A new alkalophilic isolate of Bacillus as a producer of cyclodextrin glycosyltransferase using cassava flour

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

Industrial

Microbiology

A

new

alkalophilic

isolate

of

Bacillus

as

a

producer

of

cyclodextrin

glycosyltransferase

using

cassava

ﬂour

Sheila

Lorena

de

Araújo

Coelho,

Valter

Cruz

Magalhães,

Phellippe

Arthur

Santos

Marbach,

Marcia

Luciana

Cazetta

∗

CentrodeCiênciasAgrárias,AmbientaiseBiológicas,UniversidadeFederaldoRecôncavodaBahia,CruzdasAlmas,Bahia,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24November2014

Accepted9September2015

AssociateEditor:JorgeGonzalo

FariasAvendano Keywords: Microbialenzyme Fermentation Agro-industrialsubstrates

a

b

s

t

r

a

c

t

Cyclodextringlycosyltransferase (CGTase)catalyzes the conversion ofstarch into

non-reducingcyclicsugars,cyclodextrins,whichhaveseveralindustrialapplications.Thisstudy

aimedtoestablishoptimalcultureconditionsfor␤-CGTaseproductionbyBacillussp.SM-02,

isolatedfromsoilofcassavaindustrieswastewaterlake.Theoptimizationwasperformed

byCentralCompositeDesign(CCD)2,usingcassavaﬂourandcornsteepliquorassubstrates.

Themaximumproductionof1087.9UmL−1wasobtainedwith25.0gL−1ofcassavaﬂourand

3.5gL−1ofcornsteepafter72hbysubmergedfermentation.Theenzymeshowedoptimum

activityatpH5.0andtemperature55◦C,andmaintainedthermalstabilityat55◦Cfor3h.

TheenzymaticactivitywasstimulatedinthepresenceofMg+2_,_Ca+2_,_EDTA,_K+_,_Ba+2_and_Na+

andinhibitedinthepresenceofHg+2_,_Cu+2_,_Fe+2_and_Zn+2_._The_results_showed_that_Bacillus

sp.SM-02havegoodpotentialfor␤-CGTaseproduction.

anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Global market for the enzymes ofmicrobial origin having

environmentaland industrial applicationshas signiﬁcantly

developedduring the last decades.Ithas elicited asearch

fornewmicroorganismswithbiotechnologicalpotentialfor

enzymatic production.Braziliantropicalsoilsare bestowed

with amicrobiota that exhibitsan exceptional capacity of

∗ _{Corresponding}_author.

E-mail:malulz@yahoo.com.br(M.L.Cazetta).

enzyme productions; unfortunately, to a large extent the

majority of these microbial communities are unknown.1–3

Several strains ofbacteria producing cyclodextrin

glycosyl-transferase(CGTase;EC:2.4.1.19)havealreadybeenisolated

fromdifferentenvironmentsindifferentpartsoftheworld,

including Brazil.4–8 _There _are _a _few _studies _exploring _the

soils from the State of Bahia, Brazil, for the isolation of

microorganismswithbiotechnologicalpotentialforthe

pro-duction ofthis enzyme.CGTaseisanextracellularenzyme

http://dx.doi.org/10.1016/j.bjm.2015.11.018

(2)

that catalyzes the hydrolysis of ␣-1,4-glycoside bonds and

subsequentintramolecular (cyclization) and intermolecular

transglycosylation(disproportionationandcoupling)reaction

of␣-1,4-glucane.9,10 _This_enzyme_being_amylolytic_in_nature

istheonlyonecapableofconvertingstarchintoamixtureof

non-reducingsugarsdesignatedascyclodextrins,besides

lin-earsugarsanddextrins.11_{Cyclodextrins,}_the_main_products

oftheCGTasecatalysisreaction,are cyclicoligosaccharides

madeupbyd-(+)-glucopyranoseunitslinkedby␣-1,4linkages.

Thesecompoundshavewideindustrial applicationsdueto

their capability to form water-solubleinclusion complexes

with several substances; this property modiﬁes

physico-chemical proprieties of encapsulated host-molecules.12,13

Theculturemediarequiredfortheproductionofthisenzyme

usually includes carbon and nitrogen sources obtained by

expensiveprocesses.Therefore,approachesinpursuitoflow

costsubstrates,especiallyfromagro-industrialorigins,have

beendevelopedwithanobjectivetodecreaseproductioncosts

ofCGTaseatcommercialscales.14–18_In_this_scenario,_the_use

ofcassavaﬂour(ManihotesculentaCrantz)asacarbonsource

infermentationprocessesforCGTaseproduction,hasgained

anintenseinterest.Cassavaﬂour,anagro-industrialproduct

containing60–80%starch,canbeobtainedbyasimplelow

costprocess.19_In_Brazil,_it_is_a_widespread_agronomic_product

speciﬁcallyoftheNorthandNortheastregions.20,21_Its_quality

isdirectlyrelatedtothephysicalandchemicalcharacteristics

ofthecassavaroots,plantgenotypeandage,favorable

envi-ronmental conditions, harvest period, soil characteristics,

geneticvariability,andtheprocessingmethod.22,23

Regardingthenitrogensource,corn steepliquoris

com-monly used in fermentation processes. It contains large

quantitiesofnitrogenalongwithproteins,amino-acidsand

vitamins.Itisalsoalowcostbyproductoriginatingfromcorn

processing.24_This_study _aimed_to_establish_ideal_conditions

forCGTaseproductionfromBacillus sp.SM-02bymeans of

experimentalmethodsbased onCentral CompositeDesign

(CCD),usingagro-industrialsubstrates,cassavaﬂourandcorn

steep liquorasalternative sourcesof carbonand nitrogen,

respectively.

Materials

and

methods

Bacterialstrain

Bacillussp.SM-02wasisolatedfromsoilsamplescontaining

cassavawastewaterfromacassavaﬂourfactoryinCruzdas

Almascounty,Bahia,Brazil.Thebacterialisolateswere

pre-servedincryotubescontaining2mLof20%glycerolandstored

at−80◦C.

Molecularidentiﬁcation

Apairofuniversalprimers,8F(5AGAGTTTGATCCTGGCTCAG

3)and1492R(5ACGGCTACCTTGTTACGACTT3)wasusedto

amplifythe16SrRNA.Thesequencingwascarriedout

com-merciallybyMacrogenCo.(Korea). The16S rRNAsequence

wasusedasqueryinBLASTN25_search_against_National

Cen-terforBiotechnologyInformation(NCBI)database.MEGA5.05

software26wasusedtoconstructanevolutionarymodeland

to generate the maximum likelihood tree. The 16S rRNA

sequences usedinthephylogenetic analysiswere retrieved

from the siteofList ofProkaryotic Standing Names(LPSN)

duringAugust,2014.27

PreliminarytestsforCGTaseproduction

Theabilityfor␤-CGTaseproductionbyBacillussp.SM-02was

evaluatedbythe degradationofstarchformingahalozone

insolidmediacontainingphenolphthalein.28_In_the_center_of

Petridishescontainingsolidmedia,holesof0.5cmdiameter

wereprepared.Theseholeswereﬁlledwith50␮Lofculture

containing3×108_cells._Petri_dishes_were_incubated_at₃₇◦_C

for72hbeforemeasuringthehalozone.

Fermentationassay

Inordertore-activatethemicroorganism,sub-cultureswere

preparedfromthestockcultureinsolidmediadevelopedby

NakamuraandHorikoshi,29_containing_(g_L−1_):_soluble_starch,

10; peptone, 5; yeast extract, 5; MgSO4·7H2O, 0.2; K2HPO4,

1;bacteriologicalagar,1.5;Na2CO3,10 (separatelysterilized

andaddedtotheculturemediaat60◦Ctemperature)atpH

9.8.Theinoculumwaspreparedafter72hofculturegrowth

bytransferring,withthe helpofaloop,oftheculturetoa

125-mL Erlenmeyerﬂask containing 50mL ofliquid media

supplementedwithcassavaﬂourandcornsteepliquorforcell

adaptationtothefermentationmediaatpH9.8.Afterthe

incu-bationperiodof24h,analiquotof1mLoftheinoculumwas

standardized(OD600=0.1)andtheﬁnalvolumeforeachassay

wasmadeto30mLbyusingoftheculturemedium,replacing

solublestarchofthebasalmediabycassavaﬂourandpeptone

andyeastextractbycornsteepliquor(Sigma®_)._The_cultures

wereincubatedinshakerat150rpmand35◦C.After72hof

fermentation,thesampleswerecentrifugedat5000rpmand

4◦Cfor30min.Thecell-freesupernatantwasusedto

deter-minetheenzymaticactivity;andtheprecipitatedcellmass

wasusedforbiomassdetermination.

CGTaseproductionusingtheexperimentaldesign

Concentrationoptimizationofcassavaﬂourasacarbonsource

and cornsteep liquorasanitrogensourcewascarriedout

using the response surface methodology (RSM). ␤-CGTase

activity(UmL−1)wasconsideredasdependentvariable and

substrate concentrationsasindependentvariables.A

facto-rialplanningmatrix22_was_performed_by_employing_Central

CompositeDesign(CCD)resultingin11runs.30_Two_levels_were

selected,asuperior(+1)andaninferior(−1),besidesa

cen-tral point(0)(theonlyoneperformedwiththreereplicates

todeterminemethodologicalstrength)andtwoaxialpoints

(+1.41and−1.41).Thismodelisrepresentedbyasecondorder

polynomialregressiontogenerateresponsesurface,Eq.(1):

y=b0+b1X1+b2X2+b12X1X2+b11X21+b22X22 (1)

whereyisthepredictedresponseofCGTaseactivity;X1and

X2arethecodiﬁedforms(cassavaﬂourandcornsteepliquor,

respectively);b0referstotheinsertionpoint;b1andb2are

(3)

b11andb22arequadraticcoefﬁcients.Theresultsobtainedby

thisexperimentalmethodwereevaluatedusingtheSoftware

Release7.1,Stat.Soft.Inc.,USA.

Enzymaticassay

Enzymaticactivitywas determinedbycolorimetric method

ofthecyclodextrin-phenolphthaleincomplex(CD-PHE).31The

mixturecontaining5mLofthecrudeenzymaticsolutionand

5mLofthesolublestarchsolutionat1%wasincubatedina

thermostatedreactorat55◦C.Aliquotsof0.5mLfromthe

reac-tionmixtureweretakenat0,3,6,9and12minandinactivated

inaboilingwaterbathfor5min.Toeachinactivatedaliquot,

2.5mLofaphenolphthaleinalcoholicsolution(3mM),diluted

in600mMNa2CO3bufferatpH10.5,wasadded.Absorbance

ofthesampleswasrecordedat=550nm.

Cellgrowth

After fermentation, the biomass was separated from the

supernatantbycentrifugationat5000rpmfor30minat4◦C.

Thebiomasswasre-suspendedin5mLdistilledwaterand

centrifugedagainforcellwashing.Afterremovingthe

super-natant,30mLofdistilledwaterwasaddedandtheprecipitate

was re-suspended. Then, optical density was measured at

600nmandbiomasswasquantiﬁedbycomparingwiththe

standardizedcurvebasedindrymass×opticaldensity.

Totalreducingsugarsdetermination

Determinationofreducingsugarswasperformedtrough

3,5-dinitrosalicylicacid (DNS)method ofMiller.32 Thesamples

weresubjectedtoanacidhydrolysisin2MHCl.Afterboiling

for20min,sampleswereneutralizedwith2MNaOH.

Totalproteindetermination

TotalproteinconcentrationwasdeterminedbytheBradford

method,33 _by_adding_0.2_mL_to_the_crude_enzyme_extract_to

2mL ofthe Bradfordreagent. Absorbance was recorded at

=595nm.

Crudeenzymepartialcharacterization

To evaluatethe effect of pH on ␤-CGTase activity the

fol-lowing buffers (50mM) were used: glycine–HCl, pH 2.0–3.0;

sodiumcitrate,pH 3.0–6.0;phosphate, pH6.0–8.0;Tris–HCl,

pH8.0–9.0;andglycine–NaOH,pH9.0–10.0.Theinﬂuenceof

metallicionsonCGTaseactivitywasdeterminedbyusing

fol-lowing solutions(50mM): CaCl2,FeCl3, NaCl,ZnSO4,EDTA,

KCl,MnCl2,CuSO4,BaCl2,HgCl2,andMgCl2.Optimal

tempera-tureofthereactionwasdeterminedbyincubatingtheenzyme

at50–70◦C.Thermalstabilitywasaccessedbyincubatingthe

enzymeat50–65◦C,for5hatpH5.0.Theexperimentswere

performed according to the standardizationsof enzymatic

activityaccordingtotheCD-PHEcomplexationmethod.31

Results

MolecularidentiﬁcationofstrainSM-02

TheBLASTNsearchattheNCBIdatabaseindicatedthatthe

16SrRNAsequenceofbacterialstrainSM-02is98–99%

iden-ticalto16SrRNAsequencesofBacillusspeciesindicatingthat

the strainSM-02belongstothisbacterialgenus. Therefore,

weperformedaphylogeneticanalysistounderstandthe

evo-lutionaryrelationship ofBacillus sp.SM-02to other Bacillus

speciesusing16SrRNAsequencesofallBacillusspecies

avail-ableattheLPSNsite.Theanalysisshowedthatthe Bacillus

sp.SM-02 isphylogeneticallyrelatedtoBacillus trypoxylicola

(Fig.1),axylanase-producingbacteriumﬁrstisolatedfromthe

gutofJapanesehornedbeetlelarva.34

CGTaseproductionusingCentralCompositeDesign(CCD)

Through the obtainedresults of CCD 2,2 _inﬂuences_of _the

studiedvariables(X1:carbonsourceandX2:nitrogensource)

over CGTaseproductionbyBacillus sp.SM-02 were veriﬁed.

Observedandpredictedvaluesbytheexperimentalmodelare

showninTable1.

Agradual increasewasobservedfrom648.5UmL−1(run

8)upto1087.9UmL−1(meanofthecentralpoints),achieving

themaximumproductionofCGTaseat25.0gL−1 ofcassava

ﬂourand3.5gL−1ofcornsteepliquor.Underthesamecarbon

sourceconcentrations,therewasanexpressivenegative

inﬂu-enceofcornsteepliquorovertheenzymeproduction,witha

decreaseofalmost60%,whencomparingvaluesofthehigher

axiallevel(run8)withthevaluesofthecentralpointruns(run

9,10and11).Thiscanbeconﬁrmedbytheestimatedeffects

(Table2),wherethequadratictermsoftheconcentrationof

cassavaﬂourandcornsteepliquorwerestatistically

signif-icant at95% signiﬁcance(p<0.05),showingnegativeeffects

overCGTaseproduction.Itmeanstheincreasein

concentra-tion ofthesevariables causedareductionintheenzymatic

production.Theadjustedmathematicalmodelthatdescribes

CGTaseproductionwithinthestudiedrangecanbeexpressed

byequationEq.(2):

y=1087.9+77.9X1−13.4X2+17.4X1X2−124.9X21−174.3X22

(2)

Table 3 shows the results evaluated by the analysis of

variance.TheF-valueof14.9indicatesthatthemodelwas

sig-niﬁcantattheconﬁdencelevelapplied(5%probability)witha

goodcorrelationbetweentheexperimentalandpredicted

val-ues(Table1).Therefore,consideringthep-valuescalculated

bytheanalysisofvariance,themostsigniﬁcantvariablewas

cornsteepliquorinlinearterms,followedbycassavaﬂourin

quadraticandlinearterms,respectively.

AccordingtoANOVAavariationcoefﬁcient(R2₎_of_0.8646

showsthat86.46%oftheresponsevariabilitycanbeexplained

bythe model. Consideringthe resultsshown, the effectof

the independent variables over the enzymatic production

responsewasevaluatedfromaresponsesurface(Fig.2).The

optimizationarearepresentsthebestresponseforthe

(4)

Bacillus okhensis - DQ026060 Bacillus wakoensis - AB043851.1

Bacillus akibal - 302486182 Bacillus alkalisediminis NR_114912.1 Bacillus hemicellulosilyticus AB043846.1 Bacillus bogoriensis - AY376312.1

Bacillus alcalophilus - 631251525 Bacillus pseudalcalophilus - X76449.1

Bacillus trypoxylicola - AB434284

Bacillus sp. SM - 02

Bacillus nanhaiisediminis - GQ292773.1 Bacillus ligniniphilus - JQ044788.1 Bacillus macyae - AY032601.1 Bacillus halodurans - AJ302709.1 Bacillus okuhidensis - AB047684.1

Clostridium perfrindens - 659364527 Sarcina ventricull - 5852402 100 100 58 70 62 100 100 73 52 57 100 0.1

Fig.1–Thesubcladeofamaximumlikelihoodtreebasedon16SrRNAsequencesshowingthephylogeneticrelationshipof

strainSM-02withthespeciesofBacillusgenus.Thetreewasgeneratedwith16SrRNAsequencesfromspeciesofthegenus

availableattheLPSNsite(ListofProkaryoticNameswithStandinginNomenclature–www.bacterio.net/index.html)ason

August,2014.ThephylogeneticanalysiswasperformedusingMEGA5.1softwareandemployingtheK2+G+Imodel.

Numbersabovethebranchesindicatebootstrapsupport;andthetreewasrootedwith16SrRNAsequencesofClostridium

perfringensATCC13124andSarcinaventriculi.Thebarrepresentsthenumberofexpectedsubstitutionpersitesunder

K2+G+Imodel.

lowestCGTaseproductionwasobservedattheinferior(−1.41

and−1)andsuperior(+1and+1.41)levels,wherethelowest

andhighestconcentrationsofcarbonandcornsteepliquor

arefound,respectively.

Besides, Fig. 2shows that near the optimization point,

there is a range of concentrations of the carbon and

nitrogen sources that showed optimal production of the

enzyme,between 22.5gL−1 and 27.5gL−1 and 3.0gL−1 and

4.0gL−1,respectively.Thus,any concentrationfoundinside

this optimalrange may beused forenzymatic production,

withoutinﬂuencingtheprocesswithintheoptimized

condi-tion.

Table1–CentralCompositeDesignmatrixforenzymeandbiomassproduction.

Runs Codedlevels Reallevels(gL−1₎ _Enzymatic_activity_(U_mL−1₎ _Biomass_(g_L−1₎

X1 X2 Cassavaﬂour CSL Observedvalues Predictedvalues Observedvalues Predictedvalues

1 −1 −1 20.0 2.5 797.3 741.7 1.6 1.6 2 +1 −1 30.0 2.5 930.8 862.6 2.0 1.9 3 −1 +1 20.0 4.5 743.9 680.0 2.2 2.1 4 +1 +1 30.0 4.5 947.1 870.6 2.6 2.4 5 −1.41 0 17.9 3.5 670.9 728.0 1.6 1.6 6 +1.41 0 32.0 3.5 873.3 948.3 1.9 2.1 7 0 −1.41 25.0 2.1 698.1 758.2 1.8 1.8 8 0 +1.41 25.0 4.9 648.5 720.4 2.0 2.0 9 0 0 25.0 3.5 1045.2 1087.9 2.0 2.0 10 0 0 25.0 3.5 1141.2 1087.9 2.0 2.0 11 0 0 25.0 3.5 1077.2 1087.9 2.0 2.0

(5)

Table2–Estimatedeffectsforthecyclodextringlycosyltransferaseproduction.

Factors Effects Standarderror t(5) p-Value

Mean 1087.9a _51.7 _21.0 _0.000004 Cassavaflour(gL−1)L 155.6 63.3 2.5 0.057300 Cassavaflour(gL−1)Q −249.6a _75.4 _−3.3 _0.021175 CSL(gL−1)L −26.8 63.3 −0.4 0.690033 CSL(gL−1)Q −348.6a _75.4 _−4.6 _0.005716 Cassavaflour×CSL×2L 34.8 89.6 0.4 0.713325 a _significant_at_5%_probability.

Table3–Analysisofvariancefortheregressionmodelobtainedfromresponsesurfaceexperiment.

Factors df Someofsquares(SS) Meansquares(MS) F-value

Regression 3 256,212.8 85,404.3 14.9* Residual 7 40,120.0 5731.4 Total 10 296,332.8 ∗ _signiﬁcant_at_5%_probability. 17.9 2.1 2.5 3.5 4.5 4.9 20.0 1000 800 600 400 200 0 –200 25.0 Cassava flour (g.L–1) Cor n steep liquor (g.L –1 ) 30.0 32.0

Fig.2–ContourplotfromthemodelequationforCGTase

productionofBacillussp.SM-02whileusingcassavaﬂour

andcornsteepliquor.

Partialcharacterizationofthecrudeenzyme

Thecrudeenzymaticextractshowedoptimaltemperatureof

55◦C,butretainedupto70%activityintemperaturesbetween

50and60◦C.Relativeactivitiesat50◦Cand70◦Cwere72%and

40%,respectively(Fig.3).

TheeffectofpHonCGTaseactivitywasevaluatedwithin

therangeofpH2.0–10.0(Fig.3).Theenzymeshowedabove

70%activitywithintherangeofpH3.0–9.0,with100%activity

atpH5.0.

EnzymaticactivitywasenhancedinthepresenceofMg+2_,

Ca+2_,_EDTA,_K+_,_Ba+2_and_Na+_,_with_a_maximum_observed_for

Mg+2_. _The_enzymatic _activity _was _slightly _inhibited _in _the

presenceofHg+2_, _Fe+2_,_Cu+2 _and_Zn+2_, _with_retention _of_at

least83%activity.Theenzymaticactivity ofcyclizationhad

noinhibitorysigniﬁcanceinthepresenceofMn+2_(Table_4).

Thethermalstability oftheenzymewas evaluatedina

temperaturerangeof45–60◦C,pH5.0,inthepresenceofMg+2_.

Theenzymepreserveditscyclizationactivityabove50%for

3hat45,50,and55◦C(Fig.4),andtheactivitywassteeplylost

uponincubationat60◦C. 2 0 20 40 60 80 100 120 Glycine-HClCitrate Phosphate Tris-HCl Glycine-NaOH 40 50 60 80 70 90 100 3 4 5 6 7 pH 50 55 60 65 70 Temperature (ºC) Relativ e activity (%) Relativ e activity (%) 8 9 10 11

Fig.3–EffectoftemperatureandpHonCGTaseactivityofBacillussp.SM-02grownoncassavaﬂour25.0gL−1andCSL

(6)

Table4–EffectsofmetalliciononCGTaseactivityof Bacillussp.SM-02.

Ion Relativeactivity(%)

Control 100.0 CaCl2 114.9 BaCl2 112.8 CuSO4 94.3 FeCl3 90.9 ZnSO4 88.9 HgCl2 83.2 NaCl 113.0 EDTA 107.7 KCl 114.1 MgCl2 130.4 MnCl2 99.0 Minutes Enzymatic activity (U .mL –1 ) 0 0 15 30 45 60 75 90 105 60 120 180 240 300 45ºC 50ºC 55ºC 60ºC 360

Fig.4–EffectoftemperatureonthermalstabilityofCGTase producedbyBacillussp.SM-02grownoncassavaﬂour 25.0gL−1andCSL3.5gL−1.

Table5–Production,yieldandproductivityofCGTaseby Bacillussp.SM-02incassavaﬂourandcornstepliquor.

Parameters Results

Speciﬁcgrowthrate(h−1) 0.03

Yx/s(gg−1) 0.14

Yp/s(UmL−1g−1) 67.07

Yp/x(UmL−1h−1) 474.67

Qp(UmL−1h−1) 8.56

Speciﬁcactivity(Umg−1) 21.37

In ordertovalidatethe responsesobtainedthrough the DCCR,thebehaviorofthestrainwasevaluatedbysubmerged fermentationusing25.0gL−1ofcassavaﬂourand3.5gL−1of cornsteepliquorat35◦C,150rpm,for120h.Theenzymatic activity proﬁlecoincided with the cellular growth and the enzymaticsynthesiswashigherduringtheexponentialstage ofthegrowth,attainingthemaximumactivity after72hof thebeginningoffermentation(Fig.5).Then,anabrupt

reduc-tion inthe enzymatic productionwasobserved, coinciding

withtheonsetofthedeclinephase.Thesubstratewasalmost

completelyconsumedafter120hoffermentation.Thespeciﬁc

growthrate(u)was0.03h−1andthebiomassyieldasa

func-tion ofthesubstrate wasof0.14gg−1.Theenzymaticyield

asafunctionofthesubstrate(Yp/s)was67.07UmL−1g−1and

theproductivitywas8.56UmL−1h−1,withaspeciﬁcactivity

of21.37Umg−1(Table5).

Discussion

Based on statistical fundamentals,the method offactorial

planning allied to the surface response analysis has been

designatedtostudytheoptimizationofparametersinvolved

in industrial processes with the aim of minimizing costs

and time, maximizing yield, productivity, and quality of

production.30,35_In_the_present_study,_the_use_of_factorial

plan-ningshowedthatBacillussp.SM-02hasapotentialasCGTase

Time (hours) Enzymatic activity (U .mL –1 ) Biomass (g.L –1 ) T otal protein (g.L –1 ) T

otal reducing sugars (g.L

–1 ) 0 0 200 400 600 800 1000 1200 5 0.06 0.05 0.04 0.03 0.02 0.01 0.00 0 4 8 16 12 20 24 4 3 2 1 0 20 40 60 80 100 120

Fig.5–Fermentationtime-courseforBacillussp.SM-02incassavaﬂour25.0gL−1andCSL3.5gL−1,for120h,35◦Cand

(7)

producer,exhibitingtopcyclizationactivitycorrespondingto

1087.9UmL−1,rarelyfoundinotherreportedbacterialstrains.

Pintoetal.36_reported_high_enzymatic_activity_from_the_crude

extractproducedbyBacilluscirculansATCC21783insubmerged

culturessupplementedwithﬁbrousindustrialresidueofsoy.

UsingoptimizedconditionsofpH,temperature,andaeration,

the authors achieved 1155UmL−1 activity, with maximum

yieldof 32.8UmL−1g−1. Inthe present study,the variance

analysis(ANOVA)indicatedinﬂuenceofcassavaﬂouralong

withcornsteepliquoroverCGTaseproductioninBacillussp.

SM-02,showingthathighconcentrationsofthecarbonand

nitrogensourcesnegatively inﬂuenced enzymeproduction.

Similarresultshavebeenreportedindifferencestudies,where

maximumproductionofCGTasewasobtainedatlowsubstrate

concentrations. Rosso et al.37 _optimized _production _media

forCGTase from B. circulansDF 9R,by employingdifferent

sourcesandconﬁrmedthat1.5%ofcassavastarchincreased

theenzymaticactivity.Zainetal.17_performed_optimization

studies of the production media of CGTase by Bacillus sp.

TS1−1with3.3%oftapiocastarchand0.13%ofyeastextract

achievingmaximumproductionof78.1UmL−1.Pintoetal.36

studiedenzyme productionfrom B. circulans ATCC using a

solublestarchconcentrationﬁve-timeslower thanthe

con-centrationusedbyMakelaetal.,38_and_obtained_21-times_high

enzymeproduction.Parketal.39_explained_that_the_excess_of

substrateincreasesviscosityoftheculturemedia,which

inter-fereswithoxygenabsorptionbythemicroorganism.Glucose

andmaltosemayaccumulateintheculturemediaduetohigh

concentrationsofstarchandexertacatabolicrepressoreffect,

inﬂuencingCGTasesynthesis,asobservedbyGawandeand

Patkar.40

Alreadypublishedresultssuggestthatthenatureand

con-centrationofthecarbonandnitrogensourcesused,andthe

microorganismstrains,directlyinterfereCGTaseproduction.

Gawande et al.41 _afﬁrmed _that _{concentration} _of _the

car-bonsourceisimportantduringenzymeproductionbymany

organisms,especiallywhenthissourceisessentialforenzyme

induction.Onthebasisoftheseobservations,theDCCR

sta-tisticalmethodologyappliedinthecurrentstudytargeteda

maximumenzymeactivity,andconsequentlyleadingtoan

experimentalsetuptooptimizethe associatedparameters.

DifferentpropertieshavebeenobservedforCGTaseobtained

fromdifferentbacterialstrains.Thestudiesestablishingthe

enzymeproﬁling ofthe CGTase producing microorganisms

revealedthatdifferentbiochemicalcharacteristics,regarding

the optimal temperature and pH, thermal stability, molar

massandcapacityofcyclodextrinformation,vary

depend-ingonthemicroorganism.Thestabilityofbiologicalcatalysts

isalimitingfactor whileselectingthem forindustrial

pur-poses dueto high temperaturesand extremevariations in

pHused inthe industrial processes.42 _The_enzymes _might

rapidly get inactivated. Therefore, in biotechnology,

ther-mostability of microbial enzymes has major importance

in bio-processes,43 _especially, _when _considering _storage _of

enzymesandenzyme-formulatedproductsthatrequire

ade-quatetemperatureconditions.Bacillussp.SM-02showedhigh

stabilityat55◦Cduringa3-hour incubationperiod,a

tem-peraturepreviouslyreportedforvariousspeciesofBacillus,44

Bacillusstearothermophilus,45_Bacillus_lentus,46_Bacillus_ﬁrmus,47

besidesthe species Paenibacillus campinasensis H69-3.48 _The

vast majority ofCGtaseproducing microorganisms

investi-gated so far showed stability at temperatures between 40

and 60◦C.48–53 _However, _few_bacterial _strains _possess

ther-mostableenzymeswithoptimalperformancebetween75◦C

and85◦C.54,55 _Tachibana_et_al.56_reported_CGTase_producing

species ofThermococcus showingfunctionality at90–100◦C.

OptimalcyclizationtemperatureofCGTasefromBacillussp.

SM-02was55◦C.Thesametemperaturewasalreadyreported

forBacillussp.G1,50_Bacillus_{agaradhaerens}_LS-3C,51_Bacillus_sp.

G-825-653 _and _P. _{campinasensis} _H69-3.48 _In _general,_optimal

reactiontemperatureofCGTasefromalkalophilic

microorgan-ismshasbeenreportedamong45–60◦C.18_Some_rare_strains

asThermoanaerobacterkivui.57_have_considerable_activity_under

extremetemperaturesstartingat80◦C.

CGTasefromBacillussp.SM-02showedanoptimalrange

ofpHvaryingbetween3.0and9.0forthecyclizationreaction.

TheseresultsaresimilartothosereportedforBacillus

ohben-sisC-1400,50_B._ﬁrmus_NCIM_5119,50_Bacillus_sp._7–12,58_Bacillus

sp.G-825-6,53_and_T._kivui.57_Moreover,_the_enzyme_exhibited

activity above70% inapHrange of3.0–4.0,rarelyreported

exceptthatbyMoraetal.,59 _who_observed_enzyme_activity

above70%withinthesamepHrangeforBacillussp.CGTase

fromBacillussp.SM-02wasstimulatedbyavarietyof

metal-lic ions,specially Mg+2_, _which_showed _an_enhancement_of

about30%comparedtountreatedcontrols.Atanasovaetal.42

observedthatactivityofBacilluspseudalcaliphilus20Renzyme

wasstronglyinﬂuencedbyMg+2 _at₁₅_mM._The_implication

ofstudyingtheinﬂuenceofmetallicionsoverabiocatalyst’s

performanceisrelatedtothefactthatthesesubstancesare

abletomodifyenzymereactionratesbyinhibitingor

activat-ingenzymes;therefore,thisinformationisimportantwhile

choosingsubstratesthatwillbeusedinthefermentation

pro-cess.

Conclusion

In the present study, we were able to establish optimized

conditions, i.e.,thebest concentrationofcassavaﬂourand

corn steep liquor that resulted in high CGTase production

rates, whileusingthe factorialplanning and response

sur-facemethodology.Thereportedproductivity,alliedtothefact

thataneasyavailableandlowcostagro-industrialsubstrate

wastested,supportsuseofcassavaﬂourandcornsteepliquor

inbioprocessesrelatedtothecyclodextrinproduction.

Bacil-lussp.SM-02appearstohaveanexcellent biotechnological

potentialforCGTaseproduction,underoptimizedconditions.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

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