Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

Thermostable

chitinase

from

Cohnella

sp.

A01:

isolation

and

product

optimization

Nasrin

Aliabadi,

Saeed

Aminzadeh

∗

,

Ali

Asghar

Karkhane,

Kamahldin

Haghbeen

NationalInstituteofGeneticEngineeringandBiotechnology,DepartmentofIndustrialandEnvironmentalBiotechnology,

BioprocessEngineeringGroup,Tehran,Iran

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31March2015 Accepted4April2016 Availableonline26July2016 AssociateEditor:LucySeldin

Keywords: Biochemicalcharacterization Chitinase Cohnellasp.A01 Isolation Thermostable

a

b

s

t

r

a

c

t

Twelvebacterialstrainsisolatedfromshrimpfarmingpondswerescreenedfortheirgrowth activityonchitinasthesolecarbonsource.Thehighlychitinolyticbacterialstrainwas detected byqualitativecupplateassayandtentativelyidentifiedtobeCohnella sp.A01 based on16SrDNA sequencingandbymatching thekey morphological,physiological, and biochemicalcharacteristics. Thecultivation ofCohnella sp.A01inthe suitable liq-uid medium resultedin theproductionof highlevels ofenzyme.The colloidalchitin, peptone, and K2HPO4 representedthe bestcarbon, nitrogen, and phosphorussources, respectively.EnzymeproductionbyCohnellasp.A01wasoptimizedbytheTaguchimethod. Ourresultsdemonstratedthatinoculationamountandtemperatureofincubationwerethe mostsignificantfactorsinfluencingchitinaseproduction.Fromthetestedvalues,thebest pH/temperaturewasobtainedatpH5and70◦_C,withK

mandVmaxvaluesofchitinasetobe 5.6mg/mLand0.87␮mol/min,respectively.Ag+_,Co2+_{,iodoacetamide,andiodoaceticacid} inhibitedtheenzymeactivity,whereasMn2+_,Cu2+_{,Tweens(20and80),TritonX-100,and} EDTAincreasedthesame.Inaddition,thestudyofthemorphologicalalterationofchitin treatedbyenzymebySEMrevealedcracksandporesonthechitinsurface,indicatinga potentialapplicationofthisenzymeinseveralindustries.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Chitin,a␤-1,4polymerofN-acetyl-d-glucosamine(GlcNAc), whichiswidelydistributedamongfungi,crustaceans, mol-luscs, coelenterates, protozoan, and green algae, is the second-most abundant biopolymer found in nature after

∗ _{Correspondingauthor.}

E-mail:[email protected](S.Aminzadeh).

cellulose.1,2_{Severalmilliontonsofchitinissynthesizedand} degradedeachyearinthebiosphere.3_{Thisnaturalresourceis} relativelyeasilyaccessible,e.g.,fromsourcessuchasshrimp, crab,andkrill,whichareconsideredaswaste;chitinaccounts for 20–58% of the dry weight of these wastes.4 _Chitinous wastes are also produced in large amounts in industries

http://dx.doi.org/10.1016/j.bjm.2016.07.009

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

suchasseafoodprocessingindustry,whichproducesprawn waste (containing23% chitin).5 _{These wastes may pose as} anenvironmentalthreatontheiraccumulationand dueto extremely slow decomposition.6 _{Therefore, organisms that} producechitin-degradingenzymescanbeusefulin bioremedi-ationandwastemanagementaswellashelpreleasenutrients andmaintainthecarbon,nitrogen,andotherbiogeochemical cyclesintheenvironment.5,7,8

Chitinases (EC 3.2.1.14) are present in a wide range of organisms,includingviruses,bacteria,fungi,insects,higher plants,andanimals;theseenzymesarecapableofcatalyzing thehydrolysisofchitin.9Chitinaseparticipatesinavarietyof functions,includingdefense,nutrientdigestion, morphogen-esis,and pathogenesis.3 _{Mostchitin-degrading prokaryotes} are thegliding bacteria,pseudomonad, vibrio, enterobacte-ria,actinomycete,bacilli,andclostridia.10_{Bacterialchitinases} haveasizerangeof20–60kDa.11,12_{Chitinaseshavepotential} applicationsinvariousareasofbiotechnology,biomedicine, agriculture,andnutrition.13,14

Microorganisms adapt to the condition in which they havetoliveandsurvive.Thermophiles synthesizeproteins that are thermostable and resist denaturationand proteo-lysis. Due to their ecological role and growing interests of their application in biotechnology, a large number of chitin-degradingbacteriahavebeenisolatedandtheir respec-tive genes have been cloned and characterized. However, only few thermostable chitinases have been reported in microorganisms.15,16 Thethermostablechitinolyticenzymes can hydrolyze their substrates at high temperatures and represent important advantages against their mesophilic counterparts, for example, chemical and thermal stability, decreased viscosity, increased solubility, and significantly reduced contamination risk.16 _{Therefore, researches have} beenfocusedonmicroorganismscapableofproducingsuch enzymesthatcantolerateextremeenvironmentalconditions. Several articles have been published on the classical methodofmediumoptimizationbychangingone indepen-dentparameterwhilefixingtheothersfixed.9_{Thisprocesscan} beextremelytimeconsuming,expensive,andunmanageable wheninvolvingalargenumberofvariablesaswellasit can-notdescribethecombinedeffectofallthefactorsinvolved. Severalfactorshavebeenreportedtoinfluenceenzyme pro-ductionbybacteria.17_{Optimizingalloftheseaffectingfactors} bystatisticalexperimentaldesignscanaddressthese limita-tions.ThemethodsofTaguchihavebeenusedextensivelyin experimentdesigning.18_{However,theapplicationofTaguchi} methodinbiologicalscienceisscarce.17

ThegenusPaenibacilluswasoriginallydefinedin1993by Ash et al. after an extensive comparative analysis of 16S rRNA gene sequences of approximately 50 species of the genusBacillus.19–21 _{Theywerereportedtopossessinhibitory} effectonbacteriaorfungiowingtotheircellwall-degrading enzymes.22 _{Cohnella is a member of the Paenibacillaceae} family.23 _{Earlier, we had reported the Taguchi method of} chitinase production optimization from Serratia marcescens

B4A9 _{and polygalacturonase productionfrom Macrophomina}

phaseolina.24_{Inthisstudy,weattemptedtoisolateand}

charac-terizethethermostablechitinasefromthenovelthermophilic strain,Cohnellasp.A01.Moreover,withtheobjectiveof obtain-ing accurate data and economizing the use of time and

materials, we decided to use the Taguchi method for the optimizationofculturemediuminsteadofusingtraditional method. Onlylimitedstudieshavereportedstatistical opti-mizationfortheproductionofchitinase.25_{Thepresentreport} isanattempttoformulateasuitableproductionmediumby usingstatisticaloptimizationthatcansubstantiallyenhance chitinaseproductionbyCohnellasp.A01.

Materials

and

methods

Materials

Flake crab shell chitin, 3,5-dinitrosalicylic acid (DNS), N-acetyl-d-glucosamine,andbovineserumalbumin(BSA)were obtained from Sigma (St. Louis, Mo. USA). Colloidal chitin was prepared by the modified method of Roberts and Selitrennikoff.26 _{Taq DNA polymerase, 1-kb DNA ladder,} standard proteins for molecular weight determination, T4 DNAligase,IPTG,andX-GalwerepurchasedfromFermentas (Burlington,Canada).DNAextractionkitwaspurchasedfrom Metabion(Martinsried,Germany).TheHigh-PurePCR Purifica-tionKitwassourcedfromRoche(Indianapolis,USA).Allother chemicalswerepurchasedfromMerck(Darmstadt,Germany) andwereofthehighestanalyticalgradeavailable.

Isolationofmicroorganisms

Samplescollectedfromshrimpfarmingwastewaterlocatedin Choebdeh-Abadan(southwesternofIran)andusedfor isola-tionstudiesinourlaboratory.TheclimateinAbadanisarid. Summersaredryandhotwithtemperaturesof>45◦C(average 55◦C);thesoaringtemperaturesmayadvanceto>65◦C. Screeningofthermophilicchitinase-producing

microorganism

For the directscreeningofchitinaseactivityfrom bacterial colonies,clearzoneproductionwasmonitoredat60◦C.27_The isolatedmicroorganismswereculturedonagarplates contain-ing0.5%colloidalchitin,0.07%K2HPO4,0.03%KH2PO4,0.05%

MgSO4·7H2O,2%agar,0.2%NH4NO3,0.1%NaCl(w/v),and0.1%

trace elements (pH7.8). Thecultureswere incubated for3 daysat60◦C.Onlyonechitinolyticbacterialstrain(detected byacolonyproducingahaloarounditself)wastransferred intofreshchitincontainingnutrientbrothmediumand incu-batedat60◦C,followingwhich,thestrainwaspreservedas cellsuspensionsin10%glycerolat−80◦_C.

Cultureandgrowthconditions

For chitinase production,the strain selected from the pri-maryscreeningwasculturedinapreculturemedium(trace element 0.1%, tryptone 1%, yeast extract 0.5%, NaCl 0.5%, agar 0.2%, peptone 0.03%, K2HPO4 0.07%, KH2PO4 0.03%,

CaCl2·2H2O0.013%,NH4NO30.1%,glucose0.2%,colloidchitin

0.5%,MgSO4·7H2O0.05%)for24hat60◦Conashaker

incuba-tor(180rpm).Then,4mLofthepreculture(3.5×108_cells/mL)

wasaddedto100mLoftheproductionmedium(preculture mediumwithoutglucose).Theresultantinoculatedmedium

wasculturedat60◦Cfor6daysonarotaryshaker(180rpm). Thesample(4mL)oftheculturemediumwasremovedevery 24handcentrifugedat12,000×gfor15minat4◦C.The result-ingsupernatantwasthencollectedandusedforsubsequent chitinaseactivityassays.

PCRamplificationandstudyof16SrRNAgene

A partial DNA sequence for the 16S rRNA gene (ca. 1.4-kbp fragment) was amplified by PCR with the primers f16s: AGAAAGGAGGTGATCCAGCCGC and r16s: TCTTTTG-GAGAGTTTGATCCTG. Isolation of genomic DNA and PCR amplificationwereconductedassuggestedby.28 _The ampli-ficationwasperformedbyusingtheTechne®_{TC-512Gradient}

ThermalCycler(UK)withthefollowingcyclingparameters: 94◦C for1min,followedby35 cyclesof30sat94◦C,1min at51◦C,and1.5minat72◦C,withafinalextensionof5min. ThePCRfragmentwaspurifiedbyusingtheHighPure PCR PurificationKit(Roche,Germany).ThepurifiedPCRfragment wasthenligatedintothepTZ75R(Fermentas,Canada)bythe T/Acloning procedure, and the constructwastransformed

intoEscherichiacoliDH5␣.Theamplifiedconstructharboring

the16SrDNAwasthenextracted,purified,andsequencedby Seqlab(Germany) withappropriateprimers.Thenucleotide sequenceswereanalyzedbyusingtheDNASISprogram (ver-sion3.2;HitachiSoftwareEngineeringCo.Ltd.);theobtained sequenceswerecompiledandcomparedwiththesequences inthedatabases(http://www.ncbi.nlm.nih.gov)byusingthe BLASTprogram.Othermorphologicalandphysiological char-acteristicsfortheselectedstrainwereanalyzedaccordingto theBergey’sManualofSystematicBacteriology.

Selectionofcarbon,nitrogen,andphosphorussources For the selection of the best source of carbon, nitrogen, and phosphorus for chitinase production, several carbon, nitrogen, and phosphorus sources were used in the pro-duction liquid medium. To select the carbon sources in the production medium without chitin, 1% (w/v) glucose, galactose, mannose, fructose, arabinose (as monosaccha-ride); lactose, sucrose, maltose (as disaccharide); and starch, polygalacturonic acid, cellulose, chitin plus glu-cose, and chitin powder (as polysaccharide) were added to each basic medium separately. To select the nitrogen sources, 1% (w/v) peptone, NH4HCO3, (NH4)2SO4, NH4NO3,

NH4Cl, Ca(NO3)2·4H2O,C4H11NO3,(NH4)2HPO4,and triptone

NH4H2PO4wereaddedtothebasicmediumwithoptimal

car-bonsource.Similarly,1%(w/v)Na2HPO4,NaH2PO4,K2HPO4,

KH2PO4,andNH4H2PO4wereaddedtothemediumto

deter-minetheoptimumphosphorussources.Thecarbon:nitrogen (C:N)ratioarethesameinallthemedia.

Experimentaldesignandanalysis

Wefirstdetermined the various factorsto beoptimized in theculturemediumthatcanhavecriticaleffectonthe chiti-naseyield.Factorsandtheirrelatedlevelswereselectedbased on the consensus among the design engineers, scientists, and technicians with relevant experience involved in this experiment.Basedontheobtainedexperimentaldata,eight

Table1–Variablesandtheirlevelsemployedinthe Taguchi’srobustdesignmethodforoptimalchitinase productionbyCohnellasp.A01.

Serial number

Factors Level1 Level2 Level3

1 Temperature(◦C) 55 60 65 2 pH 6.5 7.5 8.5 3 NaCl%(w/v) 0.17 0.5 1.5 4 K2HPO4%(w/v) 0.035 0.07 0.14 5 NH4NO3%(w/v) 0.5 1.0 2.0 6 Chitin%(w/v) 0.17 0.5 1.5 7 Traceelement%(v/v) 0.05 0.1 0.2 8 Inoculation%(v/v) 2 4 8

factors were consideredtosignificantlyinfluencethe chiti-naseyield.29Table1displaystheeightcontrolfactorsandtheir levelsemployedintheTaguchi’srobustexperimentaldesign. AstandardorthogonalarrayL2730_{wasusedtoexaminethis} eight-factor(sixchemicalparameters,twophysical parame-ters)system.Aruninvolvedthecorrespondingcombination oflevelstowhichthefactorsintheexperimentwereset.Thus, theTaguchi’smethodcancalculatetheconditionofleast vari-abilityfromtheSNratiosandtheconditionofthebestreaction performedbymaximizingtheoveralldesirability.31

Extractionandpartialpurificationofchitinase

Allexperimentalstepswereconductedat4◦Cinacoldroom. Theresultingsupernatant(100mL)oftheproductionmedium wasfractionatedby20–80%solidammoniumsulfate precip-itationandthencentrifugedat12,000×gfor15minat4◦C; theprecipitatedproteinsweresuspendedin20mMphosphate buffer(pH7.8)anddialyzedagainstthesamebufferfor24hat 4◦C.

Enzymaticactivity

The chitinase activity was determined by measuring the reducingend-groupN-acetylglucosamine(GlcNAc)(asafinal product) produced from colloidal chitin as a substrate. Colloidal chitin was prepared from chitin, as described previously.9_{Thestandardreactionmixturecontaining0.1mL} of1%(w/v)colloidalchitinand0.1mLofthepartially puri-fiedenzymewasincubatedat60◦Cfor60min.Thereaction was stoppedbythe additionof0.2mLofDNS,followedby heatingat100◦Cfor5min.Then,thesampleswerecooledto roomtemperatureandcentrifugedat6000×gfor15min.The concentrationofthereducingsugarwasdeterminedbythe modifiedDNSmethod.32 _{Theabsorptionofthetestsample} wasmeasuredat540nmbyaUVspectrophotometer (Beck-manDU530,USA)alongwithreactionblanks.Oneunit(U)of thechitinaseactivitywasdefinedastheamountofenzyme requiredtoliberate1mmolofthereducingsugarperminute underthedescribedconditions.

EffectoftemperatureandpHonthechitinaseactivityand stability

ThebestpHvalueofpartiallypurifiedchitinasewas deter-mined by using 50-mM mixed buffer (glycine, NaHCO3,

NaH2PO4)(pHs3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,and12.0)

byperformingthestandardassay.Thebesttemperaturevalue ofthechitinaseactivitywastestedat10,20,30,40,50,60,70, 80,and90◦Cin50-mMphosphatebuffer(pH7.8).Afterthat, theresidualactivityofthechitinasewasmeasuredbyusing thestandardassay.

Todeterminethetemperaturestability,chitinasewas ini-tiallypreincubatedatdifferenttemperatures(10–90◦C).Every 10and20min(upto90min),thesampleswereplacedonan icebathfor30minandthenthecolloidalchitinwasaddedand thestandardenzymeassaywasperformedat60◦C.Toobtain thepHstability, theenzymewasaddedtobufferswith dif-ferentpHs(3–12)for90minat25◦C,followedbythestandard enzymeassayatthebestpHvalue.

Effectofmetalionsanddetergentsonchitinaseactivity To study the detrimental effects of metal ions and deter-gents,thechitinaseactivitywasdeterminedbythestandard assaymethodinthepresenceofAg+_,Al3+_,Ba2+_,Ca2+_,Co2+_,

Cu2+_{, Fe}2+_{, Fe}3+_{, K}+_{, Ni}2+_{, Mg}2+_{, Mn}+2 _{(1,5,10,and 15mM)}

andethylenediaminetetraaceticacid(EDTA)iodoacetamide, iodoaceticacid,Tween20,Tween80,TritonX-100,andSDSat 0.1%(w/v).Therelativeactivitywascalculatedwithrespectto thecontrol,wherethereactionwasperformedintheabsence ofanyadditive.

Michaelisandrateconstantdetermination

TheMichaelisconstant(Km)andthemaximumvelocity(Vmax)

ofthe enzyme(U/mL)wereevaluated byusing asubstrate concentration of 0.0–10mg/mL. The reaction mixture was incubatedat60◦Cfor1h.AnalysisoftheMichaelis–Menten curvewasconductedbyusingtheGraphPadPrism6software todeterminetheenzyme’sKmandVmax.

Scanningelectronmicroscopy(SEM)

Inordertoobservethemorphologicalfeaturesofthe microor-ganisms and the effect of chitinase on the morphological changesofchitin,thebacteriaandchitinintheabsenceand presenceofenzymewereprovidedandphotographedunder an SEM (LEO, 1455 VP; Germany) as described in previous articles.9,33,34

Statisticalanalyses

Alldataarepresentedasmeans±standarddeviation(SD)ofat leastthreeindependentexperiments.Statisticalanalysiswas performedbyusingtheStudent’st-test.p<0.05was consid-eredasstatisticallysignificant.

Software

Qualitek-4software(NutekInc.,MI)wasusedforautomatic designing of experiments by using the Taguchi approach. Qualitek-4softwareisprovidedtouseL-4toL-64arraysalong with a selection of 2–63 factors with two, three, and four levels toeach factor. Theautomatic layout option permits

Table2–Morphological,physiologicalandbiochemical characteristicofCohnellasp.A01.

Characters Results

Form Rod

Gramstain Positive

Spore Sporecentral

andoval

Motility Positive

Catalase Positive

Oxidase Negative

Utilizationof

Glucose,maltose Positive

Arabinose,xylose,mannitol,lactose,fructose Negative

Hydrolysisofstarch Positive

Hydrolysisofgelatin Negative

Indolformation Negative

GrowthonNaCl Positive

Growthtemperature 40–60◦C

Qualitek-4 toselectthe arrayusedand toassignfactorsto suitablecolumns.

GraphPadPrism6(SanDiego,CA,USA)wasusedto deter-minetheenzyme’sMichaelis–Mentenkineticparameters,Km

(substrateconcentrationthatyieldsahalf-maximalvelocity), andVmax(maximumvelocity)valuesbynonlinearregression analysis.

Results

Isolationandidentificationofchitinolyticmicroorganisms

We isolated 12 strains from the water and waste water of Abadanshrimp farming ponds insouthwesternIran. In the initial screening, qualitative cup plate assay was per-formed for chitinase production, indicating one isolate as themostactiveone.9_{Biochemicalandmicrobiological} anal-yses were performed to characterize the screened strain

(Table2).InaccordancewiththeBergey’sManualof

System-atic Bacteriology,theA01strainwasclassifiedasabacteria belongingtothegenusCohnella.Subsequentsequence anal-ysis ofthe 16S rDNA gene confirmed the isolate as being

Cohnellasp.

Nucleotidesequenceaccessionnumber

Thenucleotidesequenceof16SrDNAfromCohnellasp.A01 wouldbemadeavailableintheGenBanknucleotidesequence databases(accessionnumber:JN208862).

Effectsofincubationtime,carbon,nitrogen,and phosphorussourcesonchitinaseproduction

Chitinase was produced in the media containing 0.5% of colloidalchitin.Duringtheearlyperiodoftheincubation, min-imal enzymeactivity wasdetectedinthe culture;however, after72hofincubation,theenzymeactivitywassignificantly increased(Fig.1A).

ThegrowthofCohnellasp.andtheproductionofenzyme by this bacterium were studied by using different carbon

0 20 40 60 80 100 Re la ti ve A c ti v it y , % 0 20 40 60 80 100 0 24 48 72 96 120 Glucose

GalactoseManoseFurctoseArabinoseLactoseSucroseMaltose

Polygalacturonic acid

Peptone

NH4HCO3(NH4)2SO4NH4NO3 NH4Cl

Ca(NO3)2 .4H2 O

Na2 HP04 NaH2 PO4 K2 HPO4 HH2 PO4 NH4 H2 PO4 C4 H11 NO3NH4 H2 PO4 Tripton Starch Cellulose Chitin Chitin+Glucose 144 Re la ti ve acti v it y % Time(h) 0 20 40 60 80 100 Re la ti ve A c ti v it y, % 0 20 40 60 80 100 Re la ti ve A c ti v it y, %

A

D

C

B

Fig.1–Effectsofincubationtime(A),carbon(B),nitrogen(C),andphosphorus(D)sourcesonchitinaseproduction.All experimentswerecarriedoutintriplicate,andeachpointillustratestheaverageofthreeindependentexperiments.For moredetails,pleaserefertothe“Materialsandmethods”section.

sources. Chitinase production was induced by chitin and inhibited in the presence of easily metabolized monosac-charides, such as glucose, galactose, mannose, arabinose, and fructose(Fig. 1B). Thesecompounds inhibited enzyme biosynthesisandpresentedwithlowchitinaseactivitieswhen thesemonosaccharideswereusedascarbonsources.Lactose, sucrose,andmaltosedetractedchitinaseproduction (approx-imately70–80%).Polygalacturonicacid,starch,andcellulose decreasedenzymeproductionbyapproximately50–60%; nev-ertheless,chitingavethehighestenzymeactivity(100%).The mediumcontainingNH4NO3 (Fig. 1C)and K2HPO4 (Fig. 1D)

showedthehighestenzymeactivityascomparedtothe oth-ers.

ResultsofTaguchidesignanalysis

Weuseddifferentmediawithdifferentinducersandunder differentconditionstoanalyzethepossiblefractionsof chiti-nasessynthesizedbyCohnellasp.Themaineffectsoftheeight controlfactorsattheirthreelevelsoncrudeenzyme biosyn-thesisare shown inFig. 2. Thecontribution ofeach factor ispresentedinTable3.Accordingtothesedata,inoculation amountand temperature were themostsignificant factors influencingthe productionofchitinaseinadditiontotrace elementsandNaClconstitution.

EffectoftemperatureandpHonenzymeactivityand stability

Theeffectoftemperatureonthecrudeenzymeactivitywas studiedatseveraltemperatures.Thebesttemperaturevalue was obtainedat70◦C (Fig.3A)and the best pH valueat5 (Fig.3B).Thetemperature stabilityin10-and20-min incu-bationofenzymeindifferenttemperatureconditionsshowed thattheenzymepossessed>50%activityuntilat80◦C temper-ature(Fig.3C).Theenzymedisplayedactivityoverarelatively widerangeofpH(4.0–11.0),i.e.,>75%(Fig.3D).

Effectofmetalions,chelators,andsomechemicalson chitinaseactivity

Thecrudechitinaseactivity wasmeasuredattheoptimum pH andtemperature inthe presenceofvarious metalions andchemicalcompounds.MetalionssuchasAg2+_andCo2+

inhibitedtheenzymeactivityupto40%at15mM concentra-tion.Ontheotherhand,Mn2+_andCu2+_{increasedtheenzyme}

activity up to25%at5mMconcentration(Table4). Iodoac-etamide and idoacetic acid (1% w/v) inhibited the enzyme activity byapproximately55%.Tweens(20and 80)and Tri-tonX-100slightlyincreasedtheenzymeactivity(110,115,and

Mean of means 65 60 55 1.0 0.8 0.6 0.4 0.2 8.5 7.5 6.5 0.17% 0.5% 1.5% .035% .07% 0.14% 2.0% 1.0% 0.5% 1.0 0.8 0.6 0.4 0.2 1.5% 0.5% 0.17% 0.05% 0.1% 0.2% 2% 4% 8%

Temp pH NaCl K2HPO4

NH4NO3 Chitin Trace element Inoculation

Fig.2–Themaineffectplot(datameans)formeansonchitinaseproduction.Formoredetails,pleaserefertothe“Materials andmethods”section.

Table3–Theanalysisofvariance(ANOVA)table.

Serialnumber Factors Degreeof

freedom(DOF)

Sumofsquares Variance F-ratio p-Value

1 Temperature 2 7.677 3.839 70.763 0.000 2 pH 2 0.101 5.056E−02 0.932 0.0399 3 NaCl 2 1.533 0.767 14.131 0.000 4 K2HPO4 2 0.117 5.836E−02 1.076 0.347 5 NH4NO3 2 7.127E–02 3.563E−02 0.657 0.522 6 Chitin 2 0.599 3.678E−02 0.678 0.511 7 Traceelement 2 7.642 0.299 5.517 0.006 8 Inoculation 2 3.472 3.821 70.443 0.006

108%,respectively),whileEDTAstimulatedtheactivityupto

120%(Table5).

DeterminationofMichaelisandrateconstant

TheMichaelisconstant(Km)andthemaximumrate(Vmax)of

theenzyme(U/mL)wereassayed atchitinconcentrationof

Table4–Effectofsomemetalionsontheenzyme activity. Residual activityat 15mM Residual activityat 10mM Residual activityat 5mM Residual activityat 1mM Metal ions 89.2 91.3 86.9 85.9 LiCl 126.1 124.6 127.1 113 CuSO4 99.1 98.9 101.2 100 AlCl3 94.9 96.9 100 100 K2Cr2O7 93.6 92.6 93.3 88 KCl 95 94.4 88.8 92.7 MgSO4 92.4 91.2 100 96.5 FeCl3 59.1 63.6 68.4 69.9 AgNO3 95 94.4 100 95.9 BaCl2 125.8 125 126.2 110 MnSO4 55.2 67.1 81.2 97.4 Co(NH2)2 93 98.8 99.2 100 ZnSO4

0.0–10mg/mL. Analysisofthe Michaelis–Mentencurve was accomplished with GraphPad Prism6. In enzyme kinetics,

Vmaxillustratedthemaximumvelocityreceivedbythesystem atsaturatingsubstrateconcentrations.Thesubstrate concen-trationatwhichthereactionvelocitywashalfofVmaxvalue representedtheMichaelis–Mentenconstant(Km).TheKmand

Vmaxvaluesoftheenzymewere5.7mgmL−1and0.87units (␮molmin−1)inthepresenceofcolloidalchitin,respectively (Fig.4).

Table5–Effectofsomechemicalcompoundsand chelatorsontheenzymeactivity.

Chemicalcompoundsandchelators(1%) Enzyme activity(%) Control 100 Citricacid 96.9 EDTA 120 SDS 87.4 PMSF 100 Iodoacetamide 45.7 Iodoaceticacid 45.5 Tween20 110 Tween80 115 TritonX-100 108

0 50 100

A

B

C

D

0 20 40 60 80 R e la ti ve a c ti vi ty % Temperature °C 0 50 100 0 20 40 60 80 R e la ti ve a c ti vi ty % Temperature ºC 0 50 100 0 2 4 6 8 10 12 R elati ve a cti v it y % pH 0 50 100 0 2 4 6 8 10 12 R e la ti v e a c ti v it y % pH

Fig.3–Temperature(A)andpH(B)profilesandtemperature(10“”and20“䊉”minuteincubation)(C)andpH(D)stability ofpartiallypurifiedchitinase.Allexperimentswerecarriedoutintriplicate,andeachpointillustratestheaverageofthree independentexperiments.Formoredetails,pleaserefertothe“Materialsandmethods”section.

SEM

TheSEMimageillustratedinFig.5AshowsCohnellasp.A01 cellsaslong,straight,rod-shapedbacilli.Toinvestigatethe morphologicalchangesinchitinafterchitinasetreatment,we performedSEMtrials.Intheabsenceofenzyme,chitin illus-trated more or less a smooth outside layer (Fig. 5B). After chitinasetreatment(Fig.5CandD),theSEMimagerevealed cracksandseveralsmallholesonthechitinsurface,withthe

[Chitin] (mg/ml)

Enzyme activity (U/ml)

0 5 10 15

0.0 0.2 0.4 0.6

Fig.4–Michaelis–Mentencurveofchitinaseactivityversus differentconcentrationsofchitinincubatedatthebest temperatureandpHvalues.TheK_mandVmaxvaluesofthe

enzymewere5.7mgmL−1and0.87units(␮molmin−1).For moredetails,pleaserefertothe“Materialsandmethods” section.

amount of pores remarkablyincreasingwith the periodof exposure(incubation).

Discussion

Theincreaseofenzymeproductsoftendependsonscreeninga largenumberofmicroorganismsforanenzymeactivity.9,35,36 In this study, a chitinolytic strain was isolated from the shrimpfarmingwastewaterlocatedinChoebdeh-Abadanand confirmed to be Cohnella sp. A01 based on its morphol-ogy,physiologicaland biochemicaltests,and the16S rRNA sequenceanalysisreports.The16SrRNAgenesequencesare largelyusedfortheidentificationofprokaryotes.37

Thispaperpresent productoptimizationofchitinaseby

Cohnella A01forthe firsttime. Otherstudies havereported

similar studies on Pseudomonas sp. TKU015,38 _Aeromonas

schubertii,39_{andBacillussp.13.26.}40 _{IntheA.hydrophilaHS4,}

maximumchitinaseactivitywasdetectedtooccurafter24h of incubation, and remained invariant up to 48h, while

A. punctata HS6 produced maximum enzyme after 48h of

incubation.41_{Theprimaryreasonsforthereductionin} chiti-nase productionmaybeduetothelackofnutrientsinthe culturemediumortheproductionoftoxicchemicalsinthe medium,eventuatingintheinactivationofsecretary machin-eryoftheenzymesorbreakupofenzymebyproteases.42

In the first stage of the medium ingredients optimiza-tion formaximum enzymeproductionby Cohnellasp.A01, theoptimumcarbon,nitrogen,andphosphorussourceswere selectedbytheonefactor-at-a-timeprocedure.Thestudyon

Fig.5–ScanningelectronicmicroscopyphotographsofCohnellaA01(A);untreatedchitin(B);treatedchitinafter1h(C);and 2h(D).Formoredetails,pleaserefertothe“Materialsandmethods”section.

theeffect ofcarbonsourcesshowed thatmonosaccharides inhibited chitinasebiosynthesis and its synthesisin small levelswithlowactivity.Inhibitionoftheenzymeefficiency inthe presenceofsimplesugars may bedue tocatabolite repression.43_{Theseoutcomesareinagreementwiththatfor} chitinaseproductionbyStreptomycesviridificans44_and

Tricho-derma harzianum,45 _{respectively. The resultsof the present}

surveydemonstratesthattheadditionofmonosaccharidesto theculturemediumreducesthechitinaseactivityofCohnella

sp.A01,whichwassimilartochitinaseproductionby

Strepto-mycessp.46_{andSerratiamarcescensB4A.}9

Theefficacy ofnitrogen sourceson enzyme production demonstratedthatNH4NO3presumablyisthemostoptimal

nitrogen source for enzyme production. It may be due to ammonium(inammoniumnitrate)istheinorganic nitroge-nousformofsimplerassimilationandalsobecauseoflower energeticcostsofmetabolization.47

K2HPO4wasrecognizedasthebestphosphorussourcefor

theenzymeproductionbyCohnellasp.A01,inconcordance withthatreportedforchitinasebiosynthesisbyPaenibacillus

sp.D148_{andVibrioalginolyticusJN863235.}49

Culturalconditionsare significantfactorsthataffectthe productefficiencyandcellgrowth.Inthisresearch,theeffects ofvariousparameters,includingincubationtemperature, ini-tialpH,NaCl,K2HPO4,NH4NO3,chitinlevels,traceelements,

and inoculation percentage on the efficiency of chitinase

productionwereevaluatedandoptimizedbytheTaguchiL27 arraymethod.Thedataobtainedfromthismethodsuggested thatthetemperatureandNaClaswellasinoculationamount weremoreeffectivethanothersontheenzymeproduction. The temperature ofincubation and inoculationpercentage were themostimportantfactors influencingthe changein theproductionlevel(Fig.2).

ThetemperatureandpHofenzymeareusuallydistinctive parametersthatdeterminewhetheranenzymeisappropriate forbiotechnologicalapplications.ThechitinaseofCohnellasp. A01wasactiveat80◦Cafter20minoftemperaturetreatment andshowedthebestactivityat70◦C(Fig.3AandC). There-fore,Cohnellasp.A01chitinasecanbecalledasathermostable enzyme.Acomparablethermostabilityhasbeenreportedfor alkalinechitinasebyBacillusthuringiensissubsp.Kurstakistrain HBK-51,50 _{BacillusthuringiensisMexicanisolates,}51

Brevibacil-lusformosusBISR-1,52_{andRhodothermusmarinus.}15_Thehighest

enzymeactivitywasfoundatpH5(Fig.3B),althoughactivity wasdetectedoverawidepHrangeof4–11(Fig.3D).A compa-rablepHoptimumhasbeenreportedforBbchit1ofBeauveria

bassianaNCIM1216.10_{ThisenzymehasitisthebestpHinthe}

acidicrangeandcanpossiblybeusedforthecontrolofplant fungalpathogens.

Thestudiedenzymedidnotshowrequirementofany spe-cificmetalionsforitsactivity,althoughMn2+_andCu2+ _had

iodoacetamideinhibitedtheenzymeactivityat1mM concen-trationbyapproximately55%,indicatingthatseveralcysteine residuesformapartofthecatalyticsiteofchitinase.Tweens 20,80,andTritonX-100stimulatedthechitinaseactivityby upto15%.Asthesurface-activereagentsmayhaveenhanced theturnovernumberofenzymeduetotheincreasedcontact frequencybetweenthesubstrateandactivesiteoftheenzyme withdisruptingsurfacetensionoftheaqueousmedium.33,43

In the test of substrate concentration against chitinase activity,theactivity ofenzymewasfoundtoincrease with increase in the substrate concentration to about 7mg/mL ofcolloidalchitin, but noeffect withfurtherincrease. The enzymeshowed a Km value of 5.6mg/mL against colloidal

chitin,whichisrelativelylowerthansomeotherreportedKm

values,suchas12mg/mLforchitinasefromBacillussp.BG-1153 and8.3mg/mLforchitinasefromSerratia marcescensB4A33_; thechitinaseofCohnellasp.A01revealedahighaffinityfor colloidalchitinandpossiblygreaterpotentialintheindustry. Studyofthemorphologicalalterationsofchitintreatedby enzymeviaSEMexperimentsexhibitedcracksandporesinthe substratesurface.Thesamemorphologicalchangeshavebeen reportedforSerratiamarcescensB4Achitinase33_andchitinase

from penicilliumsp.LYG0704.54 _{Thisdirectevidenceimplies}

thattheisolatedchitinasewasquiteeffectiveonchitin degra-dation.

Conclusions

The isolation study revealed that shrimp farming ponds areanappropriateenvironmentforscreeningofchitinolytic enzyme-producingbacteria.Ourstudyaffirmedtheefficiency oftheTaguchiexperimentaldesignforspecifyingthe most appropriatemediumcomponentstoobtain maximum pro-ductionofenzymebyCohnella sp.A01. Thiswork revealed thattheproductionofchitinaseisfeasiblefromanewly iso-latedCohnellasp.A01. Chitinaseproducedfrom Cohnellasp. A01isactiveoverawiderangeoftemperatureandpH,with goodtolerancetohightemperatures.Thegrowthofenzyme productsmostlyreliesonscreeningofextensivenumberof organismsfortheirenzymeactivitywithaspecificsetof bio-chemicalcharacteristics that suitsthe targetedpopulation. Thus,mergingenzymescreeningwithproteinengineering, directedevolution,andmetagenomestudies,novelenzymes withimprovedefficiencyunderspecificapplicationsand con-ditionscanbebuilt.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This work was supported by International Foundation for Science(IFS),COMSTECH(CommitteeonScientificand Tech-nological Cooperation) Grant no. F/4900-2, and National InstituteofGeneticEngineering and Biotechnology(NIGEB) (GrantNo:463). Wewouldlike tothankthemforfinancial supportofthisproject.

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