evolving a lipase for hydrolysis of natural triglycerides along with … · 2020. 11. 12. · 1...
Post on 01-Feb-2021
0 Views
Preview:
TRANSCRIPT
-
This is a repository copy of Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants.
White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/150864/
Version: Accepted Version
Article:
Alfaro-Chávez, AL, Liu, J-W, Stevenson, BJ et al. (2 more authors) (2019) Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants. Protein Engineering, Design and Selection, 32 (3). pp. 129-143. ISSN 1741-0126
https://doi.org/10.1093/protein/gzz023
© The Author(s) 2019. Published by Oxford University Press. All rights reserved. This is anauthor produced version of a paper published in Protein Engineering, Design and Selection. Uploaded in accordance with the publisher's self-archiving policy.
eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/
Reuse
Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item.
Takedown
If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request.
mailto:eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/
-
1
Evolving a lipase for hydrolysis of natural triglycerides along with
enhanced tolerance towards a protease and surfactants.
AnaL.Alfaro-Chavez1,Jian-WeiLiu
2,BradleyJ.Stevenson
1,AdrianGoldman
3,4andDavidL.
Ollis1.
1ResearchSchoolofChemistry,AustralianNationalUniversity,Canberra,ACT2601,Australia
2CSIROLandandWater,BlackMountain,Canberra,ACT,2601,Australia
3SchoolofBiomedicalSciences,AstburyCentreforStructuralMolecularBiology,Universityof
Leeds,LS29JT
4MolecularandIntegrativeBiosciencesProgram,UniversityofHelsinki,FIN-0018.
CorrespondingAuthor:
DavidLOllis
ResearchSchoolofChemistry
AustralianNationalUniversity
Canberra,ACT,2601,Australia
Tel:+61254377
E-mail:david.ollis@anu.edu.au
-
2
Abstract
Intheaccompanyingpaper,wedescribedevolvingalipasetothepointwherevariantswere
soluble,stableandcapableofdegradingC8triacylglyceridesandC8esters.Thesevariants
were tested for theirability to survive inanenvironment thatmightbeencountered ina
washingmachine.Unfortunately,theywereinactivatedbothbytreatmentwithaprotease
used in laundry detergents and by very low concentrations of sodium dodecyl sulfate. In
addition,allthevariantshadverylowlevelsofactivitywithtriglycerideswithlongaliphatic
chainsandwithnaturallyoccurringoils,likeoliveoil.
Directed evolution was used to select variants with enhanced properties. In the first ten
roundsofevolution,theprimaryscreenselectedforvariantscapableofhydrolysingoliveoil
whereas the secondary screen selected for enhanced tolerance towards a protease and
sodiumdodecylsulphate(SDS).Inthefinalsixroundsofevolution,theprimaryandsecondary
screensidentifiedvariantsthatretainedactivityaftertreatmentwithSDS.Sixteencyclesof
evolutiongavevariantswithgreatlyenhancedlipolyticactivityonsubstratesthathadboth
long(C16andC18)aswellasshort(C3andC8)chains.Wefoundvariantsthatwerestablefor
morethan3hours inproteaseconcentrationsthatrapidlydegradethewild-typeenzyme.
Enhancedtolerancetowardssodiumdodecylsulphatewasfoundinvariantsthatcouldbreak
downnaturallyoccurringlipidandresistproteaseattack.Theaminoacidchangesthatgave
enhancedpropertieswereconcentratedinthecapdomainresponsibleforsubstratebinding.
-
3
The object of this study was to identify lipase variants that could be used in industrial
applications. To identify suitable variants, we used directed evolution (for reviews, see
(Arnold1998;ArnoldandVolkov1999;Arnold2018;Porteretal.2016;Powelletal.2001;
Turner2009)).Thestartingpointforthisworkwasacollectionofvariantsproducedinastudy
describedintheaccompanyingpaper(Alfaro-Chavezetal.2019).Thesevariantswereevolved
from a lipase, Lip3 (wild-type -wt), fromDrosophilamelanogaster encoded by the gene
CG8823.Thepreviousstudyproduced30variantswithhigheractivitiesthanwtasmeasured
withcrudelysate.Thewtandfiveofthesevariantswerepurifiedandcharacterized.Thewt
expressed well, but had very little activity due, in part, to its poor solubility. The variants
exhibited greatly enhanced solubility and thermal stability as well as modest increases in
activitycomparedwithwt.Wedecidedthatthesevariantswereasuitablestartingpointfor
thecurrentstudy,whichaimedtoidentifyvariantsthatcouldwithstandtheharshconditions
foundinlaundrydetergents.
Laundrydetergentsvaryconsiderably incompositionandapplication.Asetof threequite
different detergents was chosen for testing variants. The first was a traditional laundry
powder,LD1,thatreliedmainlyuponsurfaceactiveagentstocleancloths.Thesecond,LD2,
wasintendedforwashingincoldwaterandcontainedanenzymethatremovedstainsthat
wererichinprotein;theenzymewasthoughttobeavariantoftheproteasesubtilisin.The
third detergent, LD3, is marketed as a liquid; it operates in warm water and is intended
primarilytoremovestainscausedby lipids.Theenzymepresent is thoughttobea lipase.
Therewerepredominantlytwotypesofsurfaceactiveagents inthesedetergents;anionic
and non-ionic. There were many types of each of these surface-active agents in the
detergents,toomanytobeusedforselectioninourexperiments.Wethereforeusedasingle
representativesurfaceactiveagent,sodiumdodecylsulphate(SDS) intheexperiments.As
willbeshownbelow,thestartingvariantsexhibitedgoodtolerancetowardssomenon-ionic
agents,whiletheyshowedpoortolerancetowardsSDS,ananionicsurface-activeagent.One
ofouraimswastoidentifyvariantsofLip3thatwerestableinSDSandthatretainedtolerance
tonon-ionicsurface-activeagents.
-
4
In addition to surface-active agents, laundry detergents frequently contain enzymes.
Proteaseswerethefirstandarestillthemostcommonenzymeadditive(Kumaretal.2008;
Maurer 2004; Saeki et al. 2007). Lipases are also found in laundry detergents (Adrio and
Demain2014;Hasanetal.2010;NiyonzimaandMore2015),butarenotusedinconjunction
withproteases thatcoulddegradethem.Acommercialpreparationofsubtilisinknownas
Alcalase®,wasoneofasetthatwereavailablefromcommercialsuppliersforuseinlaundry
detergents.Ourobjectivewastoidentifyvariantsthatcouldwithstandtreatmentwiththis
proteasealongwithsurfaceactiveagents. Inaddition,wewantedLip3variantsthatcould
degrade lipidsthatmightbeencountered inawashingcycle.Thestartingvariantsforthis
workhadenhancedactivitytowardstri-octylglyceride.Wewantedtoidentifyvariantsthat
could act on tri-acyl glycerides with long chains (C16 and C18) and natural oils that had
variablelengthchains,specificallyoliveoil(16-22C)(Quintero-Flórezetal.2015)andcoconut
oil(6-14C)(Bezardetal.1971).
Ourgoalwas to identifyvariantswithenhancedtolerance toSDSandthatwereprotease
tolerantwhileexhibitingbroadsubstratespecificity.Aschematicworkflowdiagramofthe
experimentalprocedure isshowninFigure1.Theinitialobjectivewastoproducevariants
suitableforindustrialapplications.However,itisthemethodsusedtoidentifyvariantsthat
maybeofpracticaluseratherthanthevariantsthemselves.Ourapproachcanbemodified
todealwith the featuresofaspecific laundrydetergent. If thedetergentusesaprotease
otherthanAlcalase®,ourapproachcanbeusedtoevolvetolerancetothatenzyme;and,of
course,surfactantsotherthanSDScouldbeusedinthesecondaryselectionprocess.
MaterialsandMethods
Thematerialsandkitsusedformolecularbiologyaredescribedintheaccompanyingpaper
(Alfaro-Chavezetal.2019).ChemicalsusedwereofanalyticalgradeandobtainedfromMerck
Pty. Ltd. (Australia) unless otherwise stated. Tris buffer was obtained from BioStrategy
(Australia), sodium dodecyl sulfate (SDS) from Amresco (Australia) and Bromophenol blue
fromLabChemInc.(PA).MaterialstoprepareculturemediawerefromBactoLaboratories
PtyLtd(Australia).Oliveoil,coconutoilandcommercialdetergents(LD1,LD2andLD3)were
purchasedfromaCanberransupermarket(2016).
-
5
Phase1-Initialcharacterization
Lip3wtandvariantsR7_59A(Round7,Tray5,Well9A)andR7_82Ewereselectedforinitial
characterization. These enzymes were purified as described in the accompanying paper
(Alfaro-Chavezetal.2019).Theobjectofthe initialcharacterizationwastodeterminethe
abilityoftheenzymestodegradeesterswithlongchains;foursubstrateswereused:pNP-C3,
pNP-C8, pNP-C16 and pNP-C18. This assay was essentially the same as described in the
accompanyingpaper(Alfaro-Chavezetal.2019)andwillonlybebrieflydescribedhere.All
assays were done in triplicate. In addition, the ability of wt and variants to tolerate
commercialdetergentswithagarplateswastested.
Esteraseactivity
Theesteraseactivitywasmeasuredspectrophotometricallyasdescribedintheaccompanying
paper(Alfaro-Chavezetal.2019).Alltheesteraseassaysweredonewith0.4mMofsubstrate
andthehydrolysisreactioncarriedoutin96wellplates.Thefinalvolumeofthereactionmix
was200µLconsistingof100 µLofenzymesolution–eitherpurifiedproteinorcrudelysate.
Asnotedinthepreviouspaper(Alfaro-Chavezetal.2019),thepurifiedenzymewasstoredas
aconcentrateinbufferA(seeproteinpurification)anddilutedintoassaybuffer(50mMTris-
HClBuffer,pH8).100 µLofsubstratesolutionwasaddedtotheenzymesolutionandthe
reactionfollowedat405nmfor5minutesat22oC.Eachsubstratewaspreparedasfollows:
aquantityofsubstratewasdissolvedin10mLof isopropanolbeforemixingwith90mlof
assay buffer to give twice the final substrate concentration. 20 µL/mL Triton X-100 was
requiredinthefinalsubstratepreparationtopreventtheprecipitationofpNP-C16andpNP-
C18whentheyweremixedwiththeassaybuffer(Guptaetal.2002).Theactivitywastaken
astheinitialvelocityofthereaction,asdescribedinouraccompanyingpaper(Alfaro-Chavez
etal.2019).
TolerancetoDetergent,SDSandTolerancetoAlcalase
Twoesteraseassayswerecarriedout.Inthefirst,theassaywasasdescribedintheprevious
paragraph.Inthesecond,the100µLofenzymealsocontainedeither:5g/LLD1,LD2,orLD3,
or2%(v/v)TritonX100or20g/LSDS,or0.4%(v/v)Alcalase®.Thesolutionsofenzymeand
-
6
additivewereincubatedat22°Cforonehourandtheactivitymeasuredasdescribedinthe
previousparagraphusingpNP-C16.Thetoleranceswerecalculatedbydividingtherateofthe
secondreactionbythefirst.
Tolerancetocommercialdetergentsasdeterminedwithagarplates.
Theagarplateswerepreparedinessentiallythesamewayasdescribedintheaccompanying
paper(Alfaro-Chavezetal.2019).However,inadditiontoC8-TAG,variousconcentrationsof
thecommercialdetergents(0.1%,0.5%,1%and2%)wereadded.
Phase2-Evolution
Stage1-Librarycreation
ThelibrarycreationemployederrorpronePolymeraseChainReaction(epPCR)asdescribed
previously (Alfaro-Chavez et al. 2019; Stevenson et al. 2008). This protocol used short
extensiontimessothatthegeneswouldbeshuffledwiththeStaggeredextensionprocess
(StEP)(Stemmer1994;Zhaoetal.1998).R8wasgeneratedfrom50-60ngofeachofthe30
genes isolated in R7 variants. Randomized mutations were introduced using MnCl2, high
concentrationsofMgCl2andTaqDNApolymerase(Alfaro-Chavezetal.2019).Thelibraries
forR8toR23wereproducedbymixingtheproductofthreePCRruns,eachwithdifferent
amounts of MnCl2. The amplified mixed product was cleaned using the Wizard® PCR
purificationkit(Promega)anddigestedwith20UofeachNdeIandEcoRI-HF.Thedigestion
proceededat37°CforthreehoursintheCutsmart®buffer.Thedigestedproductwasisolated
byagarosegelelectrophoresisandtheWizardpurificationkit.TheligationwassetupwithT4
DNAligaseandthepurifiedPCRproductligatedintothepETMCSIIIplasmidcutwiththesame
restrictionenzymes.Thereactionwascarriedoutovernightat4°C.Theresultantplasmids
werepurifiedwiththePromega®PCRpurificationkitandelutedinto30µLofwaterbefore
transformationintoE.coliBL21(DE3)byelectroporation.TransformantswereplatedonLB-
agarplateswith100mg/Lampicillintodeterminetheconcentrationofcolonyformingunits
beforeplatingoutallofthelibraryonselectionagarplatesat300-500coloniesperplate.
TheinitialR14librarygaveasmallerthanexpectednumberofcolonieswhenitwastested,
solibrarycreationwasrepeatedwiththeadditionofthewtgenetothemixtureofgenesto
perform this epPCR cycle. In R19 a different approach was taken. The digested gene was
-
7
ligatedintopET26btoreducetheexpressionlevel.However,thetransformationefficiency
was still lower than required. The problem with library size was eventually solved by
introducinganewprimerforsequencingandepPCR:5’-GAAGGAGATATACATATGGG-3’.
This primer included theNdeI site and was used in R19 to R23. R23 was prepared as for
previousroundsexceptthattheparentgenesusedastemplatesinepPCRwereR18_211H,
R21_112B,R21_411C,R22_73H,R22_512GandR22_311H.
Stage2-Libraryscreening
Thiswasthedonewithalargeprimaryscreenthatgaveafewhundredvariantsfortestingin
secondaryscreens.Theprimaryscreenwasdoneinthreedifferentways:A)agarplateswith
lipid(R8through17),B)96-wellplateculture(R18)andC)agarplatesreplicatedontofilters
withselectionusing1-naphthylpalmitate.Themethodsusedfortheprimaryscreenaregiven
below,followedbythesecondaryscreenmethods.
Agarplates+lipid:R8-R17.
TheagarplateswerepreparedwithglyceryltrioctanoateinR8andR9andwitholiveoilin
R10toR23,butwithdetergentsincludedinR10andR11.Asinourpreviousstudy,theLBagar
plateswerepreparedwiththelipidsincorporatedbysonication.ForR8andR9,themolten
agarsolutionconsistedof0.6%TGC8incorporatedwithvigorousagitationbeforesonication
(at≈60°C)fortwoperiodsof15minat50%powerand50%pulselengthusinganOmniSonic
Ruptor®sonicatorequippedwithanOT-T-375probeprovidedbyOmniSonic.Ampicillinwas
added in a final concentration of 50 µg/mL. The agar plates with olive oil had a final
concentrationof0.2%(v/v)andwerepreparedinthesameway.R10andR11usedagarplates
supplementedwithanenzymaticdetergent(LD2)previouslyheatinactivated(65°Cforone
hour).Thedetergentwasaddedtoafinalconcentrationof0.1%(w/v).
Therewerebetween300to500coloniesinoculatedperplate.Theselectionwasbasedon
the quality of the halos. This usually meant that colonies with the largest haloes were
selected. Not all colonies grew to the same size and so in some cases, we selected small
colonieswithlargehaloescomparedtothecolonysizeforsecondaryscreening.
-
8
96-wellplateselection:R18
AfterR17,wedecidedtoscreenlargelibrariesfortolerancetoSDS.InR18thiswasdonewith
96-wellplates.ThelibraryforR18consistedof~1.4x104variantsandwasplatedin4096-well
platesso thateachwellhad,onaverage,between3-4colonies thatgrew in200µLofLB
mediumwith50µg/mLampicillin.Theplateswereincubatedfor18hoursat37°Cand500
rpminHeidolphMicroplateShakerTitramax1000withIncubator1000.Afterincubation,25
µLofcultureweretransferredtoreplicaplates(twicetogiveplatesAandB)andlysedwith
2µgoflysozyme.
PlateAwasusedtoselectmutantstolerantto0.5%SDSandplateBwasusedtochecklipolytic
activity.70µLofSDSsolutionpreparedinassaybuffertoafinalconcentrationof0.5%SDS
wasaddedtoplateA.After15minutesofincubationatroomtemperature,theactivitywas
measured by adding 100 µL of pNP-C16 substrate mixture . Activity was followed for 5
minutesat405nminaSpectramaxM2ereaderatroomtemperature.Totestlipolyticactivity,
wepreparedamixture(0.02%v/vcoconutoil,0.02%oliveoilv/vin1mMTris-HClbufferpH
8, 0.07 mM phenol red and 0.1% Triton X-100 as emulsifier), which was sonicated for 15
minutesatmediumultrasonicpowerinSoniclean160TUltrasonicCleaner3todissolvethe
lipids.175µLofthismixturewasaddedtoplateB.
Theplateswereincubatedatroomtemperatureat100rpmfor1hourinHeidolphMicroplate
Shaker Titramax 1000 and the mutants that turned yellow were selected. The mutants
selected from plates A and B were streaked into LB amp plates with 0.2% olive oil and
incubated overnight at 37° C. the colonies with the clearest haloes were selected and
transferredtonew96-wellplateswith200µLofLBmediumwith50mg/Lampicillin(LBA)
andincubatedat37°Covernightandfurthertestedinsecondaryscreening.
Agarplates–1-Naphthylpalmitate:R19-R23.
This screen followed a similar protocol to one that had been used to select lipases for
biodieselproduction(Kormanetal.2013).Thescreen involvedreplicationofthecolonies,
their lysis, treatment of the lysate with SDS and subsequent activity determination. After
preparing the library determining the cell density, around 500 colonies per plate were
-
9
inoculated on LB agar plates with Ampicillin. The plates were then incubated at 37° C
overnight. The colonies were then blotted onto Whatman grade 3 filter paper (GE
Healthcare),lysedwith50mMTris-HClatpH8,0.1MNaCl,0.1%TritonX-100and1mg/ml
lysozyme (Sigma) and incubated for one hour at room temperature. The lysis buffer was
decantedandthelysedcellstreatedwithSDSforonehour,withtheconcentrationofSDS
increasedineachroundofevolution.Theactivityofthelysedcellswasdeterminedbyadding
amixtureof1mM1-Naphthylpalmitate,3mMFastBlueB,0.5%(v/v)TritonX-100and0.5%
(w/v)agarand50mMTris-HClpH8.Activemutantsexhibitedanintensebrownishcolour,so
thecorrespondingcoloniesontheagarplatesweretransferredto96-wellplateswith200µL
of LBAand incubated at 37 °C overnight. The variants were then tested in the secondary
screen.
Stage3Secondaryscreen
Thecoloniesofvariantsselectedintheprimaryscreenweregrowntoprovidecrudelysate
fortestingtheiresteraseactivity,andsomeoftheroundsusedotherassaysaswell.InR18
anesteraseassaywasusedtoselectvariantstogeneratethenextlibrary.Inotherrounds,
two assays were used to select variants. These dual assays employed an esterase to
determine:A)tolerancetodetergents(R8-R10),mainlySDS(R11–R17andR19–R23);B)
tolerancetoprotease(R12–R17);andC)Thermostability(R21-R23).Inthesedualassays,
variants that exhibited good activity in either assay were selected along with those that
performedwellinbothassays.
Coloniesweregrownin96-wellplateswith200µLofLBmediumcontaininganantibiotic.
Thesesolutionswereinoculatedwithcoloniesselectedfromthepreviousprimaryscreenand
were grown for 18 hours at 37° C in an incubated shaker (Heidolph Microplate Shaker
Titramax1000withIncubator1000)setat500rpm.Next,25µL(R15–R18)or50µL(R11–
R14andR19-R23)or100µL(R8–R10)aliquotsofovernightcultureweretransferredto96-
wellplatesandlysedwithlysozyme(finalconcentration80µg/mL)inassaybufferininafinal
volumeof250µL.
-
10
Esteraseactivity
Esteraseactivitywasmeasuredusingp-nitrophenylesterstoidentifythemostactivevariants.
Thisassaywasessentiallythesameasusedfortheinitialcharacterisationasdescribedabove
asintheaccompanyingpaper(Alfaro-Chavezetal.2019).Thereactionmixturecontained30
µLofcrudelysatetowhich70µLassaybufferwasadded.Thereactionwasinitiatedbythe
additionof100µLofsubstratemixturepreparedasdescribedunderinitialcharacterization.
The formation of p-nitrophenolate was followed spectrophotometrically at 405 nm. The
activitywastakenasthe initialvelocityof thereaction,asdescribed inouraccompanying
paper(Alfaro-Chavezetal.2019).
TolerancetoDetergent,SDS,orAlcalase
With one exception, the same methods were used as those described in the Initial
Characterizationsectiongivenabove.Theexceptionwasproteinsource;crudelysateswere
usedasdescribedinthepreviousparagraph.InthesecondaryscreenforR23,theesterase
activitywasmeasuredbeforeandaftertreatmentwith4%(w/v)SDSasdescribedbelow(final
characterisation).ThemosttolerantandactivevariantwascharacterizedinPhase3.
Thermostability
Lipasesampleswereincubatedat60-65°Cfor30minutesfollowedby4°Cfor20minutesin
a Bio-Rad C1000TM
before centrifuging in an Eppendorf 5804 Benchtop centrifuge for 5
minutesat300rpm.Fiftymicrolitresofeachsupernatantwasaddedtoa96-wellplatewell
and50µLofassaybufferwasaddedbeforethereactionwasinitiatedwith100µLofpNP-C8
ester.Theinitialratewasmeasuredasdescribedaboveforesteraseactivity.
Phase3-Finalcharacterization
Wecharacterisedseveralvariantstogainsomeinsightsintothecourseoftheevolution.The
variantschosenwerethebest in their respectiverounds.VariantswerechosenfromR13,
R17,R18,R20andR23(R13_44C,R17_29G,R18_211H,R20_11A,R23_110A).Theseenzymes
werepurifiedasdescribedintheaccompanyingpaper(Alfaro-Chavezetal.2019).Mostof
the assays were same as described above in the initial characterization. In addition, the
lipolyticactivityofthesevariants,wt,R7_59AandR7_82Eweremeasured(below)andthe
meltingtemperature(T50)wasdetermined.WedefinedtheT50asthetemperatureatwhich
-
11
theenzymeretainshalfofitsactivityafterheattreatmentfor45minutes(formethodssee
(Alfaro-Chavezetal.2019)).ActivityasafunctionofSDSconcentrationwasalsodetermined
fortheR7_59A,R18_211HandR23_110Avariants.Allassaysweredoneintriplicate.
Lipolyticactivity
Themethodapplied,detailedintheaccompanyingpaper(Alfaro-Chavezetal.2019),isbased
onthelipaseassaydescribedby(Camacho-Ruizetal.2015).Inadditiontothesubstratesused
inthepreviousstudy,glyceryltrioleateandoliveoilwerestudied.Themethodmeasuresa
shiftinpHthatisproduceduponthereleaseoffreefattyacidsinthelipasecatalysedreaction.
Thebufferusedwas1mMTrisHClpH=8with0.07mMphenolred.Sonicationwasusedto
incorporate the following lipids into this buffer: 1 mM tributyrin-TG C4, 1 mM glyceryl
trioctanoate-TGC8,1mMglyceryltrioleateTGC18,0.04%coconutoiland0.04%oliveoil.
Theassayrequired0.2%TritonX-100forOliveoilandTGC18.Thereactionwasinitiatedby
theadditionof10µLofenzymesolutionto190µLofsuspendedsubstrate.Thereactionwas
followedspectrophotometricallyat560nm.TheamountofacidreleasedasafunctionofpH
wasreadfromthecalibrationcurveintheaccompanyingpaper(Alfaro-Chavezetal.2019).
Theactivity/permoleofenzymewascalculatedforeachvariant.
TolerancetohighconcentrationsofSDS
The tolerance of R7_82E, R18_211H and R23_110A to high concentrations of SDS was
determined. The protein concentration used was 1 mg/ml (R7_82E) and 0.1 mg/ml
(R18_211HandR23_110A)allintheassaybuffer.AhigherconcentrationoftheR7variant
wasneededsothatitsactivitycouldbeaccuratelymeasured.WeaddedSDSstocksolution
(20%w/v)to50µLoftheproteinsolutionandmadethesolutiontoafinalvolumeof100µL
withfinalSDSconcentrationsof0.1,0.5,2,4,8and10%.AcontrolwithoutSDSwasalso
madeandusedtomeasuretheinitialactivityoftheenzymes.Thesesolutionswereleftfor
30minutesat22oC.TheSDSwasprecipitatedwith25µLofassaybufferthatcontained0.3
MKCl,andanother25µLassaybufferaddedtogiveafinalvolumeof150µL.Thesolutions
werecentrifugedfor30minutesat2235gatroomtemperatureand10µLofsupernatant
weretransferredtofresh96-wellplatestomeasureesteraseactivitywithpNP-C16.Activity
wasmeasuredspectrophotometricallyat405nmasdescribedunderinitialcharacterization.
-
12
This procedure was applied as a secondary screen for R23 with 4% of SDS as final
concentration.
Proteolyticdigestion
TheR712HwassubjectedproteolyticattackwithAlcalase®.Sampleswerepreparedfollowing
theproceduredescribedbyWu(Wuetal.1999).0.1%v/vAlcalase®waspreparedwith50
mMphosphatebufferandmixedina1:1ratiowith8mg/mlofthelipasesolution.Samples
wereincubatedat22°Cand120μLaliquotswereremovedat:1,2,3,4,6,8,10,20,30,40,
50and60minutes.40μLof10%trichloroaceticacidwasaddedtoeachsampletostopthe
reaction.Thesampleswerecentrifugedat12500rpmfor5minutesandwashedtwicewith
200μLdiethylether.Thesampletubeswerethenleftopentoevaporatethediethylether
andthepelletresuspendedin100μLofbuffer(50mMTris-HClpH8)andpreparedtorunin
a SDS-PAGE gel. The sample collected after one minute gave rise to bands in a gel that
corresponded to Alcalase along with the intact variant and a collection of low molecular
weightbands.ItappearedthatAlcalasehaddigestedmuchoftheenzymeafteraminute.A
samplecollectedafteroneminutewasbuffer-exchangedusinganAmiconUltrafilterinto0.5
mLof20mMammoniumformatepH3usingthreewashesat4°Cfollowedbycentrifugation
at10000rpmfor5minutes.Thefinalconcentrationofthesamplewas2mg/mL.Thesamples
wereanalysedattheMassSpectrometryFacilityattheResearchSchoolofChemistry(ANU)
andthecleavagesitesidentifiedusingtheFindPeptserver(Gattikeretal.2002).
Theworkdescribedherecanbedivided(Figure1)intothreephases.Inthefirstphase,we
characterizedtheabilityofthestartingvariantstodegradeavarietyoftriacylglycerols(TAGs)
andesterswithlongaliphaticchains,aswellastheirtoleranceofsurfaceactiveagentsand
proteolyticenzymes.Theresultsoftheseexperimentswereusedtodesigntheexperiments
inthesecondphase,theevolution. Theevolutionwasaniterativeprocessinwhichthree
stages were repeated till the desired variants were obtained (Figure 1). In the first stage,
librariesofmutatedgenesweregeneratedand transformed intoE. coli for the two-stage
screeningprocess.Theselibrariesvariedinsize,theaveragebeingaround5x104,fromwhich
about103variantswereselectedforsecondaryscreening,andafewtensselectedtogenerate
-
13
the next library. As will be described below, the methods used to measure activity in the
secondary screen utilized variants as produced in crude lysates so that it was not clear if
increases in activity were due to increased catalytic performance or a change in physical
properties,suchasincreasedexpression,solubilityorstability.Forthisreason,aselectionof
variants was purified and characterized; their catalytic properties were determined along
withtheirtoleranceofproteasesandsurfaceactiveagents.
Phase1InitialCharacterisation
Our startingpointwas the30variantswehadpreviously identifiedafter seven rounds of
directed evolution as being more stable, soluble and active thanwt (Alfaro-Chavez et al.
2019).WetestedtheabilityofwtandtwoselectedR7variantstodegradeesterswithlong
aliphaticchains,andtheiractivitiesafterbeingtreatedwithsolutionsofthreecommercial
laundrydetergents(LD1,LD2andLD3),SDS,ananionicdetergent,andTritonX-100,anon-
ionicdetergent.R7_59AandR7_82Ewerechosenbecausetheyexhibitedthebestlipolytic
activitywithnaturaloils(Alfaro-Chavezetal.2019).
NeitherthewtnortheR7variantsexhibitedsignificantactivityinthepresenceofSDS,LD1or
LD2(TableI).AlthoughLD2containedaprotease,itwasheatinactivatedpriortobeingused,
sothelackofactivityisnotduetoproteolysis.Thestartingvariantsexhibitedvaryinglevels
oftolerancetoTritonX-100andLD3.TheR7_59Avariantwasunaffectedby2%TritonX-100,
suggesting that tolerance to this reagent would not present problems for evolution.
Alcalase®,aproteasemarketedforuseinlaundrydetergents,almostcompletelyinactivated
thewt enzyme, but R7_82 exhibited some tolerance to it, suggesting that finding other
variants resistant to proteases should be possible. Although the starting enzymes had
reasonablelevelsofactivitytowardsC3andC8substrates,theyhadlowlevelsofactivitywith
substratesthathadlongaliphaticchains(C16andC18).Forthisreason,wedecidedtoselect
foractivitywithasubstratethatwasmid-waybetweenC8andC16,soC12esterswereused
inthefirstround(R8)oftheexperimentsdescribedinthispaper.
TheadditionofTAGstoagarmakestheplatescloudy,andtheybecomeclearinthepresence
ofanactivelipase,socoloniesproducingactivelipasehaveclearhaloesaroundthem(Lanka
and Latha 2015; Lawrence et al. 1967). Could we add detergents to the agar to test for
-
14
tolerancetothesereagents?SDSlysescellssoitwaspointlesstryingthisreagent.However,
low concentrations of LD1 and LD2 could be used while LD3 did not appear to be an
impedimenttogrowthofE.coli(TableII).WedecidedtotryLD2intheprimaryscreenforR9
andR10(TableIII),butitresultedinlowsurvivalratesandsoitwasnotusedinlaterrounds
ofevolution.Theseresultssuggestedthat,iflargelibrariesweretobetestedfortoleranceto
surfaceactiveagents,thetestswerebestdoneinthesecondaryscreen,asinR8toR17,or
byreplicatingthelibraryandlysingcells,aswasdoneinR18toR23.
Phase2Evolution
Thesecondphaseofthisworkinvolved16roundsofdirectedevolutionthatcanbedivided
intothreesectionsbasedontheselectionconditions(TableIII).Inthefirstsection(R8-R17),
selectionintheprimaryscreenwasdonewithagarplatesdopedwithTAGsoroliveoil.Asin
theaccompanyingpaper(Alfaro-Chavezetal.2019),selectionintheprimaryscreenwasdone
bypickingcoloniesthatgaverisetolargehaloes.Inthesecondsection(R18),96wellplates
were used to grow a library for primary screening and selection was done by monitoring
esteraseactivity in thepresenceofSDSornaturaloils. In the thirdsection (R19-R23), the
librarywasgrownonagarplates,replicatedonafilter,thecellslysedandactivitymonitored
with1-naphthylpalmitate.Attheendofeachround,thegenesofvariantsselectedinthe
secondaryscreenweresequenced(TableS2).
Thegenesofthebest30variantsidentifiedattheendofthepreviousstudy(Alfaro-Chavez
etal.2019)wereused,asinthatpaper,togeneratethelibraryscreenedinR8.Thelibrary
size was estimated to be 3 x 105, much higher than subsequent libraries, because larger
quantitiesofgenesfrommorevariantswereused. Insubsequentrounds, the librarysizes
variedfromabout8x103to6x10
4.ThiswasnotduetotheamountofDNAusedtogenerate
thelibrary,andnotbecausethevariantswerebecominglethaltothecells.Thelatteridea
wastestedinR19byexpressingthevariantgenesinwithaplasmidthatgavelowerlevelsof
expression(pET26b),whichonlygaveaslightincreaseinlibrarysize.Wethereforeacquired
a fresh stock of pETMCSIII and new primers for the epPCR (see methods), which led to
relativelylargelibrariesforthefinalthreerounds(TableIII).
R8-R18:Broadeningspecificityandincreasingtolerance
-
15
The objectives of the evolution varied through the course of the experiment, and so the
methods used to screen the libraries changed (Table III). In R8 through R12, the primary
screen targeted rate enhancement and broader substrate specificity while exposing the
variantstolowlevelsofcommerciallaundrydetergents.InR8,theprimaryscreenutilisedC8
TAG,withpNP-C8inthesecondaryscreen.Thiswasliketheconditionsusedtoselectvariants
inR1throughR7ofourpreviousstudy.InR12,colonieswereselectedafter72hoursbased
onhaloesformedinplatescontainingfromoliveoil.Inthesecondaryscreen,goodactivity
wasobtainedwithpNP-C12.Intheserounds,therewasanincreasingamountofSDSadded
tolysatepriortotheesteraseassay;inR12theSDSconcentrationwas0.07%,upfromthe
0.02%usedinthepreviousround.
InR13throughR17,evolutionforenhancedcatalyticpropertiescontinued.Forinstance,In
R13haloeswereobservedafter48hourswhileinR17haloescouldbeseeninjust40hours.
Inthesecondaryscreen,esteraseactivitywaseasilydetectedwithpnp-C12inR13andwith
pNP-C16inR17.InR13,0.4%Alcalasewasaddedtothecrudelysateanhourpriortoassaying.
ThesameconcentrationofproteasewasusedinR17,butthevariantswereexposedfor3
hours.VariantswithgoodtolerancetoAlcalasewereeasilyidentifiedinthesecondaryscreen,
butthesamewasnottrueforSDStolerance.Attemptsto increasetheSDSconcentration
above0.1%didnotproducemanyvariantswithdetectableactivity.Itappearedthatthesmall
secondarylibrariesweresufficientlylargetoincludevariantsthatweretoleranttoprotease,
butnottoincludethosewithincreasedtolerancetoSDS.Largerlibrarieswerenecessaryto
screenforSDStoleranceatconcentrationsclosetothetargetof2%.
InR18,thelibraryusedfortheprimaryscreenwasgrownin96wellplates,replicated,cells
lysedandtreatedwith0.5%SDSandafteranhourassayedwithpNP-C16toselectvariants
forsecondaryscreeningwithpNP-C16.Theresidualactivityofonevariantwasmeasuredas
afunctionofincreasingSDSconcentration(Figure2).ItwasclearthattheR18_211Hvariant
was considerably more active than the R7 variants, that it retained more than half of its
activity after treatment with 2% SDS for 15 minutes, and that 4% SDS was required to
completelyabolishactivity.Atthispoint,R18_211Hwaspurifiedalongwiththewt,twoR7
variants(R7_59AandR7_82E),oneR13variant(R13_44C),andoneR17variant(R17_29G).
The properties of these proteins were determined, showing that directed evolution had
-
16
resultedinasignificantincreaseinSDStolerance(TableIV).R13_44Cretainedabout60%of
its activity after 1 hour at 22 ºC with 2% SDS; R17_29G retained 70% whereas R18_211H
retainedover80%activityinthesametest.Furthermore,R18_211Hwasunaffectedby2%
TritonX-100,2%Alcalase,0.5%LD2orLD3.ItappearedthatselectionforSDSstabilitywas
sufficienttoidentifyvariantssufficientlystabletowithstandthedetergentinLD2andLD3.
R18_211H lost most of its activity when treated with LD1, but was still better than the
R17_29G;theincreaseinSDStolerancewasaccompaniedbyanincreaseinthermostability.
TheT50ofR18_211Hwas37o
Chigherthanthatofthewt,23o
ChigherthanthebestR7variant
and18o
CbetterthanR17_29G(TableIV).TheesteraseactivityoftheR18variantwassimilar
forpNP-C3,pNP-C8,pNP-C16andpNP-C18substratesanditsactivitywiththelongestchain
substrates was significantly better variants from previous rounds. This broadening of the
substratespecificitywasreflectedinthelipolyticactivityoftheR18variant,asitwasmore
activethanvariantsfrompreviousroundagainstlong-chainTAGs.
R19toR23:EvolutionforSDStoleranceandCharacterization
Our goal was to develop a high-throughput screen to select for variants with improved
tolerancetoSDS.ItwasclearfromR18thatlargelibraries(oftheorder104)wereneededto
improvetolerancetoSDS.However,makingsuchlargelibrarieswaslaboriousandexpensive
using96wellplates (>600platesneeded fora6x104 library), sowedecided todevelopa
replica agar plate assay (see Methods). In R19 through R23 variants were selected for
secondaryscreeningiftheyexhibitedgoodesteraseactivityaftertreatmentwithSDSona
replicaplate.Inthesecondaryscreen,theactivityofvariantswasmeasuredwithanesterase
assay before and after treatment with SDS so that the stability of variants could be
determined.TheSDSconcentrationwasincreasedsteadilyduringtheserounds(TableIV).In
R23 olive oil was added to the agar plates used in the primary screen, and selection for
secondaryscreeningrequiredgoodlipaseactivityaswellasesteraseactivityaftertreatment
withSDS. Inthisround,3%SDSwasused intheprimaryscreenwhile4%wasused inthe
secondary screen. We also selected for thermostability in the secondary screen (see
Methods).
-
17
Phase3Detailedcharacterization
The best variants from each of R13 (R13_44C), R17 (R17_29G), R18 (R18_211H), R20
(R20_11A),andR23(R23_110A)werepurifiedandcharacterized(TableIV).Inaddition,for
purposesofcomparison,thewtproteinandtwoofthebetterR7variantswerepurifiedand
characterized.ComparedwiththeR7variants,theyieldofpurifiedproteindecreased.For
example,theR7_82Egave55mgfroma250mLculturewhileR18_211Hgrownunderthe
sameconditionsgave43mg.ComparedwithR7variantstherewasanincreaseinesterase
andlipaseactivities(TableIV).TheR23_110Avariantwas2-3timesmoreactiveonC3andC8
shortchainesterscomparedtotheR18_211Hvariant,butabout50%lessactiveonC16and
C18esters(TableIV).ThiswassurprisingasselectioninthesecondaryscreenwaswithapNP-
C16substrate.LipolyticactivitydroppedbetweenR18andR23;theactivitiesofR23_110A
withallsubstrates1.5-3timeslessthanforR18_2117H.
SDStoleranceimprovedastheevolutionprogressed.Therewasnoobservablelossinactivity
in the R20 variant after treating with 2% SDS for one hour while the same treatment of
R23_110A resulted in an increase in the residual activity; concentrations of SDS up to 4%
activateit(Figure2).CompleteabolitionofactivityoftheR23variantrequiredaonehour
treatment with 10% SDS as is evident in Figure 2. Although the R23 variant exhibited
extremelygoodtolerancetoeitherSDSorTritonX100,itwasnotastolerantofcommercial
powdersLD1andLD2andAlcalasecomparedwiththeR18variant(TableIV).Forexample,
theR18variantretainedabout80%ofitsactivitywhentreatedwithSDSandretainednear
100%ofitsactivityaftertreatmentwithLP2orAlcalasewhereastheactivityoftheR23variant
increased by a factor of 2.6 after treatment with SDS but retained only about 10% of its
activity after treatment with LP2 and 40% of its activity after treatment with Alcalase.
However, treatment with LD3, a product formulated to accommodate a lipase, did not
diminishtheactivityoftheR18,whileitappearedtoactivatetheR23variant.
Itwasexpected that increased tolerance toSDSwouldbeaccompaniedbyan increase in
thermostabilityandallthepost-R7variantshadincreasedthermalstabilitycomparedtothe
R7variant.However,theredidnotappeartobeacorrelationbetweentolerancetoSDSand
thermostability. The R18 variant exhibited the best thermostability (T1/2 = 73o C), but the
-
18
thermalstabilityoftheR23variantwaslower,butstillrespectable(T1/2=65oC),beingclose
to30o
Chigherthanthewt.
Theabilityofavarianttotolerateaproteasereached100%inanR13variantbutdecreased
to80%inR17_29G(TableIV).However,theabilitytotolerateaproteasevariedwithinany
round(Figure3),andtherewereafewvariants inR17thatexhibited100%tolerancetoa
protease after 1 hour and maintained most of their activity after 3 hours (Table S1 in
Supplementarymaterial).Anattemptwasmadetoidentifyresiduesresponsiblefortolerance
to Alcalase. The wt protein was digested and the resulting peptides sequenced. The
proteolyticcutsitesareshownonFigure3.Intwoinstances,therewerechangesclosetothe
cutsites;theT185Asitewas4residuesawayfromthecutsiteat181;andtheA174Vsiteis3
residuesawayfromthecutsiteat171.Theremainingcutsiteswerewellseparatedfrom
variantchanges.
Analysisofsequencechanges
Thevariantsoftencontainedmorethanonenewchange(supplementarymaterial,TableS2).
Only a few persisted into subsequent rounds and some of these approached saturation
(presentin>90%ofsequencedvariants).Onaverage,aboutonechangeperroundpersisted.
The initial library for this study was generated with mutated genes of Lip3 from the
companionstudy(Alfaro-Chavezetal.2019).Thisintroducedfourmutationsthatsaturated
the R7 final selection (V201A, N268I, P291L and M321L). By R17 there were seven more
saturatedmutations(M20V,M59L,A174V,T185A,S210T,D230GandL351Q)andbyR23an
additionalthree(E46K,W107L,C202Y)andonereversion(S210T).Ofthesethreesaturations,
onlyE46KwaspresentinR7(TableV).Incontrast,thereweremutationsthatrevertedtowt
astheselectionconditionswerealtered.Forexample,theY40Fchangewaspresentin23%
ofthevariantssequencedinR7,allvariantssequencedinR17hadrevertedtoYatresidue
40.E250Gwaspresent in45%ofthegenessequenced inR7and47%atR17butwasnot
presentatR23.
AsisevidentinFigure3,alittleoverhalfofthechangesobservedinR23(55%)werefoundin
theCapdomain,whichcontainsjustonethird(35%)oftheresiduesintheprotein.Ofthe
changesobservedinR23,sixwereinthecoredomain.Fourofthese(M59L,W107L,M321L
-
19
andL351Q)werefoundintheinterfacewiththeCapdomainandtwo(M321LandL351Q)
wereinloopsthatcontainedtriadresidues(Figure3).WithintheCapdomain,mostofthe
changesobservedinR23werefoundintheLidportionorveryclosetoit(V201A,C202Yand
D230G).OnechangeinR23(A174V)wasfoundintheregionlinkingtheCapdomaintothe
Coredomain.TheremainingchangesobservedinR23(T185A,N268IandP291L)werefound
inhelicesoftheCapdomain(Figure3).OfthesaturatingchangesobservedthatinR23,four
ofthe13resultedinchangesofsulphurcontainingresidues.Threeofthefourchangesoccur
inthecoredomainwithone(M20V)closetotheN-terminus.
Thestartingvariantsforthepresentworkemergedduringsevenroundsofevolution(Alfaro-
Chavezetal.2019). Inthisevolution,crudelysatewasusedtoselectvariantsassessedon
theirabilitytodegradepNP-C8estersandC8TAGs.Theseactivitiesareessentiallythesame
andsotheevolutionisone-dimensional.Largelibraries,oftheorder104,werescreened,and
increases in activity occurred because the amount of active enzyme in the crude lysate
increased.Directedevolutionthusselectedforvariantswithincreasedstabilityandsolubility,
notnecessarilyincreasedspecificactivity.Thepresentworkwasmoreambitiousasselection
was for four properties: increased activity, broader substrate specificity, tolerance to a
proteaseandtolerancetodetergents.
Atthestartofthiswork,thequestionarose:whatwasthebestapproachtoimprovingthree
quitedifferentenzymeattributes?Oneapproachwouldbetoevolveoneattributeatatime.
There were two reasons for not taking this approach. First, we were concerned that the
improvementsobtainedinoptimizingoneproperty(say,substratespecificity)wouldbelost
intheprocessofoptimizingotherproperties(say,surfactantstability)insubsequentrounds;
thatis,substratespecificitywouldbelostassurfactantstabilitywasenhanced.Second,we
had already developed high throughput screens to improve substrate specificity (Alfaro-
Chavezetal.2019),butnottoimprovetolerancetoproteasesandsurfaceactiveagents.We
thereforedecidedtoselectforsubstratespecificityinalargeprimaryscreenfollowedbya
smaller secondary screen where tolerance for another attribute (stability in surfactants
and/ortolerancetoaprotease)wasselected.Inthisway,asinglecycleofdirectedevolution
-
20
couldbeusedtoimproveallthreeattributesofselectedvariants.Thisapproachwouldreduce
thenumberofcyclesrequiredtoreachthetargetconditions,providedthatusefulvariants
couldbeidentifiedinthesmalllibrariesexaminedinthesecondaryscreenandthatthetwo
properties being selected were not completely mutually incompatible – something we
thoughtunlikely.ThisapproachtoevolutionresultedinaslowincreaseintolerancetoSDS.
ItwasthoughtthatamorerapidincreaseinSDStolerancecouldbeachievedbyscreening
largerlibraries;thiswascarriedoutinthelastsixroundsofevolution.
Phase1Initialcharacterization
TheobjectoftheseexperimentswastodeterminethepropertiesoftheR7variants,toselect
conditionsforscreening.TheR7variantshadlittletoleranceofSDSwhileone(R7_59A)was
stableinaTritonX-100solution,andanother(R7_82E)exhibitedsometolerancetoAlcalase
(TableI).Asaresult,littleattentionwaspaidtoTritonX-100inthesubsequentevolution,and
selectionfortolerancetoAlcalasewasdelayedtillthevariantshadacquiredsomelevelof
tolerancetoSDS.TheR7variantshadlowlevelsofesteraseactivitywithsubstratesthathad
longaliphaticchains,suggestingthatthispropertymightbedifficulttoalter.Asaresult,the
firsttworoundsofevolutionselectedfortheabilitytodegradeC8substrates.Theabilityto
detectlipaseactivityinthepresenceofcommerciallaundrypowderswasalsotestedwithan
assaythatutilizedanagarplateasusedintheprimaryscreens(TableIII).Itappearedthat
activitycouldonlybedetected invery low levelsof LP1andLP2. It subsequentlybecame
apparentthatthisresultwasduetothepoortoleranceofE.colitotheseproducts.Cellscould
be grown in LP3, but this product already contained a lipase so there was little point in
evolvingtolerancetoitinLip3variants.
Phase2Evolution.
Themethodusedtogeneratethelibrariesremainedthesameinalltheroundsofevolution,
even though the library varied in size (see Results). By way of contrast, the selection
conditionschangedagreatdealinresponsetotheresultsobtainedinthepreviousround.
Selection inR8throughR17wasforalteredsubstratespecificityalongwithtolerancetoa
proteaseandSDS.IntheprimaryscreenofR8throughR18,librariesoftheorder104were
usedtoidentifyvariantswithincreasedactivityonoliveoil,whichhaslongaliphaticchains.
Theability todegrade these substrates improved slowly (Table IV). Forexample, thebest
-
21
variant in R13 exhibited lipase activity with olive oil that was enhanced by a factor of
approximately4.5comparedwiththewt,whilebyR17thishaddecreasedtoabout2.8.The
secondary screens were carried out with libraries of the order 103 and selection was for
tolerance to SDS and proteolysis. Tolerance to proteolytic attack was achieved easily by
scanning the small libraries of the secondary screen. Tolerance to SDS did not improve
significantly;0.1%SDSwasusedtoselectvariantsinR17.Itappearedthatlargelibrarieswere
neededtoevolveforchangesinsubstratespecificityandimprovedtolerancetoSDS.
FromR18toR23,largelibrarieswereusedtoidentifyvariantswithtolerancetoSDSwhile
maintainingactivitytowardssubstrateswithlongaliphaticchains.ToleranceofSDSimproved
dramatically.R23_110A,forinstance,exhibitedactivitythatwasincreasedbyafactorof2.6
upontreatmentwith2%SDSforanhour;itwasactivatedbySDSconcentrationsupto4%.
However,thisvariantexhibitedreducedperformanceinsometests;forexample,itslipolytic
activitywaslessthanthepurifiedR18variant.HowcouldthescreensusedinR19through
R23bemodifiedtoenablebetterall-roundvariantstobeidentified?Thisrequiresthatthe
SDStolerancebemaintainedandactivitywithlongchainsubstratesbeimproved.Thiscould
be achieved by placing greater emphasis on activity. For example, the secondary screen
selectioncouldbebasedonactivityaswellastolerancetoSDS.InR23,variantswereselected
iftheyexhibitedhighlevelsoftolerancetoSDSwithoutexceptionallyhighlevelsofactivity.
A better all-round variant could be obtained by selecting variants that have good SDS
toleranceandcatalyticactivity.
Phase3FinalCharacterization
AcasualinspectionofTableIVpromptsthequestion:whichisthebestvariant?Itisdifficult
to answer this question as no one variant is best in all the assays. The properties of the
variantschangedasselectionchangedandthecatalyticpropertiesdidnotshowacleartrend.
TheR18variantwasthemostactivelipasewitholiveoilwhiletheR20variantwasthemost
activelipasewithglyceryltrioleate.ThecatalyticpropertiesoftheR23variant(TableIV)were
inferiortovariantsobtainedinpreviousrounds,butitwasmuchmorestableinSDS.Similarly,
Alcalasetoleranceshowsnocleartrendastheevolutionprogresses.OneR7variantshows
sometoleranceofAlcalase;byR13Alcalasetoleranceisnear100%;inR17,itdroppedto80%;
inR18andR20,itincreasedto100%;andinR23,itdroppedagainto41%.Itappearsthat
-
22
tolerancetoAlcalaseiseasilylostandacquired.Itshouldbenotedthatouruseoftheword
“tolerance”impliesstabilityinsolutionforafewhours(aswasthecaseinourassays).Wedid
nottestforlongertimeperiods.
TheonepropertythatsteadilyimprovesduringevolutionisthetolerancetoSDS.Thisreagent
easily denatures thewt and R7 variants. The R13 variant retains 59% of its activity after
treatmentwithSDSwhiletheR20retains100%afterthesametreatment.TheR23variant
(R23_110A)isactivatedbySDS.Ifavariantwasrequiredtocatalysereactionsinthepresence
ofSDS,theR23_110Aactivities(TableIV)shouldbemultipliedby2.6.Suchacorrectionwould
maketheR23variantthebestchoiceforinclusioninadetergent–exceptthatR23_110Ais
susceptible to proteolysis. As noted above, tolerance to proteolytic attack was readily
acquired through selection with small libraries produced in secondary screens. The R23
variantusedtogeneratethedatainTableIVwasselectedbecauseofitsabilitytotolerate
SDSandnoconsiderationwasgiventoitstoleranceofAlcalase.Itisveryprobablethatother
R23variantsweretolerantofAlcalaseand,ifthiswerenotthecase,tolerancecouldeasilybe
regeneratedbyscreeningsmalllibraries.
In the early rounds of evolution, commercial detergents were used in selection. These
products were not ideal for evolution because their composition is not always publically
availableandtheycanchangeatthediscretionofthemanufacturer,sowedecidedtouse
selection with SDS instead. Nevertheless, purified variants were tested for tolerance to
commercial products. Interestingly, the R23 variant was not the variant most tolerant of
commercial powders. In going from R18 to 23 the tolerance to SDS increased, but the
tolerance to LD1 and LD2 decreased. It is probable that SDS is not present in any of the
commercial product and that in concentrating on SDS stability in R18 through R23 we
weakened the ability of variants to tolerate the detergents present in LD2. The R18_21H
variantshowedthebestoveralltolerancetocommercialpowders,butitstolerancetoLD1of
only16%couldbedescribedasmarginal.However,theR18_21Hvariantwasunaffectedby
thedetergentsinLD2thathadbeenformulatedtocontainaprotease.Itisnotclearthatthe
proteaseinLD2isAlcalase,butifitwerethenR18_21Hcouldbeaddedtothisproductsothat
enzymescouldactonbothproteinsandlipids.
-
23
Comparisonwithotherstudies
Directedevolutionhasbeenusedtoidentifydetergentstablevariantsofotherproteins,but
themethodsusedforselectionwerequitedifferenttothatusedhere(ScottandPlückthun
2013; Yong and Scott 2015). In these studies, the Cellular High-Throughput Encapsulation
SolubilisationandScreening(CHESS)methodwasused(YongandScott2015).Inonestudy,
very large libraries (1010
) were screened with an instrument (FACS) that detected a
fluorescencesignal.Thevariantproteinsweretestedwith2%SDSinabuffered10%glycerol
solution.OnevariantthatexhibitedenhancedtolerancetoSDS,hadonlytwochangesfrom
thestartingprotein.Bywayofcontrast,ourlibrariesweremuchsmallerandrequiredmultiple
roundsofevolutiontogiveenhancedstability,buttheselectioninourexperimentswasfor
multiple properties, not just SDS tolerance. When our experiments were focused on
enhancingSDStolerance(R18-R23),thetolerancequicklyroseandselectioninvolvedtreating
theenzymewith4%SDS,withnoglycerolpresenttoenhancestability.Wearguethatour
methodsaremoreversatileandmorethanadequatetoidentifythesmallnumberofchanges
requiredtogiveenhancedtolerancetoSDS.
Tothebestofourknowledge,directedevolutionhasnotpreviouslyusedtoenhanceprotease
tolerancein lipases,thoughotherapproacheshavebeen.Toenhanceproteaseresistance,
Grbavčićandcoworkersusedalipasefromthesameorganismasaprotease(Grbavčićetal.
2011).Thisassumesthatthelipasehasevolvedtobetolerantoftheprotease:eveniftrue,it
is unlikely that both enzymes will be suitable in all other respects for use in a laundry
detergent.Traditionalproteinengineeringapproacheshavealsobeenused(Frenkenetal.
1993;Markertetal.2001).Inonesuchapproach,proteasecleavagesiteswereidentifiedand
site specific mutagenesis used to alter the protein sequence near the cleavage site. This
approachislabourintensiveandassumesthatthechangeswillnotdegradetheactivityofthe
enzyme.Finally,previousstudies(Eceetal.2015)haveshownthatincreasedthermalstability
cangiverisetoincreasedtolerancetosurfaceactiveagents,butevolutionwithselectionfor
tolerance to surface active agents has not been described in the literature. Overall, we
expectedthatdirectedevolutionwouldbeamoreefficientwaytogeneratetolerancetoboth
proteasesandsurfaceactiveagents.Directedevolutionhasbeenusedtogenerateprotease
toleranceforsmallpeptidesusingadifferentmethod(Fiaccoetal.2016)tothatusedinthe
presentstudy.
-
24
Directedevolutionhasbeenusedinlipasestoidentifyenzymevariantswithalteredsubstrate
specificity (Bornscheuer2008;KlausLiebetonetal.2000b;Prasadetal.2011;Reetzetal.
1997).Ourstudydiffersfromthosecitedinthatsubstratespecificitywasalteredalongwith
otherproperties.
Sequenceanalysis
Studies involvingdirectedevolutionoftengivesome insight intotherelationshipbetween
sequenceandvariousphysicalproperties.Thepresentstudyisunusualinthatselectionwas
madeforthreeattributes,buttherearemanyotherpropertiesthatmightbeaffectedbythe
evolution.Forexample:enantioselectivity (Engströmetal.2010;K. Liebetonetal.2000a;
Reetz et al. 1997), stability in organic compounds (Dror et al. 2014; Korman et al. 2013),
thermostability(Augustyniaketal.2012;Liuetal.2017;Yuetal.2012;Zhangetal.2003),
substratespecificity(Fujiietal.2005).
Thechanges insequencethatoccurduringevolutioncouldprovide informationaboutthe
role of specific residues in catalysis as well as the physical properties of the enzymes.
Unfortunately,changesinthesequencewerenotmadesinglyagainstaconstantbackground
soitisdifficulttoknowtheeffectsofasinglemutationonthepropertiesofavariant.InR1
toR7(Alfaro-Chavezetal.2019),selectionforincreasedactivityresultedinenhancedstability
andsolubilitywithmodestgainsinactivity.FromR8onwards,selectionwasbasedonmore
thanonepropertyandonlyasmallsubsetofvariantswasthoroughlycharacterized,making
the task of assigning a role to specific changes more difficult. For example, it has been
suggestedthatsurfactantstabilitycorrelateswiththermalstability(Eceetal.2015;Salameh
andWiegel2010).Inthisstudy,theSDSstabilityincreasedasevolutionprogressed,butthe
same cannot be said for thermos-stability. The R23 variant exhibited greatly enhanced
tolerancetoaheatshockthatthewtortheR7variants,butitwaslessstablethantheR18
variant.Ourresultsgivesomesupporttothepropositionthatsurfactantandthermalstability,
however, they also suggest that this proposition may breakdown as SDS concentration
increases;moredataarerequiredtoconfidentlyanswerthisquestion.
-
25
Understandingtherelationshipbetweensequenceandfunctionisusuallydonewiththeaid
ofoneormorestructures.WewereunabletoobtainuseablecrystalsofLip3orofvariants
identifiedinR7orR17.Wethereforeadoptedahomologymodellingapproach(Alfaro-Chavez
etal.2019)andmappedthelocationsofthechangesontoaschematicofthestructure(Figure
3).ThreeofthechangesfixedintheR7variants(Alfaro-Chavezetal.2019)revertedtowtby
R17.Thisisbestexplainedbytheshufflingprocessinlibrarygeneration,ratherthansequence
changesintroducedbytheerrorpronepolymerase.SomeofthegenesfortheR7variants
wouldhavehadwild-typesequencesatthesepositions,andsoshufflinggavewtfragments
thatweremoreviableunderthenewselectionconditionsthantheR7variantsequences.One
ofthesereversions(greensquaresinFigure3)occurredinthecapdomainthatcoversthe
activesite:wepositthatitisimportantforsubstratespecificity.UptoR7,selectionwasfor
C8substrateswhileinsubsequentroundsitwasforsubstrateswithlongerchains.Thetwo
otherchangesoccurredatthestartandendofthesequence,atpositions40and347(green
squaresinFigure3).Inthesecases,thechangesoccurredinapieceofstrandthatlinkedtwo
elementsofsecondarystructureandweresolventaccessible.Wespeculatethatthesetwo
changes enhanced stability in the absence of detergents, but did not stabilise – or were
destabilising–intheirpresence.OnlyfourchangesobservedinR7(TableVandgreencircles
inFigure3)werepreserved inR17; thesewere residues201,268,291and321.Asnoted
above and explained more fully in our previous paper, three of these four changes
presumablyenhancethestabilityandsolubilityoftheenzyme.
AttheendofR17(TableV),wesequenced11variants.11changesweresaturated,ofwhich
fivewerefromR7(foursaturatedandonenotsaturatedinR7).TheM20VandM59Lchanges
mayreflectoxygensensitivity(seebelow).D230isinthelidofthecapdomain,suggestinga
role incatalyticenhancement.Otherchangesare in surfaceaccessible loops thatconnect
secondarystructureelementsandarepossiblyresponsibleforenhancingtolerancetosurface
activeagents.ThereweretwochangesthatsaturatedinR17(T185AandL351Q)wereclose
toproteasecleavagesites(seeFigure3).Bothchangescouldhavealteredproteasetolerance.
Asexpected,therewerenochangesintheinteriorofthemoleculecore.
At theendofR23, therewere13saturatedchanges; threewerenew,butpresent inR17
variants(E46K,W107LandC202Y,bluecirclesinFigure3A)andonethatwassaturatedinR17
-
26
andlostbyR23(S210T,redsquareinFigure3A).Threemutationssuggestthatchangesin
chargedresiduescanimproveSDStolerance(E46K,reversionofE347toKandD230G).The
reversechange,K347E,occurredin30%ofvariants inR7,butdisappearedwhenselection
involved SDS. Similarly, the D230G change that saturated in R17 removed a negatively
charged residue from the surface of the protein. The W107L change could give increased
stabilitybyreplacinganexposedlargearomaticsidechainwithasmallerone.
TheC202Yisdiscussedbelow.
AttheendofR23therewerethreechangesinwhichmethionineresidueswerealtered.The
M21LchangesaturatedinR7.ThechangesthatwereintroducedbetweenR7andR17include
threechangestomethionineandcysteineresidues:M20V,M59LandC202Y,withtheC202Y
changesaturatinginR23.WesuspectthattheoxidationsensitivityofMetandCys(Means
andFeeney1971)mayexplainthesechanges.M20andM321arelocatedonthesurfaceof
ourmodelstructure,butthereareothersulphurcontainingresiduesthatareaccessibleto
solventandnotmodified,sooxidationsensitivitycannotbethesoleexplanation.TheM59L
andC202Ychangesaffectburiedresidues.M59Lmaystabilizebygivingbetterpacking,but
thisisprobablynottrueofC202Y,asthisresidueisinthecapdomainclosetothehingepoint
ofthelid,suggestingthatthechangemayenhancecatalysis.
ConcludingRemarks
Perhaps the most important aspect of this work has been the development of screens to
selectforvariantswithenhancedproperties.Itmaybepossibletoadaptthesescreensfor
tolerance to surface-active agents other than SDS and proteases other than Alcalase. The
primarygoaloftheworkwastoproducevariantswithenhancementsinthree,potentially
orthogonal, properties and to develop methods to achieve this end. In this aim, we have
succeeded.Variantshavebeenidentifiedthatdegradeoilsandthattoleratesurfaceactive
agentsandproteases.Ourprotocolscanbeusedinthefuturetoevolveotherenzymesfor
applicationsinlaundrydetergents.However,thepresentworkalsoillustrateshowproteins
canevolvetosurvivewhatwasinitiallyanextremelyhostileenvironment.Intwostudies,a
relatively small number of changes were required to convert an enzyme that had poor
solubilityandweakactivityinwatertoonethatwasnotonlysoluble,butexhibitedenhanced
-
27
tolerance to heat treatment and that showed preferential activity in detergent solutions,
conditionsnotlikelytobeencounteredbytheorganismfromwhichtheenzymewasfound.
Acknowledgments.
WeacknowledgethestaffattheBRFforperformingtheDNAsequencing.Theauthorswish
to thankCSIRO forprovidingproteinpurification facility.A.A. is supportedbyaSENESCYT
scholarship from the Ecuadorian government. AG is supported by the BBSRC
(BB/M023281/1), the Academy of Finland (1286429) and by a Royal Society Wolfson
fellowship(alltoAG).
-
28
References
Adrio,JoseL.andDemain,ArnoldL.(2014),'MicrobialEnzymes:ToolsforBiotechnological
Processes',Biomolecules,4(1),117-39.
Alfaro-Chavez,Ana,etal.(2019),'Improvingonnature’sshortcomings:evolvingalipasefor
increased lipolytic activity, expression and thermostability', Protein Engineering,
DesignandSelection.
Arnold,FrancesH.(1998),'DesignbyDirectedEvolution',AccountsofChemicalResearch,31
(3),125-31.
--- (2018), 'Directed Evolution: Bringing New Chemistry to Life', Angewandte Chemie
(Internationaled.inEnglish),57(16),4143-48.
Arnold, Frances H. and Volkov, Alexander A. (1999), 'Directed evolution of biocatalysts',
CurrentOpinioninChemicalBiology,3(1),54-59.
Augustyniak, Wojciech, et al. (2012), 'Biophysical characterization of mutants of Bacillus
subtilis lipaseevolvedforthermostability:Factorscontributingto increasedactivity
retention',ProteinScience,21(4),487-97.
Bezard, J., Bugaut, M., and Clement, G. (1971), 'Triglyceride composition of coconut oil',
JournaloftheAmericanOilChemists’Society,48(3),134-39.
Bornscheuer,UweT.(2008),'Alterationoflipasepropertiesbyproteinengineeringmethods',
OCL,15(3),184-88.
Camacho-Ruiz, María de los Angeles, et al. (2015), 'A broad pH range indicator-based
spectrophotometricassayfortruelipasesusingtributyrinandtricaprylin',Journalof
LipidResearch,56(5),1057-67.
Celniker, Gershon, et al. (2013), 'ConSurf: Using Evolutionary Data to Raise Testable
HypothesesaboutProteinFunction',IsraelJournalofChemistry,53(3-4),199-206.
Dror,Adi,etal. (2014), 'Proteinengineeringbyrandommutagenesisandstructure-guided
consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in
methanol',Appliedandenvironmentalmicrobiology,80(4),1515-27.
Ece, Selin, et al. (2015), 'Improving thermal and detergent stability of Bacillus
stearothermophilusneopullulanasebyrationalenzymedesign',ProteinEngineering,
DesignandSelection,28(6),147-51.
Engström,Karin,etal.(2010), 'DirectedEvolutionofanEnantioselectiveLipasewithBroad
Substrate Scope for Hydrolysis of α-Substituted Esters', Journal of the American
ChemicalSociety,132(20),7038-42.
Fiacco, Stephen V., et al. (2016), 'Directed Evolution of Scanning Unnatural-Protease-
Resistant(SUPR)PeptidesforinVivoApplications',ChemBioChem,17(17),1643-51.
Frenken,L.G.,etal.(1993),'Pseudomonasglumaelipase:increasedproteolyticstabilityby
proteinengineering',ProteinEng,6(6),637-42.
Fujii,Ryota,etal.(2005),'DirectedevolutionofPseudomonasaeruginosalipaseforimproved
amide-hydrolyzingactivity',ProteinEngineering,DesignandSelection,18(2),93-101.
Gattiker,Alexandre,etal.(2002),'FindPept,atooltoidentifyunmatchedmassesinpeptide
massfingerprintingproteinidentification',PROTEOMICS,2(10),1435-44.
Grbavčić, Sanja, et al. (2011), 'Production of lipase and protease from an indigenous
Pseudomonas aeruginosa strain and their evaluation as detergent additives:
Compatibility study with detergent ingredients and washing performance',
BioresourceTechnology,102(24),11226-33.
-
29
Gupta,Namita,Rathi,Pooja,andGupta,Rani(2002),'Simplifiedpara-nitrophenylpalmitate
assayforlipasesandesterases',AnalyticalBiochemistry,311(1),98-99.
Hasan, Fariha, et al. (2010), 'Enzymes used in detergents: Lipases', African Journal of
Biotechnology,9(31),4836-44.
Korman,TylerP.,etal.(2013),'Dieselzymes:Developmentofastableandmethanoltolerant
lipaseforbiodieselproductionbydirectedevolution',BiotechnologyforBiofuels,6(1),
70-70.
Kumar, Dinesh, et al. (2008), 'Microbial proteases and application as laundry detergent
additive',ResearchJournalofMicrobiology,3(12),661-72.
Lanka,SuseelaandLatha,J.NaveenaLavanya(2015), 'Ashortreviewonvariousscreening
methods to isolate potential lipase producers: Lipases-the present and future
enzymesofbiotechindustry',InternationalJournalofBiologicalChemistry,9(5),207-
19.
Lawrence, R. C., Fryer, T. F., and Reiter, B. (1967), 'Rapid Method for the Quantitative
EstimationofMicrobialLipases',Nature,213(5082),1264-65.
Liebeton,K.,etal.(2000a),'Directedevolutionofanenantioselectivelipase',ChemBiol,7(9),
709-18.
Liebeton,Klaus,etal.(2000b),'Directedevolutionofanenantioselectivelipase',Chemistry&
Biology,7(9),709-18.
Liu, Yihan, et al. (2017), 'Improvement in thermostability of an alkaline lipase I from
Penicilliumcyclopiumbydirectedevolution',RSCAdvances,7(61),38538-48.
Markert,Yvonne,etal.(2001),'IncreasedproteolyticresistanceofribonucleaseAbyprotein
engineering',ProteinEngineering,DesignandSelection,14(10),791-96.
Maurer,Karl-Heinz(2004), 'Detergentproteases',CurrentOpinioninBiotechnology,15(4),
330-34.
Means,GaryandFeeney,Robert(1971),ChemicalModificationofProteins(USA).
Niyonzima,F.N.andMore,S.S.(2015),'Microbialdetergentcompatiblelipases',JOURNAL
OFSCIENTIFIC&INDUSTRIALRESEARCH,74(2),105-13.
Porter,JoanneL.,Rusli,RukhairulA.,andOllis,DavidL.(2016),'DirectedEvolutionofEnzymes
forIndustrialBiocatalysis',ChemBioChem,17(3),197-203.
Powell, Keith A., et al. (2001), 'Directed Evolution and Biocatalysis',Angewandte Chemie
InternationalEdition,40(21),3948-59.
Prasad, S., Bocola, M., and Reetz, M. T. (2011), 'Revisiting the lipase from Pseudomonas
aeruginosa:directedevolutionofsubstrateacceptanceandenantioselectivityusing
iterativesaturationmutagenesis',Chemphyschem,12(8),1550-7.
Quintero-Flórez,Angélica,etal.(2015),'TheFattyAcidCompositionofVirginOliveOilfrom
DifferentCultivarsIsDeterminantforFoamCellFormationbyMacrophages',Journal
ofAgriculturalandFoodChemistry,63(30),6731-38.
Reetz, Manfred T., et al. (1997), 'Creation of Enantioselective Biocatalysts for Organic
ChemistrybyInVitroEvolution',AngewandteChemieInternationalEditioninEnglish,
36(24),2830-32.
Saeki,Katsuhisa,etal.(2007),'Detergentalkalineproteases:enzymaticproperties,genes,and
crystalstructures',JournalofBioscienceandBioengineering,103(6),501-08.
Salameh, Moh'd A. and Wiegel, Juergen (2010), 'Effects of Detergents on Activity,
Thermostability and Aggregation of Two Alkalithermophilic Lipases from
Thermosyntrophalipolytica',Theopenbiochemistryjournal,4,22-28.
-
30
Scott, Daniel J. and Plückthun, Andreas (2013), 'Direct Molecular Evolution of Detergent-
Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells', Journal of
MolecularBiology,425(3),662-77.
Stemmer,W.P.(1994),'RapidevolutionofaproteininvitrobyDNAshuffling',Nature,370
(6488),389-91.
Stevenson,BradleyJ.,Liu, Jian-Wei,andOllis,DavidL. (2008), 'DirectedEvolutionofYeast
Pyruvate Decarboxylase 1 for Attenuated Regulation and Increased Stability',
Biochemistry,47(9),3013-25.
Turner, Nicholas J. (2009), 'Directed evolution drives the next generation of biocatalysts',
NatureChemicalBiology,5,567.
Wu,Chenyi,etal.(1999),'Proteolysisofnativeproteins.Trappingofareactionintermediate',
JournalofBiologicalChemistry,274(2),1108-15.
Yong,K.J.andScott,D.J.(2015),'Rapiddirectedevolutionofstabilizedproteinswithcellular
high-throughputencapsulationsolubilizationandscreening (CHESS)',Biotechnology
andBioengineering,112(3),438-46.
Yu,Xiao-Wei,etal.(2012),'EnhancedthermostabilityofaRhizopuschinensislipasebyinvivo
recombinationinPichiapastoris',MicrobialCellFactories,11(1),102.
Zhang,Ningyan,etal. (2003), 'Improving toleranceofCandidaantarctica lipaseB towards
irreversible thermal inactivation through directed evolution', Protein Engineering,
DesignandSelection,16(8),599-605.
Zhao,H.,etal. (1998), 'Molecularevolutionbystaggeredextensionprocess (StEP) invitro
recombination',NatBiotechnol,16(3),258-61.
-
31
Listoffigures:
Fig1.Schematicdiagramtoillustratethestagesinevolution
Fig2.ToleranceofvariantsoflipaseLip3toSDSafterseveralroundsofevolution
Fig3.A.SchematicdiagramoflipaseLip3.Arrowsrepresentβsheets,barrelsα-helices.
CatalytictriadS145,D318andH350areingreenandresiduesoftheoxyanionholein
orange.MutationssaturatedinR7andretainedthroughoutevolutioningreencircles.
MutationssaturatedinR17andretaineduntilfinalevolutioninredcircles.Mutations
saturatedinR23inbluecircles.Mutationsthatweresaturatedbutdisappearedarein
squares.Alcalase®hydrolysissitesareindicatedbyasterisks(*).B.ModelofLip3.Inthe
leftthecompletestructure,coredomaininblue,capdomaininbrown,lidinred.Inthe
rightgreenspheresshowmutationssaturatedinR7,redspheressaturationsinR17,blue
spheressaturationsinR23andblackspherescutsitesofprotease.
-
TableI.Initialcharacterizationofwild-typeandvariantsfromR7.
wt R759A R782E
DetergentEffect
%remainingactivityafter60minutesofincubation
% % %
SDS2% 1±2 1±1 2.3±2.5
TritonX-1002% 20±3 99±1 37±1
LD10.5% 5±1 2±1 1.9±0.1
LD20.5% 2.2±0.3 1.9±0.2 2.5±0.2
LD30.5% 30.7±0.3 64.7±0.2 36.6±0.4
Alcalase®2% 4.0±0.4 6.5±0.5 48.8±0.4
SpecificActivity
µM!-nitrophenol/min/µgprotein
pNP-C3 15.9±0.02 99.9±0.1 74.1±0.1
pNP-C8 96.6±0.1 147.56±0.02 148.14±0.01
pNP-C16 1.635±0.002 3.06±0.02 0.782±0.004
pNP-C18 1.566±0.003 4.16±0.01 0.89±0.01
-
TableII.Testofgrowthinagarplateswithdetergents
Plates CG8823wt R9library
LBamp ++ ++
LBampTGC8 ++ ++
LBampTGC4 ++ ++
0.05%LD2 ++ ++
0.1% + +
0.5% - -
1% - -
2% - -
0.05%LD1 ++ ++
0.1% + +
0.5% - -
1% - -
2% - -
0.5%v/vLD3 ++ ++
1% + ++
1.5% + ++
2% + +
(++)>2000colonies
(+)~500colonies
(-)nogrowth
-
TableIII.Experimentaldetailsofthecontinuousgenerationduringdirectedevolution
Rou
nd!
PrimaryScreening! SecondaryScreening
Library! Variants1! Selection
2! Selection
3! Variants
4
8 2.72*105
820 TGC8/18hours pNP-C12/Detergenttolerance! 10
9 2.4*104
620TGC8/oliveoil/
18hours/72hours!
pNP-C12/
DetergentLD1!19
10 6*104
620oliveoiland
detergent/72
hours
pNP-C12/0.01%SDS! 14
11 6*104 880
oliveoiland
detergent/72
hours
pNPC12/0.02%SDS! 20
12 2.4*104 350 oliveoil/72hours pNP-C12/0.07%SDS! 15
13 6.4*104 530 oliveoil/48hours
pNP-C12/0.1%SDS/0.4%protease1
hour!12
14 1.4*104 260 oliveoil/48hours
pNP-C12/0.1%SDS/0.4%protease3
hour!20
15 8*103 180 oliveoil/48hours
pNP-C12/0.1%SDS/0.4%protease1
and3hours 10
16 2.1*104 260 oliveoil/40hours
pNP-C16/0.1%SDS/0.4%protease1
and3hours/0.02%tritonX-100/oils 20
17 2.4*104 260 oliveoil/40hours
pNP-C16/0.1%SDS/0.4%protease1
and3hours/0.1%tritonX-100/oils
mixture+phenolred17
18 1.4*104 350
96-wellplate
pNP-C16/SDS
0.5%/oilsmixture
pNP-C16 14
19 3.3*104 440
LBkanplates
(pET26b)/1%SDS-
naphthylpalmitate
pNP-C16/0.5%SDS 6
20 3.3*104 180
LBampplate(new
primer)/2%SDS-
naphthylpalmitate
pNP-C16/0.5%SDS 3
21 6*104 450
LBampplate(new
primer)/2%SDS-
naphthylpalmitate
pNP-C16/0.5%SDS/
thermo65!C30’11
22 6*104 720
LBampplate(new
primer)/2.5%SDS-
naphthylpalmitate
pNP-C16/0.5%SDS/
thermo65!C30’9
236*10
4
(150
col/plat)
540LBampplate+olive
oil/3%SDS-naphthyl
palmitate
pNP-C16/4%SDSremovedwithKCl/
thermo65!C30’10
1Thenumberofvariantsselectedintheprimaryscreen.
2Selectionwasdoneforlipolyticactivityinagarplates.ForR9,twosetsofagarplateswereused,one
waspreparedwithTGC8incubatedfor18hoursandtheotherwaswitholiveoilandincubatedfor72
hours;timerequiredforhaloformationdecreasedfrom72to40hoursforoliveoilandtheincubation
temperaturewas37°Cinallrounds.3Selectionwasinteractiveinthesecondaryscreening.Thetemperatureatwhichtheselectedcolonies
weregrownin96-wellplatesforalaterassayforesteraseactivity4Thenumberofvariantsselectedinthesecondaryscreen.
-
TableIV.Characterizationofmutantsforlibraryofsurfactanttolerance.
wt R7_59A R7_82E R13_44C R17_29G R18_211H R20_11A R23_110A
DetergentEffect
%remainingactivityafter60minutesofincubation
SDS2% 1±2 1±1 2.3±2.5 59±1.5 69±1.5 84±10 102±2 260±3
TritonX-1002% 20±3 99±1 37±1 101.5±0.4 95.1±1.4 98±2 98±1 118±4
LD10.5% 5±1 2±1 1.9±0.1 1.8±0.1 7.2±3.2 16.9±0.2 1.2±0.2 11±1
LD20.5% 2.2±0.3 1.9±0.2 2.5±0.2 98±3 72.7±3.1 98±1 44±6 11±1
LD30.5% 30.7±0.3 64.7±0.2 36.6±0.4 98±2 122.3±5.2 97±2 102±1 140±5
Alcalase®2% 4.0±0.4 6.5±0.5 48.8±0.4 96±4 80.3±2.3 101±2 100±3 41±1
SpecificActivity
µMp-nitrophenol/min/µgprotein
pNP-C3 15.9±0.02 99.9±0.1 74.1±0.1 106.7±0.3 320.1±5 131.6±0.4 476.7±0.1 366.7±0.2
pNP-C8 96.6±0.1 147.56±0.02 148.14±0.01 145.61±0.03 857.6±6 146.7±0.02 460.81±0.03 697.6±0.1
pNP-C16 1.635±0.002 3.06±0.02 0.782±0.004 56.6±0.1 21.3±2.2 139.09±0.05 295.32±0.01 95.56±0.01
pNP-C18 1.566±0.003 4.16±0.01 0.89±0.01 65.98±0.12 29.7±1 160.2±0.1 152.66±0.01 94.2±0.1
Lipolyticactivity
µMacidreleased/min/µMprotein
Tributyrin 4.5±0.3 23±1 16±3 65±1 30±366±1 71±8 40.2±0.3
glyceryltrioctanoate 9.5±0.2 60±1 57±1 59.4±0.1 78±1 55±1 36.4±4.1 21.7±0.4
coconutoil 3.1±0.2 14.5±3.5 14±2 13±1 12.4±2 20±2 19±2 10±1
glyceryltrioleate 0.2±0.2 2.4±0.4 2±1 17.3±0.3 14±0.2 21±1 23±2.0 13.3±0.4
oliveoil 0.08±0.01 1.3±0.3 1.2±0.5 3.7±0.3 2.2±0.3 16.9±0.4 15±1 5.1±0.3
Thermostability
[°C]
T50,45min 37.3±0.1 45±0.4 49.6±0.7 57.1±0.2 55.8±0.7 73.8±0.3 58.2±0.4 65.1±0.8
-
TableV.Alterationsinaminoacidsequences
ID
20
40
46
59
107
174
185
186
201
202
210
211
225
230
234
242
250
268
291
305
321
347
351
act
Subs
(pNP)
WT
MàV
YàF
EàK
MàL
WàL
AàV
TàA
RàP
VàA
CàY
SàT
NàH
TàA
DàG
NàS
YàH
EàG
NàI
PàL
KàG
MàL
KàE
LàQ
1
0.0
5
C8
C16
R7
(30)
%
10 23 3 10 0% 10 3 0 100 0 30 0 23 7 0 0 47 93 100 3 93 30 3
104-
222
C8
R17
(11)
%
91 0 55 100 27 100 100 0 100 9 91 18 9 100 18 18 45 100 100 27 100 0 100 0.94-
1.15
C16
R18_211H V K L L V A A Y G I L L Q 1 C16
R23
(8)
%
100 0 100 100 100 100 100 63 100 100 0 0 0 100 13 0 0 100 100 0 100 0 100 0.2-
1.2
C16
Consurf*
E E E B E B B E B B E E E E e/f b e/f B E E B E B
Solv.
Access.
(model)
28.
9
131.
4
135.
4
13 136.
8
2.7 23.
5
205.
9
93.
6
32.
6
59.
8
80.
3
120.
5
72.
1
61 129.
5
92 0.0
3
3.6 141 10.
2
127.
8
16.
1
*(Celnikeretal.2013)
-
SupplementaryMaterial
TableS1.Characterizationofvariantsas indicatedby residualactivity for tolerance to
surfactants,detergentsandAlcalase®
Time
[min]wt R7_9A R7_82E R13_44C R18_211H R20_11A
% % % % % %
SDS1%
30 7.98±0.29 29.5±0.1 57.3±0.1 85.02±0.05 101.63±0.02 100.3±0.1
60 1.98±0.05 31.3±0.1 58.83±0.02 86.1±0.1 98.94±0.03 102.8±0.1
90 0.9±0.1 30.1±0.1 59.78±0.01 82.82±0.03 99±3 98±5
120 0.6±0.5 28.9±0.4 54±1 78±2 97±2 100±1
SDS2%
30 4.39±0.02 2.3±0.1 3.8±0.4 19±1 21±6 92±5
60 1±2 1±1 2.3±2.5 59±17 84±13 102±2
90 1.1±0.4 0.3±0.5 6±6 0.5±0.1 5±1 58±8
120 0 1±1 2.5±0.5 3±2 2±1 0.4±0.2
TritonX-
1002%
30 80±1 128±2 87±1 100±3 100±1 98.3±0.2
60 20±3 99±1 37±1 101.6±0.4 98±2 98±1
90 18.2±0.4 89±1 34±1 100±3 99±1 99.02±0.39
120 16.5±0.6 75.8±0.5 25±2 101±1 98±2 101±2
LD10.5%
30 5.95±0.42 1.7±0.1 0.1±0.4 25.9±0.4 74±5 50±2
60 5±1 2±1 1.9±0.1 1.8±0.1 16.9±0.2 1.2±0.2
90 1.1±0.2 0.2±0.1 0.5±0.2 0.9±0.1 6.6±0.1 0.18±0.01
120 0.5±0.3 0.03±0.02 0.2±0.2 1.2±0.2 5±1 2±2
LD20.5%
30 13±3 0.8±0.5 15±1 98.1±0.4 102±2 97±1
60 2.2±0.3 1.9±0.2 2.5±0.2 98±3 98±1 44±6
90 1.5±0.2 0.6±0.1 2.6±0.1 97±1 76±1 9±3
120 2.5±0.4 0.6±0.2 1.5±0.1 95±1 45±1 2.2±0.1
LD30.5%
30 83±2 125±42 85±1 101±3 100±1 100±2
60 30.7±0.3 64.7±0.2 36.6±0.4 98±2 97±2 102±1
90 21.1±0.3 52.3±0.4 30.6±0.1 99±2 100±1 101±2
120 21±1 45.9±0.4 24±1 98.4±0.5 99±3 102±1
Alcalase®
2%
30 2.±3 5±1 39±2 99.7±0.2 100±2 99±1
60 3.97±0.41 6±1 48.8±0.4 96±4 100.5±2 100±3
90 7±2 5±1 50±1 86±2 95±2 99±3
120 6±1 4.6±0.3 41±1 59.3±0.4 76±2 66±1
p2 main p2 Table Ip2 Table IIp2 Table IIIp2 Table IVp2 Table Vp2_Figure 1p2_Figure 2p2_Figure 3p2 Supplementary Material
top related