Partial Purification of Bacterial Chitinase as Biocontrol of Leaf Blight Disease on Oil Palm

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Researdl Journal cl MicroOOlogy is a monthly international peer-reviewed journal dedicated to publish and disseminate signitlcant research In ttie field of mlaobiology. Scope IX the journal includes: structure and function, blochemlstry, enzymology,

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Editor-in-Chief: Sang tie<: Lee

rtlal Puritkatlon of ｾ｣ｴ･ｲｩ｡ｬ＠ Chltinue as Blocontrof of leaf 81ight Oisea5e on Oil Palm

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ｾ＠ If! Htf:xt ･｡ｾＺ＠ Ｈｒ･Ｇｾｲ･ｯ｣･Ｇ｜Ｉ＠

tiblotk: Resistance and Surviving Percentage of Lactic Acid Bacteria and Bifldobacterium spp.

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>latlon and ldentlticatlon of Mid-Gut Bacterial Community of llactrocera dorsalls (Hendel) (Dlptera: phritidae)

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Researdl Journal of Mierobiology is a monthly international peer-reviewed journal dedicated to publish and disseminate si9nifkant research In the field of microbiology. Scope of the journal includes: structure and function, biochemistry, enzymology,

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ｾｴｮｲＭ｡＠ ｴｴｮｲｯｮｾＢＧｋｃ＠ R •:1pjOPz""-1.an

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Research

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ISSN 1816·4935 I DOI: 10.3923/jm.2014.

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Partial Purification of Bacterial Chitinase as Biocontrol of Leaf

Blight Disease on Oil Palm

Muhammad Asril, Nisa Rachmania

Mubarik

and Aris Tri

\iVahyu<li

Department of Biology, FaculLy of Mathematlcti nnd Natmal Sciences, Bogor Agricultural lJn1ver1'\1ty,

Jalan Agatis, Dramaga. Bogar, 16680, Indonesia

Corresponding Author: Nisa Rachmania Muborih, Department of Biology, Faculty of Mathematics and Natural

Sciences, Bogor Agricultural University, Jalc111 Agati11, Dramaga, Bogar, 16680, Tndone.'lia 1el !Fax: +6'2-251 ·8622833

ABSTRACT

Clutin is a maJor component of fungi cell wall, mycelia, stalks and ｳｰｯｲｾ＠ which can be

hydrolyzed by chitmase. This study was conducted to measure the ability of chitinase producing li::icteri::i m d1:1f,,'1'ncl mg ch1ti 11 of f 1111g-11 l 1rntl1 ogens such as (1m •11lrtria a//i 11i8 r-rncl

Colletotrich11rn ｾｬｵｮＱＱＺＱｰｵｲｩｵﾣ､･ｳ Ｎ＠ ｔｨｾｳｴｾ＠ pul.hog'f'rts cuusPll ant rac.:h nost> , lr•:tt' liltghL aml roll.mg· on

ml palm leaves. Chit.inase producing ＱｩｾＱ｣ｌｦ＾ｬＧｩｈＬ＠ ｦＳ｡｣ｩｬｬｷｾ＠ Owringir>n.<:is SJ\ I Ii\ 12 08 1so)ale w<'>rP nsPd

111 this study. SAi IA l2 08 showed maxm1um chitinasP w1t.h spPCific Hd.1v1ty (7.8D6 1 J mg-1 pl'OLP1n)

,,t 60 h mcubat.J.on ｾｬ｡ｸｩｲｮｵｭ＠ lempernLur•' ;.rnd pH of chitinase adiVlty w01·1> :35°C and 7.0.

respectively. Clutmase was pa1t1ally pu1ified by

30°0

ammonium sulphate prec1p1tat10n could

increase

2 ::S5 fold

th on

the

specific ad 1 v1Ly

Thl'

ad 1 v1Ly

of

partwlly p11nficd clnl 1nase \Vas <•pt1mnl

at 45°C and 7.0, r cspect1vdy This ch1lrnase was stable at optimum tcmpcraLure for 180 min

1ncubat10n On ｓｄｓＭｊ＾ａ｣ｈｾＺ＠ :.snalys1s, lhF! e11zy111P had n'olt>cuhir wP1ght. nf 107, 102, 82, 6:J f\:1, ·l•'l :11lcl '1-1 kDa from 2y111ob'1 am analysis only 82 k DH prol.Arn band showr>cl ch1trnnsP nctw1ty.

In

t•itrn

and detached

lco.f

bioussay showed thaL ch1Li11asP of SAH.A. 12.08 had antagonist act1v1ty and

liiocontrol effica(:y t.o C:. rL{fi.nis and

r:.

gloMt::(lof'inidM in oil pnlm 101..tvl's

Key word s: AnLagonist1c activity.

CollPtotric}111 m

gloeosµorioidPs, C11n·ulari1L

a/j'inis.

birwontrnl

f ffsc::wy, chi Ii •wlytic had. "l'H"l

ｬｾｔｒｏｄｕ ｃｔ ｉ ｏｉＧ｜＠

Chit.in 1s a poly s1-1cclrnride cons1stmg of P· 1. 1-:\ -acelylgl ucosanu ne (Glc:NAc) residues t.h:tt. are

highly cross·hnked by hydrogen bonds Ch1trn is one of the most abundant renewable natmnl resou rce after c"llulos0. Chitin is widely distributed m nature as 1t is found in Lhc cut1cl1's nf 111sPCts, sl1ells of crnst.aceans, nPrnaLud"'s and <:Pll Wt-llls of most fnng1 (CJohel el ril. , 200fi,

ｦｾｨｵｌＱＮ｡｣｜Ｑ｡ｲｹ｡＠ el ul, 2007; Arnnnz el 1d., ＲＰｾＡ ＩＮ＠

Curuulciria rt//inis tine! CollPtntrichttm glMo.<1porioides are 1'11ng-1 c:n11sir1g ll-'af hlight :incl le;1f spots disease on oil palm nursery which rli:>cri>ase lf.s £>cononuc valut>. ln lndo1ws 1H I hi> cl1sPr sP was

slill categorized as secondary diseases but 111 Thruland it seems to be scnous disease in plant when'

•' 1.01°0 causc-d by Gunularir.L sp and

22°0

causl'd

by

Colletotrichum sp. (i\.itt1morakul et u.l .. 20la>

1 h us. I.ht> st.11<ly nho11t. 1 ·m ly prevention uf tl11s dts<'HS<' 1s r<>quuro. Ch1t.m 1x11.t.n11wd m 1 lic c<'ll walls mycelium, stalks and spor·'S ofpathogcmc funKI are able to degraclt> h\' ch1tinase Ａ＿ＮＺＭｴﾷｾｲＮ＠ 2Ci<_l5>

ChiL111ase 1s a b"l'Ollp of i:>nzymes that can dPgradt> chit.in polymer ｷｩｴｨｾ＠ slng-Ps Endocl11t inasP

'8C 3.2.1. l l) dflgrn<lP the p olymer into ｯｨｾｯＱＱＱｐ ｲｳ＠ Lhen degraded t o monomers by exoch it 1nAsP

Reo.

J.

Microuiol., 2014

bacteria (Liu et al., 2010), aclinomyeet.es (Gherbawy et al. 2012), fungi (Lee et al., 2009), insects

(Bansode trnd BaJekal, 2001:>) and plants ＨｾＱ｡ｴｳｵｳｨＱＱｮ｡＠ el

oL

2006) 1'he usP of ch1trnase some this

decades is increasing along with the wide range application of this enzyme. One of them is used

as a bioconlrol againsL v11rio11s types of fungal pathogens (L)e la Vega ＼ｾｉ＠ al., 2006) which is

expected t.o reduce

the

USP of synthetic fungi.c1des. Those efforts are <:onlmue<l Lhroughout

the

world

wide to increase the production of clutinase from various bacterial isolates. The most species is often

used as b1ocontrol agent from group Bacillus. Vanous chitinase pmclucing Barillus species have

been reported among

B.

cere1ts

(Huang

et al.,

2005),

B.

thuringien.<1is

(De la Vega

et

al.,

200()) and

B. li<:heniformis

(Kamil

et al.,

2007). Some of the latest characteristics of bacterial chitinasc has

been \\'idely reporte<l (Dhaltacharya el al., 2007). However, therP were still no reports about the

purification and characterization of indigenous bactenal chitinase which used as a b1ocontrol agent

of C.

affinis

and C.

gloeosporioides.

The objectives of the study were to evaluate paitial purified

and cl1araclPl'i?.e ･ｸｴＮｲ｡｣ｾｬｬｵｬｴｩｲ＠ ch1tmase of ｂ｣ｵＺｩｬｬｷｾ＠

lhuringiensis

SAlli\ 12.08 and det.erminE> ll s

potenC'y ns b1ocontrol of

C.

alfinis

nncl

C.

[!loPo:::;porioirJes.

M A'l'E RI ALS .1-\J\l ) ).1E'J' llO DS

Microorganis m straiu n nd growth conditio n s: Bacillus

thuri11g1e11s1s

SAHA 12.08 was

previously isolated from soil in ,J amb1, Indonesia (1 luryanto, 2013). Cultur0 was grown and

lllHtnt.anwd on sol11l lllfo'cllllrn d11t 111 ai;:ir at ;{/<lC. Th!:' t'1111i;al pathoi;ens

r.

affini.'I

ｾｭｊ＠

C. gl.oi'.'usporioides are is•Jla!Pd from mf•'Ct.:..'Cl c.1} polm leaves (Hurynnto. 2013; Ptu·namasan, 2013).

ThP isc-.1:-il.Pcl fungi were grown 011 'ot ;1to Oi>xtrose 1\gur iPDA.1 pl1Jtrs ancl rnc·11hat,ed at 28"C for 1-6 days.

Tlw

both of isolate m'f' oolle<:L1><l ::it. IPB Cu lt.uri> CoUect 1011, DepartmPnL of D1ology, f ucu lly

of Mathcmot1cs and Naturnl Scic1wcs. l3ogor Agricultural U111ve1·sil.y. Indonesia.

l\Jodclli n g bacterial growt h c u rYc fro m isolat e'-a n d c h iti n ase produ ctio n : lnr>culat10n of

2-4 loopful of selected 1so at"S mt<• 50 mL nutrient brnth \>18) suppl2mcntC'd with ＰＮＳｾｯ＠ chitin c:olloi(h1l Arnl incubale<.l HI ［ｾＷ｣｣＠ for lG h T11 amount of Jllb c11lt111'1> (JO· cells mL-) was mocul11Lecl rnt.o 100 ml. NB wit.h eol loi(J:.il !'!iii 111 which used P11zy111e prnd11ct.io11 m""'.hun1. Ench c11lt.1u·t> was

incubatPcl at :n°C. 120 qrn1 and r•ollf'l'INI f?ve1y t) 1m11l 72 Ii :nul t.l1Pir opt.ical dens1t.y were

lllPAS 1 r1>;\ SJl"Ctrophol rn 11rt 1·11 :-ii Iv it. 1)()() nm TI ., c11 l:.111·r!' W""I., t.h..,n •·entr fuf.. :it. l)()()i') qHn

(centnfugc Hermie with rotor 220 07) for 20 1mn tr1 obtrun t ht' ｣ｮ ｴ､ｴｾ＠ e:-.."tracl of e>..trnccllular

ch1tin .. ｾｳ･＠ enzyme.

ｾ ｦ ･ Ｚｴ ｳｵｲ･ ｮ ｷ ｵ ｴ＠ o f c hi t in ns1! acfi\. il y a n d p rote i n 1·0 11 cen t ra tion: C lut.11111SP acl1v1t.y wns

111Pas 11 <>d hy Sprncller ( l '.)fl'iJ ｔｨｾ＠ en HIP 1 •xl l'acl of ｐｘｌＱﾷｷｐｬｬＱＱｬｾＱＱ＠ ｦ＾ｬｬｾＩＧｬｮｐ＠ w: • ._ n1ld1>d 111 () 3°0 c111loid;il c:h1Lin and 0 I J\.f phosplrntP bu fff't 111. :37"C, pH 7.0 and ng1LatPcl at I '.?O rpm . The mixturP was rncub;_1led at :J0°C fol' 30 min. A.fhr 1x· nt.rifugat1ou at 8.l(J() g for 5 min, lhe Jiltru Le was ad<l0d to

750 µL tlist1Uotcd wat.er and 1500 pL ｾＳ｣ｬｲｮＱ｣ｳ＠ reagent (1\-Fernsrnnida and 0.5 M ｾ｢

Ｒ

ｃＨ｜Ｉ＠ and the

P11xtu wus lio1lf"J at lO(J°C for 10 n1111 Enzy1m .. 11d1v1t._\. was dPt·nr11wd by m1•11s1111ni:;

absorbancf" <1l. ｾ＠ 20 nm anJ using <_;1.·-'.\l,1C'I as a st:.mda1 d P1·ot 1?111 MOC•'ntratwn was detci mine.J

by the Bradford ( 1971)) 11s111i; buvi1,.. sv1·11111 albumrn :is :1 standard.

Parti:l I purifi catio n o f c h iti nasc" l 'a11.wl purifical10n was ｰﾷｾｲｦｯｲｭ･､＠ using ammonium sulphat:.l' .

Crud., .-nzyn P obl.a11wd \\·ns p1ec11 1tnh I with n11ir11on11irn snlphal e c. nee 11.rflt.ion 0 -100, up to

130-70°0 (Scopes, 1!1\:1.ti. Pr0c1p1tat .. s nrc stored at 1°(' for ｾﾷＱ＠ h and ＧＢｾｮｬｮｴＧｵｧＭ･､＠ at 6000 rpm

(centnfuge Hormlc with rotor 220.t)7) for 30 min at -!°C. Tho pnitern pellet was added to

Res. J. Microbiol., 2014

Electrophorf>.s is and :1y m ogram : ThP protein pellet was adcle<l by phosplrnte buffer (0 1 Ml

pH 7. Electrophoresis was carried out under dennlured prot ems (SOS-PAGE) 'lnd zymO{,ri·:.:im. Chitinase solution from crude enzyme and ammonium sulphalo prf'cip1tation was viewed using Sodium Dodocyl Sulphatc-Polyacrylamidc Gel ElecLt·ophoresis (SOS-PAGE) (La<'mmh, 1970) w1t.h 4% acrylam1de concentrat10n rn the stflcking gel ancl 10% acrylarmdc m the scporol1on gel. After electrophorcs1s, gels were stained with coomassie brilliant blue G-250. The chitrnase activities were

also detected on gels by using

SDS-PAGF.

and zymoE,'1'am supplemented with

O.a

0

o

_{colloida) d11tin.}

C ha1·acterization of o hitinase activity: The crude ex-tract and partially punficatJ.on was characterized at its optimum pH and temperature w1th iLs stability wns determin<l al optimum pH

and temperature. The optimum pH for chitinasc activity was determined

by

measurements at

different pH values (4.0-10.0) using colloidal clutm as a substrate under standard assay conditions.

The buffers used were HS follows: 0. 1 M citrate buffer (pl I 4..0-6.0), 0 .1 M phosphate buffer

(pH 7.0·8.0) ancl 0.1 M glycme-NaOH buffer (pH 9 0-10.0). The optimum temperat.ure was determmed by incubHt.i11g Lhe react.ion mntures al <lifferent rnngP of lempPrnt.uri> from 2R·•30°C with 5°C 111l.1>1val from thP optimum pl I obt.runed fm111 the previous Pl<per1111t>11t.. The stahi11t.y t•f

dutmasc was investigated by incubal111g the enzyme with optimum pH and opt.rnrnrn ｴ･ｭｰＩｬＧｾＧｴｵｲ｣＠

for 180 mm.

Effect of c hitinase 011 fungal patl1ogcn s by in vitro t est : The effect of isolot•' S;lliA 12.08 •vas

t..;-st..ed ai;mnsl C. affillis 1-111d C. gloeosJ>oriuides on f>I);\ Anlagornst 11· adiv1ly wns tPst.e1.L by 11s111g

ct>ll culturf>, c1 ude chil.rnase and partial p1mficat.io11 Pnzyme of LlH' st.ram with ni;ar wf>ll diffusion

method. Well containing

200

µL

cultur e or crude chit.inase was at distance of:..; cm frum margin of

PDApla1.e Opposit.P t.lw well, at a clistancn of' aL I Nist. :'I cm, suspc'Clc<l pathogenw f1111g1 Wf>n' placNI.

,\fter incubation for 6 <lays at 27°C rnh1bit10n of the pathogens dcvdopm('nl was ossesso<l with param1.::tcr. The percentage of mhib1t1011 of radial gmwth can be estimated by follcwrng e<"JUat.ion:

lnlub1ti on L'f radial growlh (0_{o) -} r, -_{r, l 00}

i:

where, _{1 1} ts le11brl.h of rachal growt.h Lowards plate tnnrgrn (3 cm) and l'z is l!engt h nf 1·adial gt owt.h t.owarcls antagonistic (Fokkoma, 1D73).

Biocoutrol n.ssa.y by detached leaf tes t : The 60 h cell culture, crude clutmasc and part.ml purification clntmas(' \.''Cl'f"' tested to b10control effcrlivencss (Ile tJ>t al, 2003) . Oil palm kaf\.\'CIS washed using st.AriJe aquodt•sL. Treul.rni>nl. consists of t10 h cell c:ult.111·P, ernclP Pll'l.yrnr-s ond pnrl.1nl punf1cut11m fmzyrni>. For nPgaL1ve co11Lrnl oil pa lm 1P1wes wen' 111oculat.ed w1tli funi;t ptilho!-fl'JIS

only, 11smg C. a//inis and C. gloeosporioides. The wholP trcatnH'llLs were co11d11ct.ed fom· t imPs.

P..<ich leaf was treated, cut. up to size of I x;3 cm ThPreaO er the IPaf wns dipp,,d 11110 t.J ｾ｡ｴＮＱＱＱ･ＱＱｴ＠ s for 30 min th0n 3u· dned The leaves put and placed mlo a petn clish cc:inl..umng ,, wet filler papr1 to

maintain thl" humichty. After 1-2 h , 50 µL of ｦｵｮｧｾｴｬ＠ pathogens Ill a concentratwn uf 4x 10" sporns

m l, l Lu Lli!:-' ()flP. end of Lhe surface

ur

I.he leaves mid then Jlll:ltbHt.Pd i'or i dnys lll thL· thll'k

cond1tions ol room temp1'rature. Pe1 contage of b1oconlrol ｯｦｦｊＮ｣｡ﾷｾｙ＠ of treatnwn' LBC) and D1s•.'ose

Incidence (DI) calculated 11smg the eciuat1on ofChancha1chaov1vat <>f rd <20071 ·

T-A

BC(%) =-->< LOO

and:

Where:

BC

=

Bioconl.rol pfficacy

Res. J. kf icro ln:ol., 2014

DI("'•) -ｾｶ＠ 100

T

T

= No.

of

infected spots inoculated with pathogens only

A

=

No. of in fed.eel spots inoculated wiLh ontogomsL and pathogens

DI = Disease incidence

S tati c ti cal a na lysis: Data was analysed usrng :MS.Excel Software.

R ESULTS

G r o w t h c u rve a tul c h iti n ase act ivity o f i!·m la t e: The isolnt.es wer't' [;l'Own 1n !\B \.\'as enriched

by collmdal ch1L111 (pH 7 Ol al 37°C. The gruwt.h rate of S;\HA 12.08 1sc1laL£' wns nscendetl fru111

0 -24 h of incubation ttnd LhE>n grov.th was rdat1vdy stabk up to GO h 111cubation and descended

ofter 72 h 111c11bot1on (P1g. 1) . The r.h1hnM;F wns produced from

:m

h 111c11bntion and relot1vely

increased unLil f)O h 111cubaLion. The highest chiLrnasc actn'lty wus fcund at the 130 h incubation,

after 1-36 h 0f mcuhnt 1011 th., aclivily was nol l'ou ncl \Fri::" I)

Par tial purific•a t ion o f c h itinase: The purtr;il pt1l'ificaLio1 procP<lun:>s oft.hi.> ch1t.inase secreti.>d

hy Lhf' SA H:\ 12.0R isolnl" ｡ｮｾ＠ summanz0<l 111 Tubk 1. The rPs111ts or ｰＱｾﾷ｣ＱＱｈｴ＠ ul.1011 ＱＱｳＱｮｾ＠ om111011111111

sulphate 0·10% 11p lo ti0-70% (w/v) sl10wed i.liat. ::I r.once 11Lnitio11 of ＺｾＰＥ＠ 11. wns able Lo produr.E:> :1

'l'ablc 1: SlUnmru·y of cht111moc partially pwification producoo h.v 8. ＯＯｷｲｩＱＱｰｩ･ＱＱｳｾ＠ SM-1.i\ l !! 08

Steps 'l'ota\ protPin (mp.) 'l'ot1I ·11·t1v1ty {l!) ｾｰ･ｮ＠ fir Mllv1ty (ti mg) Purificatfon Cfoldl Yiclrl I" • l

ｃｮｸｬ･･ｸｴｲ［ｯｾｉＮ＠ 1D.ll80U 142150 7 2 10 1.00 IM

ＺＩＨｦｾ＠ am.ntC'll.lWll SuJph&tlP VJ'f'<.'ll)J_la_ll_On _ _ __ o_.08'_1:! _ _ _ _ _ J_ll _ _ __ _ J_7_l_'>_J _ _ _ _ _ :...._> a_s _ _ _ _ l_.o_J_

••S ')

r'

(,

•Jl

ＭＭｾ＠ C) ｾ＠

UI

0 _'>.t•

Ｎｾ＠

ｸｾ＠

H

ｾ＠

--0--1 .... L<ll

-0-\ricllK' ch1lina.,_ •<II\ II\ 11 ｭｾ＠ 'rrnWn)

{ ＾ＭＭｾＭｾ＾ＭＭＭ＼ｽＭＭＭｻｽＭＭＮＮＮＮＬＮＮＮＭ ... ＭＭＮＭＭ ＭＮＮＮ Ｍ ｾＭＭＭＮＭＭＭＮ ＭＭ＼Ｉｦｬ＠

"

ｉｾ＠ ｝ｾ＠

"'""' lh

ｆｩｾＮ＠ 1 Growth a11d chit 11rnse acl1v1Ly of Lh P

n.

tli11riugiens1:.. 8Al1A 12.08 1solal<: on produd.1011

Res.

J .

Microbiol., 2014

mruamum clutmase specific activity 17 .061 U mg-1 _{(Fig. 2) and increase the purit.y upt.o 2.3n fold.}

Furthermore, ammonium sulphate at 30°0 (w/v) was used for prPcip1tal.lon of protein 111 cr11cfo

enzyme.

Chitmase from precipitation method was analyzed by SDS-PAGE to obtain the number of

protein bands And mohx:ulal' weight Zymogrom was analyzed using- 0.3% colloidal dutu. us a

substrate (Fig. 3) The result of SDS-PAGE usrng Coomassie Brilliant Blue G-250 (CBB G-2 50)

showed at ｨ ｾ｡ｳ ｴ＠ seven bands (molecular) of SAJ fA 12.08 chit.inns!> prol.1>in fracLion of 30%

Rmmonium sulphato wit.h an estimated ｭｯ ｬ ｎｾｵｬｮｲ＠ weight viz 107, 102, 82, 63, 5G, 46 and 44 kDa.

Zymogram result using 0.1 % Congo red showed one protein molecule which had chitinase acL1VlLy

with molecular weight of 82 kDa . It was calculated on the basis relative mobility of standard proteins.

18 o <;upcmaiant

i!f

16 0Proc1pit.ltC

2, I;

f' ;. ｉｾ＠

Ｍｾ＠ 10

... "" ;:; R

ｾ＠ (>

" ｾ＠

..

!:

ｾ＠

5

0 <Htl

i\mmorulQJl sulphAI< Cl1n.:cnrration ｉｾＩ＠

Fig. 2. Effect of ammoniu m s ulphate conccntratii:.n on precip1tat1on of B. i h uringir>n.c;is SAHA 12.! 18

dnlmase Barn represent sLandard f>rror

70 l<lla

l\OkD•

so Hla

ｾｯ＠ to.

M

107 ill•

11>' Uh

x2 t na

61 U>a

SS k.Da

4C> kl)a 44 LUa

F'1g. 3· SDS·PAGE and zymogram of chitmase SAHA 12.08 Al: Molecular wc-1ght ｳｬ｡ｮ､｡ｲ､ｾＮ＠

1: SDS PAGE crude e nzyme, 2: SDS PAGE of 30°0 (NJ14')₂S(j₁ prec1p1tat1on, 3: Zymog-ran1

[image:10.618.38.515.269.671.2]

Res. d . Microbial., 2014

C h ara c t e riza t.ion o f c hitinas e a cti vity: Maximum pH of the crude enzyme and partially

punficat1on enzyme isolate, SAHA 12.08 was max'imurn at pH 7.0 with the actlv1ty 0 697 an<l

0.721 U

mL-.

respectively (Fig. 4). Among the different temperature tested, the chitmase

activity of crn<l<> <'llZ) me was most active al ｾＵﾰｃ＠ bemg 0.722 U mL-1 bul maXJmum activity of

partially purification of enzyme was observed at 25, 45 and 6D°C being 0.722, 0.723 and

0 719 U mL-1, respPclively (Fig. 5).

To

<lelemune its slabilily. Lhe crude ･ｮｾｹｭｐ＠ and pnrt.ial

purification enzyme was incubated at optimum temperature for a period of

3 h.

Doth chitinase were

stable at maximum plI and temperature up l.o 180 min of incubaLion (F'ig. 11).

Effect of chitina.se o n funga.J pathoge ns b y in vitl"o test : lnhibit10n of the growth of fungal

pathogens i P , C. affini.<J and C. gloeosporioides was observed

in

Pilro by using 60 h cell culture,

crude chitinase and partial purification ch1tinasc on agar well-diffusion method (Fig. 7). lnhibit.J.on

of those fungal pathogens by SAHA 12.08 1solnlf' ehit.inase showl.'Ci that 1t. was able to rnh1b1t

C. affinis better than

C.

gloeosporioides after 5 ､｡ｾ＠ s mcubat1on. PaTt1al purification chitinasc could

0 l ruck cnr1nc

c Paru;1lly pnnht'tl ･ｮｾＱＱＱＱＮＡ＠

1111

I

,...l

E

ｾ＠

C·

Ｚｾ＠ ()I.]

ｾ＠

ﾷｾ＠

ｾ＠

.§

:c

(_.

0"1

J Cl 5 0 60 7 1• 80 l>.O 10.0

f 11;;". 4 Effrd. uf pH vnl ul's on eh1t 11rns0 art.1 v1t.y

"r

c11Hh• Pnzyrne and pm·t.1Hlly p11nf1ed Pnzyme of

R.

tl111ri11git>11.<iis SlJ IA ｬｾｊ ｜ＸＮ＠ ThP :id 1v1ty --::is nwasurPd at :l'i°C Bars l'PpresenL sL:i11dard

Pl 1'111 S

ll.?i Cl\Klc <"lr:l.:I

Partially pwiiiro cnJ";•n""

--

_0.7}

-:::.

ｾＮ＠ 0 71 .?.

i

ＱＱｾﾷＱ＠

G 11.1>7

111

Fig. 5 Effed oft,• 111pPrat.11rp on cl11tm11se ocl.1v1L.' of <:r11df> enzymf> nnd port 1aJly p111ified Pnzynw

of B. tlwringiensis SAHA 12.08. The activity was measurcJ at pH 7.0. Bars n:prcscnl

[image:11.618.93.520.340.476.2]

Res.

J.

A!icrobiol., 201./

0. 74 -0-Cnidc cmzymc

-l::r-hrual punlkarion enzyme

ＰＮＶＴＫＭｾｾ ｾｾｾｾｾｾ ｾＭＮＭｾ ｾ ｾｾＭＭＮＮＭｾｾＮＭＭｾＭｲｾｾＮＮＮＭｾ ＭＭＬＭｾ ｾＭＮＭｾＭＭＮ＠

0 15 JO 60 75 90

ｮｭｾＨｭｩｮＩ＠

\OS 120 ISO \6) 18(1

Fig 6: StabiliLy of crude chitinase and partially punfied ch1linase al opt.imu m pl l 'lnd temperoture

of

B.

thuringiensi.9 SAHA 12 08. Bars represent st1111d11rd error

Fig 7(a ·h)· E:ffoct1venPss of antagonist.

B.

lh11ringi1'1tsi . .; SAIIA 1'..l 08 at;nrnsl

r·

r1/fi11is 11nd

C. gloeosporioides in antagomst assoy aft.1'r 7 days incub:\t1011 on PDA medium . For

C. Affinis (a) 60 h cell culture, (b) Crud0 enzyme, (c) Partial punfic:ition ｾｮｺｹｭ･Ｌ＠

td) Control.

F'01

C. glo<>o1>porioides (e) 60

Ii

c ....

JI

c:ullui e cell.

(fJ

Crud,.. t>nzymP, (g)

Pm Lrn1 pur1f1c:nlion enzyme and (h) Contrnl of ofl "r 7 day 1ncubat1011

10l11lHt t.he growth of C. uffinis and ('. gloeosporioirle:; bHt t.t>r l han 1-r11de clut 11wse and ()() h cell culture for abo .1t. 39.89 and 35.80%, rcspecti vely aft.('r 5 cla;.·s moc ulal10n. Obse1 vations for 9 days

showC'd that culture cell 60 h, capable to 111hib1L C. a(finis up t.u 9 days whiJe th1 crude C)..i.ltlcl has

Llw h 1 ghPst rnl 11lnt.1on of fu llKnl pall1ogrns tiL Lhc• <'n rl y 1111 J<'lll nl1on and I I 1e ncli v II v I<' 11< l t 11 cl Pi ｾｬＧｐｮｳ｣＠

unl1l 0 days ThC' same result was also showed by pnrtial punfication enzyme, th L ab1ht.y t0 mh1b1t

WflS decreasf'd . T Towevt>r, il was still higher t hrm ll1P olhe1· l 1·ealnit>nt.s r'F1g 811). Whe1 >'llS the

mh1h1t.10n of C. glur,ospo ru>ides among !.hose t.hrPf' t.iPaln11•11ts showed t.lit> snrnJm prit.t ·rns 111 "hi di

th1'rf"I wns a dcclrning of thP nctivity alung-tlw 111n1bot.1<11i P•'l'lod <Fir;. fib)

Biocontrol assay by detached leaf test: B10<..-ontrol C'fficacy test. used twn tr·'atmcnt.s, as

cell culture 60 h and crude enzyme. However, partiol punfi.cation of cnzym(' was nol done

berausE> the i>nzymf' cfln clPst.ruct. LhP testi:>rl ]P:lVPS f.ff.,,,, ivi:> t.i:>c;I nf th• b1· .co11t.rnl from

[image:12.624.16.545.90.561.2]

ｾｯ＠ 1a)

o 6() h ｣ｾｬｬ＠ cultun:

':' 4o o Crude e•trnct

e • rart1:ll _{purified ･ｮｾｹｭ｣＠}

c

.g 'O

SI

ｾ＠

'9 ｾＮＱ＠

ｾ＠

I()

Res.

J.

Microbiol., 2014

Fig 8(t1-b): Growth reduction of (a) (;ryptocoryne a/finis nncl <b) CollPlolrirhum gloeosporioidPs in

t.)11" ｰＱﾷｐｳｐＱＱＱｾｐ＠ of B. thuri ngiensis SAflA l 2.08 ch if i rrnses

fig. 9(a·1): Effcct1vf•ness of antagonist B. thurin{!iensis SAHA 12.08 agamst leaf blight

d1SPHSP 1•;1l1SP.J by C. tl{fini.<> <lfld (';, (Jf<W08f>OI'ioir/t>,q lll b10assay using oil pAlm

lf>HVPS as for r', affins, (:11 60 Ii t:Pll c:11lt 11r1>, (h) C'rudi> • nzyme, (c) Control Ｎｾｭ､＠ for

C. gloen.<:porioides, (d) 6(1 h cell, 10) Crude 0nzym0 ;md (f) Control

e:1ch l.r?11t.mf'11ts showed that the cull.11rf' cell 60 h and tho crude chiti1rnse have the ability to inhibit

[image:13.616.44.537.109.663.2] [image:13.616.108.538.128.419.2]

Res. J. Al icrubiol., 2014

120 _{o Biocontrol cfficocy (DC)} oDiscase incidence (DI)

100

..

80

"°

g

82.90 ｒＶＴｾ＠ 83.60

ｾ＠

Sl.63

"

..

60 I

l:!

&:'.

40

20

0

ti

0 0

l

l

l

l

2 3

Trea1mcnts

Fig. l 0: Effectiveness of Lhe treat.men Ls B . lhuringiensiH SAHA 12 08 in reduct.ion of disease

incidence m C.

affinis

and C . gloeosporioides. 1. 60 h cell culture cell

+

C.

uffinis.

2. Crudo enzymc>

+

C. alfl.nili, :1: 60-h coll cult.urc

+

C. glm101>porioidP.'1

1 1: CrudP Pnzymc

+

C. gloeosporioides, 5: Con t rol of C. a/finis and 6: Conlrol of C. glot>o1:q>orioid<N'I after

5 days incubation

leaf bhght caused by C. ｡ｦｦｩｮｩｾ＠ liPl.Le1· t.h::in thP cell cu)ltffe 1)0 h wit It the pffeC'f 1veness val11e of

b iocontrol for about 86. 15% or w1th d isease incidence for about 13.5%. \Vhercas , tho rcducl1on of leaf blight rF111sPd by C. glot>osporioidP.'I showPcl by uc;inr; 1)0 h ci>ll cul1 ure WAS bPll er lhHn c:rnde

enzyme for abouL 83.6% m· with d1sease i11cide1WP for aboul 16 36% (F15 10). The rPsulls indtN\U>d

that ther e was correlation between tho act1vity of in vitro antagonist and biocontrol efficacy bY

detAched lea! assAy Lest.

D I SCUSSIO N

fsolate cf

n.

lhnringiensi.s SAi I/\ 12.08 p rnd11rPd exlrncellular d11l.111asP whtr.h ATP cap11hle to hydrolyze substrates suc•h as c0Uoulr1l ch1Lin The> specifu; ｡｣ｬＮ Ｑｶｩｌｾ＠ or t hP ･ｮｺｹｭｾ＠ began lo f111d al Lhc stationary phase when ｴＮｨｾ＠ n11mlwr of c..-11 lend t.o dc<!rt>asP Thr h 'hhcst spPcif1c acLi v1t" 1f Lht->

enzyme occunP<l 111 I.hi> l)Q h of rnc11bF1t.ion. Tht> n1glrnst. t>nzynw prc><l11ct1011 WH'> also prevwusly

reported by Nfll'ayana 11nd Vij11yHh1kshmi (2009), lhe mnx11rn1111 chiLirrnsP producl1t)ll of

Str.?ptom:yces sp. ANC 6277 observ1'<l 3t. 60 h of mcubat.1011 ancl L.1111led to dC>Crcase d1w t.•> the

mcri>asmg of mcubat.1011 per iod. Almost all ch1l 1 nase from hactena h.1s shown rnaximum 1w1.1v1ty

at 30-96

h

of incubation (7.hu et al., 2007; F'anirnnrzi (Jl cil., 2009; t\unlPbyandarn Pf nl., 20 10).

Crude exLrucLs of en:1.yrn0 obt.111 rwd from l.ltl' highes t f'llll)'rne pn1d 11ct.ion coulc.I be prec1p1t rtl.lKI

wtth ammomum sulphatE-:30% (w/v) and capable l.o 111et'PHSP Lhe punt.y ••

r

2 35 fold. Tlte percent.agP

saturation of mnmonium sulphat.P for @acl1dull11flse from d1ffPrPnl 1sol.1tes was 110L the sa111P The

p1>r ccmtago soluraLion of iltn m on111m s1 1lphuLP f'ot each cl11t.inas0 f'1·n111 ､Ｑｦｦ･ｲＱｾＱＱｴＮ＠ i-;olat.Ps w:is not.

same The salurall.on levPI of pr@r1p1totio11 are r:rnged from 30-85% (Zhung el al. 2001; Kun f'/ <Li,

20();3; Rabeeth el al. , 2011 ). Previous stud res reporLed t.hnl clnLi1rns0 fn ,m B. c·irc11lm1s \\.1,- 1 ｾｷ｡ｳ＠

cnpoblc to pnx:1p1Lal.P with 10°0 amn1r.mun1 s11lpl1:1 l.P (\•VatnnHhc et 1d, l'.)91). Tl11' 111olPc11lm· W•'1gl1t of I.hf' prnt ern was fou 11d l.o be flhou t 107 , 102, 82, 63, RR, 116 and ·l I k IJa by 10°o SDS PArn·: and showf'd a single band 82 kDa 011 J0°o zymogrnrn analysis rntbc:1t1111:: thi> ch1trn:1si> achvtly The

molecular we1ghL of dut.mase of H1u:ill1Ls sp 1.1 . B. thuringiPm.is was analyzcJ by SDS-1'.\GE,

had morP than one prolern molecul f> 111 the range of 32-125 kDa (IJe la VPgA el al., 2006;

[image:14.624.111.548.123.274.2]

Res .• T. Micru/Jinl., 2014

The highest aclivit.y of both enzymes was at pH 7.0. Chitinase isolates both crude enzyrn"' nnd partial enzyme showed a wide range pH from 4·10. Wang and Chang (1997) suggested that the optimum dutrnase of most bacteria arc varieties from acidic pH to alkaline. The highest activity

of both cHzymes wos nt.

pH

7.0. The Slimt' results also reported lhot chitinasc of B. thuringiensi.s

(Gomaa, 2012) and

Bacillus

sp. (Mubarik et

al.,

2010) bad the highest activity at pH 7.0. The

enzyme tlcl.ivit.y was dPt.ermined by the presence of donor protons an<l donor Acceptors on t.he

degree of ioruzation, obtained at pH optimum pH (Wilson and Walker, 2005). Proton donors and

flcceptors contained many degrees of ionization on Lht> sites of catalytic enzyme to support optimum

conditions, moreover, the

pH

also affects the solubility of Lhe substrALe. Extreme

pH

caused changes

in the structure of the enzyme and then decrease the e ffectiveness and efficiency of enzyme activity (Farnbee, 2001 ).

Crudo enzyme showed the highest activity at

35°C.

\Vhile the pa1tial enzyme showed the

highest range ucliv1ty at. a Lcmperaturc of 25, 45 and 60°C. MosL of the chilinase ofbactcna have

maximum activity at a wide tempe1alure. Various rcpo1ts mchcatcd that the max:imum activity was

al. 30-7n°C Wrandberg and Schurer, 1994; Sakai Pl al.., 1998; Barboza-Corona et al., 2008;

t..lubarik l' I ul , 2010, Tluagarajan et al., 2011; Jlaggag and Abdallh, 2012; Margino et ed., 2012).

f3oLh of crudf' chitinasf' and partial chil.inase st.<ible at optlmlltn ｴ･ｭｰ･ｲ｡ｴｵｮｾ＠ for 180 mtn of

rncubat.1on Kuzu et al \2012) also report.ed the s1Jrne tlnng on

R.

lhttringi<•nsis ssp. ku.'lrlaki

H BK-51 whu:h rem ams slable and active for 3 h at. ｾＰＭ I 200C.

Ch1l11111s<' Pnzyme is ctu-rently us<'d in ｢ＱｯｬｯｾｊＱＺｮｬ＠ control of fungal put.hog011s because of 1ls

ability to degrade clutrn rn fungal coll walls (Ulhoa and Pcberdy, 1991). This study was conducted

t.o analyzp Llw ahil1 Ly of 60 h cell cullurc. crude e nzyme and pa1t1ol enzyme of B. ｴｨ｡ｲｩｲｩｧｩ｣ｭｾｬｾＧｬ＠

SAIIA 12.08 isolate to 10lub1t the growth of leaf bbght pathogens (C.

affinis

and

r.

gloeosporioid1>s)

by in ntm t .... <;t The t nree ch1trnnsP t reatment.s wPre capable Lo mh1b1t t.he growth of hot.h

pathogenic fungi in vitrn. inhibition by partial ch1Lrnase was bf'Uer than crude onzymc and 60 h

cP.11 cult.11ro agni nst these fungal pal.l1ogens. ThosP results were probably ca11sed by the length of

Lune ｭｾ･､｣＾､＠ h.\ L11e cell ＱｾＱＱｬｴＮｵｲ･＠ to pro<lu<.-e chitmase while pmtial clnt.rnase an<l crude Pnzymi> WPre capab]P t.o hydrolyze ch .t.m found in fungal cell walls directly D1fferE>nces mllong the ab1lit.y of

rnh1b1t.11 11 ｵｾ＠ 1111st. ｦｵＱＱｾ｡ｬ＠ pathogens ｷｾｮＮ ＮＬ＠ causi>d h) t.l1e conccnl1 ot.1011 of hyw·ulyt1c Pnzy11m or

socondary mc-tobohc compounds found in partial enzyme or crude enzyme (Propai;dce et al., 2008).

Effect of tl'catment of chitinaso agrunst leaves infected of fungal pathogens by detached leaf

test has b0cn conducted and the results are presented 111 Fig 10. Crude enzyme has inhibitory

agamsl C. affinis, beUPr than 60 h <x->11 culture I lowever, different things 11re shown tigmnsl

C. gweosporioides 60 h roll culture capable of inh1b1trng better than crude enzyme. The results

indicated t.lrnt. LhffP wns correlation beLween the Activit.y of

irt

11itro anlagonist. and biocontrol

efficacy h.v int iuu lest. The l'esults causC'Cl by t.lw syrwrgism of action of b10contrul mcchmusms of

rell cult 11n s111:h us ant1l11ol 1c pr0duct1on and induct.um of plant resistance wllh genes a<.:Lival1011

such as dntinasc\

P

1. :.-l·r;lucanase, pel'Oxydases and phenylalanine ammonia lyase (Chani; <?t al.,

2007). AJthough there are no reports on oil palm leaf bhght b10control using chitinaso from

Bacillus sp. Several

Bacill11s

species have been widely used as b1ocontrol agents pothoge111c fungi

both of cdls, crude onzymi: and partial chit.rnase mclud.rng the gcrnnnation of seeds protected with

n.

liche11i/orr11is and B. I

/11tringiensis

for si-ed mfestPd w1lh A.

/tiger

(Gomaa, 20121 and ch1lrnnse

of

B.

tlwringiensis ssp. uizawai able to protect of bean seeds infested with six phytopathogenic

fungi (DP 111 V<>i;a el CLl., ｾＰＰＶＩＮ＠

Res.

J.

Microbiol., 2014

CONCLUSION

Bacillus

thuri11giensis SAHA 12.08 produced chitinase at 60 h incubation. This clntinase was

purified 2.35 fold with 30% ammonium sulphate. Crude dulrnase and partially chitinasP had

maXlmum pH al. 7.0, 35 and 45°C, respectively with molecular weighL of 107, 102, 82, 63, 55, 46

and

44

kDa on SOS-PAGE analysis and 82 kDa on zymogram.

The

both

of

ch1tinasc

were

stable

at

optimum temperature and pH for 180 min . This chiLinasP could

better

inh1b1L C. af[inis t.han

C'. gloeosporioides in uitro and detached leaf assay and has potential application as b1ocontro]

agents

for C.

a/finis and

C.

gloeosporioides.

ACKNOWLEDG MENT

This

research was funded by

BOPTN

scheme from Directorate

General

of

I hgher

Education

RPpublic of Indonesia in 2013 and ADS Funds from colhboratJ.on CRC IPB-University of Got.t.mgen

rn 2014 to Nisa Rachmania Mubank

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l\lengibar R.

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I

Panos nnd

8

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C.L.

Jao, ｾＰＰＷ＠ Ant1fu11gal act1v1Ly mid P11huncemenL

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