diethy-laminoethyl(DEAE)Sepharosefastflowwaspurchasedfrom GEHealthcare.Allchemicalsusedwereofanalyticalgrade. Isolationandscreeningoflipaseproducingorganisms Thelipaseproducingorganismswereisolatedfromadistillery unit.Theliquidsample(1mL)wassuspendedin9mL steril-izedwater,seriallydilutedandspreadonselectivemedium containingsesame oilasthe sole carbon sourceand incu-bated at 37◦C for 24h. This selective medium contained 2.0g/Lpeptone,5.0g/LNaCl,20 (v/v)sesame oil(emulsified with 0.01% Triton X-100), and 15.0g/L bacteriologicalagar. Toscreenforlipaseproduction,individualbacterialcolonies werestreakedontoplatescontainingtributyrin1.25g/L (emul-sifiedwith0.01%TritonX-100)and15g/Lbacteriologicalagar. Theplateswereincubatedat37◦Cfor24h,andcoloniesthat formedazoneofclearancewerelipolyticpositivestrains.The thirteenpositiveisolateswerefurtherscreenedformaximal secretionofextracellularlipasebyassayingthelipaseactivity inliquidcultureusing4-nitrophenylpalmitateasasubstrate at37◦C.TheAcinetobactersp.AU07strain,whichshowedthe highestactivity,wasselectedforfurtherstudy.Thisstrainwas maintainedinglycerolstocks(50%,v/v)andstoredat−20◦C.
Identificationofthelipolyticstrain
The taxonomy of the isolated strain was examined using Bergey’sManualofDeterminativeBiologyandconfirmedby 16S rDNA sequencing. A BLAST analysis of the 16S rDNA sequenceidentifiedthestrainasAcinetobactersp.AU07.The
800bp16SrDNAgenesequenceoftheAcinetobactersp.AU07 strainhasbeensubmittedtotheGenBankdatabase,withthe accessionnumberHQ914215.
Optimizationofmediaandcultureconditionsforlipase productionbyresponsesurfacemethodology
One-factor-at-atimestrategy
TheproductionoflipasebyAcinetobactersp.AU07was per-formedusingdifferentvegetableoilsasinducersviz.castor oil,palmoil,coconutoil,sesameoil,andoliveoilat(1%,v/v), with1%inoculumat30◦Cfor20hinarotaryshaker(150rpm). Themediacontainingoilswereemulsified with0.25% gum acaciaandwereadjustedtopH7.0.Theindividualeffectsof pH,temperatureandinducersweremonitoredandoptimized. Thecell-freesupernatantwasrecoveredbycentrifugationat 12,500×gfor10minat4◦Candusedtodetermine extracellu-larlipaseactivity.
Thenutrientmediumcontainingcastoroilenhancedlipase secretionandwasthereforeselectedforfurtheroptimization oflipaseproductionbycentralcompositedesign(CCD)and responsesurfaceanalysis.ThevariablesutilizedforRSMwere asfollows:temperature,pH,agitation,inducerconcentration, andinoculumvolume.Allfivevariablesweretestedatthree levelsanddesignatedas−1,0,+1.ThedatafromCCDwere analysed bytheleastsquaresmethod.Atotalof26 experi-mentswereconducted.Theresponsevalues(Y)ineachtrial weretheaverageofthethreereplicates.Thestatistical soft-warepackage‘DesignExpert’software(Version8.0,Stat-Ease Inc.,Minneapolis,USA)wasusedtoanalysetheexperimental designs.
Spectrophotometricassayforlipaseusing4-nitrophenyl palmitate
The lipase activity was measured spectrophotometrically using4-nitrophenylpalmitate(4-NPP)asthesubstrate.First, 400l of the enzyme was equilibrated with 50l of 1M Tris–HClbuffer(finalconcentration50mM),anddilutedwith 530lsterilewater.Next,20lof4-NPP(finalconcentration 1mM)was added, and thereaction wasincubated at30◦C for10min.Theconcentrationofreleased4-nitrophenolwas measuredat410nm.Theproteincontentwasdeterminedby Bradford’smethodusingtheBio-Rad assayreagent,10with
bovineserumalbumin(BSA)asastandard. Purificationoflipase
Theculturewasgrownfor16handcentrifugedat12,500×g for 15min at 4◦C. To precipitate the proteins the culture supernatant wasaddedwithsolidammonium sulfate(60% saturation)withcontinuousstirringandincubatedat4◦Cfor 24h.Afterincubation,thesamplewascentrifugedat4◦Cfor 15minat12,500×g,andtheresultingpelletwasdissolvedin 10mMTris–HClbuffer(pH8.0) anddialyzedwiththe same bufferfor12hat4◦C.Thedialyzedproteinsamplewasthen loadedon-toaDEAESepharoseanionexchanger,whichhad been pre-equilibratedwith20mMTris–HClandelutedwith abufferedNaCl(0.1–1M)concentrationgradient.Thelipase containing fractionswere pooled and dialyzedwith 10mM
Tris–HClbuffer. Theresulting dialyzed proteinsamplewas thenlyophilized.
RP-HPLCanalysisofpurifiedlipase
ThepurityoftheenzymewasanalysedbyRP-HPLCusingan Agilent1100HPLCsystemwithaC-18column(ZorbaxC-18, 4.6mm×250mmi.d.,5mparticlesize;Agilenttechnologies). Thecolumnwaselutedwith0.1%(v/v)trifluroaceticacid(TFA) inwaterand0.1%(v/v)TFAinacetonitrile.Thebound pro-teinswere elutedwithanincreasinggradient of2to100% acetonitrilefor40minataflowrateof1mL/min.
MolecularweightdeterminationbySDS-PAGE
Thelipasepurifiedbyionexchangechromatographywas sub-jectedtoSDS-PAGE(4%and10%,v/vacrylamidestackingand separatinggel).11 Next, the resulting gel was stained with
CoomassieBrilliantBlue,andthemolecularweightwas deter-minedbycomparingtheproteinbandwithastandardprotein markercontainingmixturesofphosphorylase(97kDa), albu-min(66kDa),ovalbumin(45kDa),carbonicanhydrase(30kDa), trypsininhibitor(20kDa)and␣-lactalbumin(14kDa). Massspectrometricsequenceanalysisoflipasefrom Acinetobactersp.AU07
MALDI-TOFMSisasensitivetechniqueusedtoanalyse pro-teinsequencesbyscreeningthemassesofpeptidesandsmall proteins.Bymeasuringthemassesofpeptides,itispossible todeterminethesequenceoftheprotein.Thepurifiedlipase wasexcisedfromtheSDS-PAGEgelandsubjectedto proteoly-sisbytrypsin,andtheresultingpeptideswereanalysedusing MALDI-TOF.Themassoftheresiduesofthepurifiedlipasewas obtainedandusedtosearchagainsttheMASCOTdatabasefor peptidemassfingerprinting.
BiochemicalcharacterizationofpurifiedAcinetobactersp. AU07lipase
Effectoftemperatureonlipaseactivityandstability
Theoptimumtemperatureforlipaseactivitywasmeasured byincubatingaliquotsofpurifiedenzymewith4-nitrophenyl palmitate (4NPP) (1mM final concentration) and 20mM sodium-phosphate buffer attemperatures ranging from 30 to80◦Cfor10min,andtherelativeactivitywasdetermined followingincubation.
Thethermalstabilityofthelipasewasmeasuredby incu-batingaliquotsofpurifiedenzymeat40◦C,50◦C,60◦Cand 70◦Cfor5h,afterwhichtheresidualactivitywasdetermined usingthefollowingequations:
ln(A)=ln(A0)−kdt (1)
whereA0istheinitialactivity,Aistheresidualactivityintime
tandkdisfirst-orderdeactivationratecoefficient:
t1/2= ln(0.5)k d
(2)
From Eq. (1), the thermal deactivation profiles were determined,12 andtherespectivedeactivationrate(k
d)
esti-mated.FromEq.(2),thehalf-life(t1/2)ofthebiocatalystwas
estimatedfromthepreviouslydeterminedkdvalue.
EffectofpHonlipaseactivityandstability
The optimal pH was determined by assaying the puri-fied enzyme at different hydrogen ion concentrations using the followingbuffers: citric acid/sodium acetate(pH 3.0–5.0),sodiumphosphate(pH6.0–7.5),Tris–HCl(pH7.0–9.0), glycine–NaOH(pH9.0–10.0)andphosphate–NaOH(pH11.0).
ForthepHstabilitystudies,thepurifiedenzymewas incu-batedwiththerespectivebuffers(50mM)for1hat30◦C,and thentheresidualactivitywasdetermined.
Effectoforganicsolventsonlipasestability
The purified lipase was incubated with 30% (v/v) organic solvent at30◦C with constant agitationat 150rpmfor4h. Aliquots ofthe suspendedenzymewere taken atdifferent intervals,andthesolventwasevaporated.Theenzyme activ-itywasthenmeasuredat30◦CandpH8.0.Thestabilityofthe lipasewascalculatedbycomparingtheresidualactivitywith thecontrol,whichwasincubatedwithouttheorganicsolvent.
Effectofmetalionsonlipaseactivity
Various metalsalts (ZnCl2, MgSO4·7H2O,FeCl3,CaCl2·2H2O,
AgNO3,MnSO4·H2O)wereaddedtothepurifiedlipaseat
con-centrationsof2mM,5mM,and10mMandincubatedat30◦C for30min.Therelativeactivityoftheenzymewasmeasured asthechangeintheactivityoftheenzymeinthepresence ofmetalsaltswhencomparedtotheenzymeactivitywithout metalsalts.
Effectofinhibitors,detergents,andoxidizingagentonlipase stability
Thestabilityofthepurifiedlipaseinthepresenceofinhibitors, detergents and oxidizing agent was evaluated by incubat-ing the purified lipase with the inhibitors-DTT, EDTA and PMSFatafinalconcentrationsof2mM,5mMand10mM;the detergents-Tween80,TritonX-100,SDSatafinal concentra-tionof0.25%,0.5%and1%;andanoxidizingagent-H2O2ata
finalconcentrationof0.5%,1%,1.5%and2%.Theincubated enzyme(withoutinhibitors,detergentsandoxidizingagent) wasusedasareferencetocalculatetherelativeactivity.
Determinationoflipasekineticsusingdifferentsubstrates
The hydrolytic activity of Acinetobacter sp. AU07 lipase was investigated using different substrates, such as 4-nitrophenyl butyrate(4-NPB), 4-nitrophenyl laurate (4-NPL), 4-nitrophenylmyristate(4-NPM)and4-nitrophenylpalmitate (4-NPP),atvarioussubstrateconcentrations(0.2–2mM).The Michaelis–Menten enzyme kinetic constants Km and Vmax
weredeterminedfromtheLineweaver–BurkandWoolf–Hanes plots.
Table1–ExperimentaldesignsusedinRSMstudieswithtwoindependentvariablesshowingtheobservedandthe predictedvaluesoflipaseproduction.
Std Run Factor1 Factor2 Factor3 Factor4 Factor5 Observedactivity (U/mL) PredictedLipase activity(U/mL) 1 26 40.00 8.00 100.00 3.00 0.25 6.20 6.36 2 3 40.00 6.00 200.00 3.00 0.25 6.60 6.76 3 8 20.00 8.00 200.00 1.00 0.75 4.40 4.56 4 11 40.00 8.00 200.00 1.00 0.25 6.00 6.16 5 1 40.00 8.00 100.00 1.00 0.75 8.65 8.81 6 13 40.00 6.00 100.00 3.00 0.75 5.32 5.48 7 5 20.00 6.00 200.00 3.00 0.75 4.13 4.29 8 12 20.00 8.00 100.00 3.00 0.75 5.40 5.56 9 22 40.00 6.00 200.00 1.00 0.75 4.60 4.76 10 4 20.00 8.00 200.00 3.00 0.25 7.73 7.79 11 25 20.00 6.00 100.00 1.00 0.25 2.42 2.74 12 10 20.00 7.00 150.00 2.00 0.50 4.32 4.02 13 14 40.00 7.00 150.00 2.00 0.50 8.40 8.10 14 24 30.00 6.00 150.00 2.00 0.50 6.12 5.82 15 15 30.00 8.00 150.00 2.00 0.50 9.40 9.10 16 9 30.00 7.00 100.00 2.00 0.50 11.50 11.20 17 19 30.00 7.00 200.00 2.00 0.50 12.32 12.02 18 20 30.00 7.00 150.00 1.00 0.50 8.42 8.12 19 21 30.00 7.00 150.00 3.00 0.50 11.00 10.70 20 18 30.00 7.00 150.00 2.00 0.25 4.20 4.00 21 7 30.00 7.00 150.00 2.00 0.75 13.20 12.90 22 2 30.00 7.00 150.00 2.00 0.50 14.50 14.65 23 23 30.00 7.00 150.00 2.00 0.50 14.50 14.65 24 6 30.00 7.00 150.00 2.00 0.50 14.50 14.65 25 16 30.00 7.00 150.00 2.00 0.50 14.50 14.65 26 17 30.00 7.00 150.00 2.00 0.50 14.50 14.65
Thetableincludesthefactorsinfluencinglipaseproduction.Factor1:temperature(◦C),Factor2:pH,Factor3:agitation(rpm),Factor4:inducer concentration(%).
Results
and
discussion
Isolationandidentificationofbacteriaproducing extracellularlipase
Thebacterialstrainsthatwereisolatedfromdistillerywaste werescreenedforlipasesecretionbyselectionontributyrin agarplates.TheAU07strainthatshowedalargerzoneof clear-ancearoundthecolonywasselectedforfurtherstudies.The morphologicalandbiochemicalcharacterizationrevealedthat theAU07strainisaerobic,gramnegativeandnon-motile.The strainwasconfirmedasAcinetobactersp.AU07by16SrDNA sequencingwhichisinaccordancewithBergey’sManualof DeterminativeBiology.
Optimizationofcultureconditionsforlipaseproductionby responsesurfacemethodology
A maximal lipase activity of 331.16U (specific activity of 38.64U/mg)wasobtainedbyculturingthebacteriumat30◦C andpH7.0for16h.Thelipaseproductionreachedamaximum duringthestationaryphaseandthengraduallydecreased.The fermentationtimetoobtainmaximumlipaseactivityis simi-lartothatoflipaseproductionbyAcinetobactersp.BK44where maximallipaseactivityoccursafter12hofincubation.13
Thoughthelipaseproductionfromthisstrainis constitu-tive,theincorporationofoilsinthe mediumincreasedthe
enzymeproduction.14–16 Earlierreportsdemonstratedlipase
productioninamediumcontaininglipidicsubstratesasthe sole carbon sourcewith an organic nitrogen source,14 but
sometimes, lipaseproduction wasrepressed bylong chain fatty acidesters.15Castoroilandsesameoilinducedlipase
productionwhencomparedwithotherlipid/oilsources (Sup-plementalFig.S1).Castoroilisthesourceofricinoleicacid whichinduceslipaseproduction.17Theoptimizationof
cul-ture conditions one factor at a time is time consuming; therefore,responsesurfacemethodology,whichisa statisti-calmethod, wasusedtodetermine theoptimalconditions for lipase production.18,19 Theanalysis of variance for the
response surface quadratic model for lipaseproduction is showninTable1.The2Dcontourplotsand3Dresponse sur-facearegraphicalrepresentationsoftheregressionequation. They wereplottedbasedon themodelregression equation toevaluatetheinteractionsamongthetestedvariablesand determinetheoptimumlevelofeachfactortomaximizelipase production(Fig.1).
Thecontourswereplottedconsideringonevariable con-stantatitscentrallevel, whiletheother wasvariedwithin theirexperimentalranges.Thedatafromthe2Dcontourplots of lipaseproductionby Acinetobactersp. AU07 (Supplemen-talFig.S2)suggestasignificantincreaseinlipaseproduction resultingfrom allthefactorsinvestigated:0.5%(v/v) inocu-lum size, 30◦C fermentation temperature, pH 7, 2% (v/v) inducerconcentrationandagitationat150rpm.Theoptimal temperature forlipaseproductionobservedinthis studyis
6 8 10 12 14 16
Lipase activity Lipase activity
Lipase activity Lipase activity
Lipase activity Lipase activity
Lipase activity Lipase activity
Lipase activity Lipase activity
8.00 7.50 7.00 A: Temperature A: Temperature A: Temperature A: Temperature B: pH B: pH B: pH B: pH C: Agitation C: Agitation C: Agitation C: Agitation E: Inoculum volume D: Inducer concentration D: Inducer concentration D: Inducer concentration D: Inducer concentration E: Inoculum volume E: Inoculum volume E: Inoculum volume 20.00 25.00 30.00 35.00 40.00 16
B
D
F
H
J
14 12 10 8 6 4 200.00 175.00 150.00 16 14 12 10 8 6 4 0.75 0.65 0.55 16 14 12 10 8 6 4 3.00 2.50 2.00 1.50 16 14 12 10 8 6 4 3.00 2.50 2.00 1.50 16 14 12 10 8 6 4 0.75 0.65 0.55 0.450.35 0.25 1.00 1.50 2.00 2.50 3.00 1.00 100.00 125.00 150.00 175.00 200.00 1.00 6.00 6.50 7.00 7.50 8.00 0.45 0.35 0.25 20.00 25.00 30.00 35.00 40.00 125.00 100.00 20.00 25.00 30.00 35.00 40.00 6.50 6.00 16 14 12 10 8 6 3.00 2.50 16 14 12 10 8 6 4 200.00 175.00 16 14 12 10 8 6 4 0.75 0.65 0.55 0.450.35 0.25 16 14 12 10 8 6 4 0.75 0.65 0.55 0.45 0.35 0.25 100.00 125.00 150.00 175.00 200.00 6.00 6.50 7.00 7.50 8.00 150.00 125.00 100.006.00 6.50 7.00 7.50 8.00 2.00 1.50 1.00 20.00 25.00 30.00 35.00 40.00I
G
E
C
A
Fig.1–ResponsesurfaceplotsoftheimpactofvariousfactorsonoptimalproductionoftheAU07lipase.Eachfiguredepicts theimpactofspecificfactorsonlipaseproduction.Thevariablesinvestigatedweretemperature,pH,agitation,inducer concentration,andinoculumvolume.(A)TemperatureandpH,(B)temperatureandagitation,(C)temperatureandinducer, (D)temperatureandinoculumvolume,(E)pHandagitation,(F)pHandinducer,(G)pHandinoculumvolume,(H)agitation andinducer,(I)agitationandinoculumvolume,and(J)inducerandinoculumvolume.
similartothoseofAcinetobacterradioresistens20and Acinetobac-tercalcoaceticusLP009.21Ourstandardizedconditionsgavean
optimumlipaseproductionof14.5U/mL(Fig.2A).
The model predicted a maximum lipase production of 16.08U/mL at 34◦C, and pH 7.8, with castor oil as the
inducer (2.3%, v/v), at an inoculum volume (0.44%, v/v), and with agitation at 199rpm. To validate the predicted response, the lipaseproduction was tested experimentally underoptimalcultureconditions,andthemaximumlipase activitywas15.84U/mL.Thelipaseproductionfromthisstrain
Table2–ResultsoftheANOVAfortheresponsesurfacequadraticmodelforlipaseproduction.
Source Sumofsquares Degreesoffreedom Meansquare F-value p>F Adj.R2
Model 395.68 20 19.78 67.86 <0.0001 0.9816
Residual 1.46 5 0.29
Lackoffit 1.46 1 1.46
Pureerror 0.000 4 0.000
Fig.2–GrowthcurveandlipaseproductionfromAcinetobactersp.AU07inshakeflaskandbioreactor.(A)Growthcurveand lipaseproductionfromAcinetobactersp.AU07inashakeflask(30◦C,pH7.0,0.5%,v/vinoculumsize,2%,v/vinducer concentration,150rpm).Thelipaseactivitywasmeasuredusing1mM4-NPasthesubstrate.Thereactionwasincubatedat 30◦Cfor10min,andtheabsorbancewasmeasuredat410nm.TheODwasconvertedtolipaseactivity(U/mL)andthegraph wasplotted.Eachdatapointisthemeanofthreereplicates.(B)GrowthcurveandproductionoflipasefromAcinetobactersp. AU07inabioreactor(3L)(30◦C,pH7.0,0.5%,v/vinoculumsize,2%,v/vinducerconcentration,150rpm,1.5vvmaeration). Thelipaseactivitywasmeasuredusing4-NP(1mM)asthesubstrate.Thereactionwasincubatedat30◦Cfor10minandOD wasmeasuredat410nm.TheODwasconvertedtolipaseactivity(U/mL)andthegraphwasplotted.Eachdatapointisthe meanofthreereplicates.
cultured under optimized conditions is similar to that of A. calcoaceticus.22 The significance of the response surface
quadraticmodelwasanalysedbyANOVA(Table2),andused togenerateaquadraticequation forlipaseactivity(Y).The adjustedR2(adj.R2=0.9816)ishighandconfirmedthemodel
is significant. Additionally, the statistical coefficients con-firmedthat the modelis significant and canbeapplied to increaselipaseproduction.Theproductionoflipaseincreased to48U/mLwhencultivatedina3Lbioreactorundercontrolled conditions(Fig.2B). Y(Lipaseactivity)=+14.70+1.12×A+0.90×B+0.23×C + 0.71×D+2.44×E+1.19×A×B +0.96×A×C+0.70×A×D−0.92×A ×E+1.39×B×C+1.20×B×D−0.73 ×B×E+1.66×C×D−1.38×C ×E−1.44×D×E−2.60×A2−2.18×B2 −0.93×C2−1.59×D2−1.88×E2
Theequationabovegivestherelationshipbetweenlipase activityandthetestedvariables,whereAisthetemperature, BispH,Cisagitation,Disinducerconcentration,and Eis inoculumvolume.
Purificationoflipase
The Acinetobacter sp. AU07 lipase (ASL) was partially puri-fiedbyammoniumsulfateprecipitation(30–60%saturation)
and further purified to homogeneity by anion exchange chromatography. Thelipase wassuccessfully purified from cell-freecrude supernatantwithanoverallyieldof36.04%, witha2.2foldpurificationandaspecificactivityof83.47U/mg. ThepurificationresultsaresummarizedinTable3.The Acine-tobactersp.AU07lipasewaselutedinanincreasinggradient ofNaCl(0.1–1M).Themaximallipaseactivitywasobtainedin thefractionelutedwith600mMofNaCl,indicatingthestrong bindingoftheproteintotheion-exchangeresin.Thepurityof theenzymewasanalysedbyRP-HPLC.Thelipasewaseluted asasinglepeakwitharetentiontimeof20.537minasshown intheSupplementalFig.S3.
SDS-PAGEofion-exchangepurifiedlipase
Thepurifiedenzymeresolvedintoasinglebandonan SDS-PAGEgel(Fig.3)hadanestimatedmolecularweightof45kDa, whichisthesameastheA.radioresistensCMC-1lipase.23 Massspectrometricsequenceanalysisofthelipasefrom Acinetobactersp.AU07
Thelipaseinthisstudyhas55%homologytothelipasefrom A.calcoaceticusPHEA-2followedbythelipasesfrom Acineto-bacterpittiandAcinetobacterbaumanii.Thepartialaminoacid sequenceisshowninFig.4.Themolecularmassandthe iso-electricpoint (pI)were predicted tobe41.196kDaand7.14, respectively. Thepeptide masses and the matchingamino acid sequences ofpeptides are listed in the Supplemental Fig.S4.
Theactivityofavarietyoflipasesprimarilydependson the catalytic triad typically formed by Ser, His, and Asp
Table3–PurificationtableofAcinetobactersp.AU07lipase.
Step Totalprotein(mg) Totalactivity(U) Specificactivity(U/mg) Yield(%) Purification(fold)
Supernatant 8.57 331.16 38.64 100 1
Ammoniumsulphate 4.30 232.09 53.96 70.10 1.4
Ion-exchangechromatography 1.43 119.36 83.47 36.04 2.2 Atwo-steppurificationofthelipasebyammoniumsulfate(60%saturation)andionexchangechromatography(0.1–1MNaClelution).Theassay wasperformedbyincubatingtheenzymeat30◦Cfor10minwith1mM4-NPasthesubstrate.Theproteinconcentrationwasestimatedbythe standardBradfordassayprocedure.Eachvalueistheaverageofthreereplicates.
Fig.3–SDS-PAGE(10%)ofthepurifiedlipasefrom Acinetobactersp.AU07.LaneA:Molecularmassmarker proteins:phosphorylase(97kDa),albumin(66kDa), ovalbumin(45kDa),carbonicanhydrase(30kDa),trypsin inhibitor(20kDa)and␣-lactalbumin(14kDa).LaneB: Purifiedlipase(10g)afterion-exchangechromatography showingsingleband.
residues.ThistriadwaspresentintheisolatedAcinetobacter sp.lipasesequence(SupplementalFig.S5).Partialaminoacid sequencingconfirmedthattheenzymeisolatedinthisstudy possessesthecharacteristicsofthelipaseenzymefamily.
Table4–Half-lifeandthermaldeactivationconstantsat differenttemperatures.
Temperature(◦C) kd(h−1) t1/2(h)
40 0.50 1.38
50 0.64 1.09
60 1.03 0.67
Thehalf-lifeandthermaldeactivationconstantsweredetermined byincubatingaliquotsofpureenzymeatvarioustemperaturesfor 5h,afterwhich,theresidualenzymeactivitywasdetermined.
BiochemicalcharacterizationofpurifiedAcinetobactersp. AU07lipase
Effectoftemperatureonactivityandstabilityoflipase
The optimum temperature for lipase activity was 50◦C (Fig.5A),whereasapproximately75%and25%activitywere observedat60◦Cand70◦C,respectively.Theoptimum tem-peratureforlipaseactivitywassimilartothatoflipasefrom A. calcoaceticusLP009.21 Thestability ofthe lipaseat
differ-enttemperatureswasdeterminedbyincubatingtheenzyme atdifferenttemperaturesfor5handassayingtheenzymeat 50◦C.After5h,theenzymeretained78%activityat40◦Cand 72% activityat50◦C (Fig. 5B),whichdemonstratesthat the enzymeisthermo stable.Alinearincreaseinthe deactiva-tionconstants(kd)wasobservedwhenthetemperaturewas
increasedfrom40◦Cto60◦C.Thehalf-lifeoftheenzymewas 1h38minat40◦C,1h9minat50◦C and 1h and 7minat 60◦C.Thecorrespondingthermaldeactivationconstants(kd)
andhalf-life(t1/2)ofthepurifiedlipaseareshowninTable4.
EffectofpHonactivityandstabilityoflipase
MostofthelipasesfromAcinetobactersp.arealkalistable,24,25
losingtheiractivityatlowpH.Thiscouldbeduetotheloss ofthecoordinatingmetalionCa2+metalionfromtheactive
Fig.5–(A)EffectoftemperatureontheactivityofAcinetobactersp.AU07.Thereactionmixturewasincubatedatdifferent temperatures(30–80◦C),andtheenzymeactivitywasassayedunderstandardassayconditions.(B)Effectoftemperatureon thestabilityoftheAcinetobactersp.AU07lipase.Todeterminethethermalstability,theenzymewaspre-incubatedat varioustemperatures(40–70◦C)for5h,andtheenzymeactivitywasassayedunderstandardassayconditions.(C)Effectof pHontheactivityandstabilityoftheAcinetobactersp.AU07lipase.Fortheenzymeactivityassays,thereactionwas assayedatvariouspHlevelsunderstandardassayconditions.Fortheenzymestabilityassays,theenzymewas
pre-incubatedwithvariouspHbuffers(50mM)ofpH(5–11)at30◦Cfor1h,andthentheresidualactivitywasdeterminedby assayingunderstandardassayconditions.
siteatlowpH.26Thepurifiedlipaseinthisstudyshowedthe
highestactivityatpH8.0whichissimilartotheAcinetobacter sp.CR9lipase27andtheAcinetobacterbaylyilipase.28TheA. radioresistensCMC-1lipasehadapHoptimaat10.5,23whereas, A.calacoaceticus1–7hadapHoptimaof9.29Incontrast,the
lipasesfromA.calcoaceticusLP009andAcinetobactersp.13,21had
pHoptimaof7and6respectively.ASLpresentedarelative activityof65%atpH9,26%atpH10,andonly11%atpH11. Theactivitywasnotreducedsignificantlyafterincubationfor 1hat30◦C(Fig.5C).
Effectoforganicsolventsonlipasestability
Thestrippingofwaterfromanenzymeprimarilyoccursin thepresenceofpolarsolventsandtoalesserdegreeinthe presenceof hydrophobic solventsdetermined bylogp val-ues.Forinstance,methanoldesorbedapproximately60%of thewaterboundtotheenzyme,whilehexaneonlydesorbed
0.5%.30 Solvents withhigh logp valuescause less
inactiva-tion ofenzymethansolventswithlowlogpvalues.31 Many
researchershavepreviouslyreportedthesolventstablenature oflipases,24,32–34 thus thestability ofAcinetobactersp.AU07 lipaseindifferentsolventswasstudied(Table5).Thepolar solvents(lowlogp)reducedtheenzymeactivity,whereasthe non-polarsolvents(higherlogp)increasedtheenzyme activ-ity.Amongthepolarsolvents,DMSOincreasedtheactivity1.9 fold.Tolueneanon-polarsolvent,increasedtheactivity 3.5-foldwhenincubatedfor2hand2.8-foldwhenincubatedfor 4h.Whenincubatedfor4heachat30◦C,othernon-polar sol-ventsalsoincreasedtheactivityconsiderably,viz.,n-hexane 1-fold,dichloromethane0.4-fold,andn-heptane0.7-fold.
Effectofmetalionsonlipaseactivity
Metalionsdecreasedtheactivityoftheenzyme.Thelipase retained only 3% of its initial activity with a 10mM Zn2+
Table5–EffectoforganicsolventsontheAcinetobacter sp.AU07lipase.
Solvents Residualactivity(%) Incubation(2h) Incubation(4h) Control 100.00 100.00 Methanol 60.40±1.10 44.15±1.85 Ethanol 56.25±3.95 35.60±4.40 1-Propanol 9.80±2.50 8.60±3.40 1-Butanol 25.20±1.00 14.50±0.50 Acetonitrile 44.85±0.15 39.0±1.00 1-Hexane 137.15±3.05 109.15±2.95 Toluene 356.30±5.90 281.10±5.85 Dichloromethane 113.50±1.50 46.25±2.75 1-Heptane 106.10±3.90 77.60±0.40 Dimethylsulfoxide 190.70±4.30 181.00±1.00 Theenzymewaspre-incubatedfor2and5hwithdifferentorganic solvents(30%,v/v)whileshaking(150rpm)at30◦Candassayed understandardassayconditions.Thestabilityofthelipasewas calculatedbycomparingtheresidualactivitywiththatofthe con-trol,whichwasincubatedwithouttheorganicsolventandwhose enzymeactivitywastakenas100%.Thestandarddeviationforall thevaluesis<±5.0%.
Table6–Effectofmetalionsontheactivityofthe Acinetobactersp.AU07lipase.
Metalions Residualactivity(%)
2mM 5mM 10mM Zn2+ 5.58±0.08 4.92±0.12 3.26±0.04 Mg2+ 51.00±1.00 34.20±1.20 16.45±1.45 Ca2+ 19.90±0.90 10.05±0.75 3.81±0.21 Ag2+ 62.00±0.40 47.85±0.45 18.25±0.45 Fe3+ 56.72±0.42 32.70±0.30 21.20±0.60 Mn2+ 36.10±0.80 11.97±0.47 0.82±0.11
Thepure enzyme was incubated with metal salts of different concentrations,andtheresidualenzymeactivitywasdetermined understandardassay conditions. The enzymeactivity without metalsaltswastakenas100%.Eachvaluepresentedhereisan averageofthreereplicates.
concentration (Table 6). Several authors observed similar resultsusingZn2+,23,35–39althoughtheinhibitoryeffectisnot
yetknown.Thedecreaseintheactivityoftheenzymeinthe presenceofmetalsionsimpliesthattheAU07lipasedoesnot requiremetalionsforitsactivity.
Effectofinhibitors,detergents,andoxidizingagentson lipaseactivity
Phenylmethylsulphonylfluoride(PMSF:aninhibitorofserine lipase)inhibited thelipaseactivity (88%inhibitionat2mM concentration).Similarresultswereobservedinthelipases fromA.radioresistensCMC-123andBacillusstearothermophilus
P140 suggestingthe involvementofaserine residue inthe
activesite.TherewasminimalinhibitionbyEDTAandDTT (Fig.6).Thedecreaseintheenzymeactivityinthepresenceof metalionsandtheabsenceofinhibitionoftheenzyme activ-itybyEDTAindicatethatmetalionsarenotrequiredforthe lipaseactivity.Theseresultssuggestthatthislipasemaybe amemberofserine-enzymefamily.Theeffectofdetergents
Fig.6–EffectofinhibitorsonAcinetobactersp.AU07lipase activity.Theenzymewasincubatedwithdifferent inhibitorsat30◦Cfor1h,andtheenzymeactivitywas assayedunderstandardassayprocedureconditions.The enzymeincubatedwithoutinhibitorswasusedasa referencetocalculatetherelativeactivity.Eachvalue presentedhereisanaverageofthreereplicates.
suchasSDS,Tween80andTritonX-100ontheenzyme activ-itywerestudied.SDSinhibitedtheenzymeactivity;however, therewasminimallossinenzymeactivity whenincubated with0.25mMTween80or TritonX-100 (Table7).Oxidizing agents(hydrogenperoxide)hadminimaleffectontheenzyme activity,andtheenzymeretained94%(at0.5mM)and73%(at 2mM)ofitsenzymeactivity,respectively.
Determinationoflipasekineticsusingdifferentsubstrates
Thekineticparameters(KmandVmax)ofhydrolysisof
differ-entfattyacidestersweredetermined.TheKmwashigherfor
4-nitrophenyl butyrate(shortfattyacid esters),and lowKm
valueswereobtainedfor4-nitrophenyllaurate,4-nitrophenyl
Table7–Effectofdetergentsandoxidizingagentonthe activityoftheAcinetobactersp.AU07lipase.
Reagents Concentration(%)(v/v) Residualactivity(%)
Control 0 100 Tween80 0.25 87.20±1.20 0.5 67.35±1.35 1 38.10±1.10 TritonX-100 0.25 94.60±0.60 0.5 43.50±0.50 1 25.65±0.65 SDS 0.5 11.98±0.98 1 10.81±0.31 H2O2 0.5 93.74±0.74 1 86.44±1.44 1.5 83.53±0.53 2 73.27±1.27 Theenzymewasincubatedwithdifferentdetergentsandoxidizing agentsofvariousconcentrationsat30◦Cfor1handthenassayed understandardassayconditions.Theenzymeincubatedwithout detergentsandoxidizingagentswasusedasareferenceandwas usedtocalculatetherelativeenzymeactivity.Eachvaluepresented hereisanaverageofthreereplicates.
Table8–KmandVmaxvaluesoftheenzymedetermined
byLineweaver–BurkandWoolf–Hanesplotsfordifferent substrates.
Substrate Km Vmax Vmax/Km
Lineweaver–Burkplot
4-Nitrophenylbutyrate 0.51mM 16.98U/mg 33.29 4-Nitrophenyllaurate 0.15mM 15.44U/mg 99.81 4-Nitrophenylmyristate 0.17mM 14.61U/mg 102.93 4-Nitrophenylpalmitate 0.19mM 13.59U/mg 71.53
Woolf–Hanesplot
4-Nitrophenylbutyrate 0.54mM 17.45U/mg 32.31 4-Nitrophenyllaurate 0.13mM 15.13U/mg 116.38 4-Nitrophenylmyristate 0.18mM 14.94U/mg 85.94 4-Nitrophenylpalmitate 0.17mM 13.45U/mg 79.11
myristateand4-nitrophenylpalmitate(moderatechainlength fatty acid esters) indicating that the Acinetobacter sp. AU07 lipaseismorespecifictosolublefattyacidestersofmoderate chainlengths.TheseresultsareconsistentwithAcinetobacter baumanniiBD5lipase.41AlowK
mvalueindicatestheenzyme
hasahighaffinityforasubstrate,whereasahighKmvalue
indicatesalowaffinity.Thecomparisonofkineticconstants (KmandVmax)obtainedforvarioussubstrates(4-nitrophenyl
esters)suggestthatthehydrolyticactivityofthislipasewas specifictomoderatechainfattyacidesters.Thislipasewas foundtohavealowerKmvaluefor4-Nitrophenyllauratewhen
comparedwiththeother substrates.Thissuggeststhatthe lipasehasahigheraffinitytowards4-nitrophenyllaurate.
TheVmaxvaluesfor4-NPL,4-NPMand4-NPPwerehighand
weresimilarinrange(Table8).TheVmax/Kmratiowasdifferent
forallsubstratesused,butformoderateandlongchainfatty acidesters,itexceededtheratioforp-NPB.TheKmandVmax
valuesdeterminedbytheLineweaver–BurkandWoolf–Hanes plotsforthedifferentsubstratesareshowninSupplemental Fig.S6.
Conclusion
A thermostable organic solvent tolerantlipase from Acine-tobacter sp. AU07 has been purified and characterized biochemically.Theproductionwasimprovedbyoptimizing the culturingconditions using responsesurface methodol-ogy.Thepurifiedenzymeshowedseveralimportantproperties suchasactivityinalkalinepH,resistancetosurfactantsand oxidizingagentsthatwouldproveusefulinindustrial appli-cationsandinthedetergentandpharmaceuticalindustries. The biofilms of the lipase secreting Acinetobacter sp. AU07 maybeusedforbioremediationtotreatwastewater contami-natedwithoils.Thethermostableandorganicsolventtolerant natureofthelipasecanbeusedtoperformsyntheticreactions inthepresenceoforganicsolventsandathightemperatures.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Special thanks to the Department of Biotechnology, Government of India, for funding Senior Research fellowship (to P. Gururaj) and programme support (BT/01/COE/07/01)toGautamPennathur,S.Ramalingam,and G.Nandhinidevi.
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2015.04.002.
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