This document describes a study that characterized the physicochemical properties of indole acetic acid oxidase (IAA oxidase) from Alternaria cepulae. The researchers found that the optimum pH for IAA oxidase activity was 5.5, and the optimum temperature was 40°C. Gel chromatography determined the molecular weight of IAA oxidase to be 30,000 daltons. The study provides information on the purification and characterization of IAA oxidase from A. cepulae which is involved in leaf blight disease of onions.

1. J. Ecotoxicol. Environ. Moni.l l(2) 147-148 (2001)
O Pilani Paramount Publications-Printed in India
ISSN: 0971 -0965-l I -01- 1 47
PHYSICOCHEMICAL CHARACTERISTIZATION OF
INDOLE ACETIC ACID OXIDASE TRQM- A-LTERNAKIA
CERULAE
B ANNADURAtr*, M SHANMUGAM** AND D B MOTLAG*
*DEPARTMENT OF BIOCIIEMISTRY AND MOLECULARBIOLOGY
UNIVERSITY OF MADRAS, CIIENNAI 600 025 INDIA
**DEPARTMENT OF APPLIED ZOOLOGY, KWEMPU UNIVERSITY
JNANA SAHYADRI, SHIMOGA DISTRICT, KARNATAKA, INDIA
ABSTRACT
The physicochemical properties like change of pH, temperature of the purified IAA oxidase from
Alternaria cepulae were investigated. Molecular weight was estimated by Gel chromatography of Andrews
method. The optimum pH of the enzyme was found to be 5.5. The optimum temperature of IAA-oxidase
activity was at 40oC. The molecular weight was determined as 30,000 daltons.
Key words: Alter:naria cepulae, pll, IAA oxidase, Molecular weight kit.
INTRODUCTION
IAA oxidase (EC1.11.17) was reported to be produced by various pathogens.
Themould Omphaliaflavida w'asreportedlAAoxidasewhileinfectingonCoffeaarabica
and Coleus blumei (Sequeira & Steeves 1954). Similarly IAA oxidase is reported to be
produced in the following cases ofpathogenesis viz Albugo candida while infecting on
Brassica napus (Srivastava et al 1962). Verticillium dahliae on Capsicum annum and
Fusarium oxysporum onMangifera indica (Kumar et al1980). Marasmius perniciosus
whiie infecting on Theobroma cacao (Krupasagar & Sequeira 1969); Protomyces
macrosporus onCoriandyum sativum (Tayal et al l98l),Verticillium dahliae while
infecting on Lycopersicum esculentum Reuveni & Ferrira 1985 and Plasmodiophora
brassica whole infecting on grapes @oemer & Ajarrg 197 4). The auxin content is reduced
in the host tissue due to the aetion of IAA oxidase.
IAA oxidase with acid pH optima has been isolated and purified from several plants
( Psenakova & Kolek 1973;Miyata et al 1981). This enzyme was isolated and purified
from downy mildew fungus Sc/e rospbra graminicola infection on pearl millet (Arora et al
1986), from Erisiphe graminis infection on barley (Fric 1975), from Pseudomonas
solanacearum infection on tobacco (Evident e et al 1986), IAA oxidase is also isolated
from virus infected plants (Gaborj anyi et al 197 3).

2. 148 BANNADURAIETAL
The characterization of IAA oxidase were. reported in many cases (Denckeva &
Klisurska 1986; Talwar et al 1985;Panel & Greppin l97Z;Mazzaet al1968;Mennes
1974; Laurema 1974). The characterization ofIAA oxidase can be altered by naturally
occurring compounds such as Phenols, Coumarins, Manganese salts and plant acids. There
were reportsthatmonophenols act as cofactorswhileparadihydricphmols andpolyphenols
act as inhibitors forIAA oxidase (Pilet 1964; 1966; Runkovaet al1972). Manganese ions
have been found to be a cofactor for IAA oxidase systems (Sequeira & Mineo 1966),
Scoioletin, a naturally occurring coumarin has been shown to inhibit IAA oxidase at higher
concenfrations, here as lower concenffations ofscopoletinwere stimulatory fimbert & Wilson
1970). The physicochemical properties ofthe pudfied IAA oxidase have been invesfigated
and presented.
MATERIALSAND METHODS
Enzymepreparation: CrudeenzymewasobtainedfromthemediumsuggestedbyRay(1956)andthepurification
was carried out according to the procedure of Annadurai ( 1 987; 1 998; 1 999; 2000).
Estimation of IAA oxidase activity: IAA oxidase activity was estimated according to the method suggested by
SequeriaandMineo(1966). TheactivityoflAAoxidasewasdeterminedbytherateofdisappearenceoflAA. The
reaction mixture contained 0.25 ml of 1 mM DCP, 0.75 ml of mixture of I mM IAA and 0.5 ml MnClr. HrO, 3.5 ml
of 0.02 M WV cihate buffer pH 5.5 and 0.5 ml protein. The mixture was shaken and incubation for I hour at 32
+ l"C Aftertheincubationwasover, I mlof Salkowskireagentwasaddedto5ml ofincubationmixture. Thepink
colour developed was read at 530 nm in a shimadzu spectrophotometer. The control treatments were carneci out in
an identical manner except that the enzyme was added after the addition of Salkowski reagent.
IAA oxidase enzyme unit was defined as the one micromoles of IAA destroyed in one Hour{lmber &
Wilson 1972).
Determination of Molecular weight
Gel filtration: The molecular weight of IAA oxidase was determined by gel filtration method according to
Andrews (1964). Sephadex G-100 was allowed to swell in 0.05 M phosphate buffer pH 7.2 for three days. The
solution was decanted and the swollen gel was further washed in 0.05 M Phosphate buffer (pH 7.2). The fine
particles were decanted and packed in a column (2 x 60 cm). The column was equilibrated with 0.05 M TrisI{ Cl
buffer containing 0.,I M NaCl at pH7.2. The column was run at a flow rate of ml,/hr and 5 ml fractions were
gollected. The elution volume (Vo) for each protein was determined by measuring the absorbance at 280 nm. The
void volume (Vo) was determined using Blue Dextran 2000. The standard proteins used were BSA (MW 68,000),
Ovalbumin (MW 45,000), Chymotrypsinogen (MW 325,000) and cytochrome C (MW 12;000). The Kav value
was calculated using the following relationship:
Ve-Vo
Kav = --------------
Vt- Vo
Where Vt is the total bed voiume. The Kav values of standard proteins were plotted in semilogarithmic graph paper
( on the scale) against the corresponding molecular weight (on the logarithmic scale). The straight line obtained was
used for the determination of molecular weight of purified enzymes.
Journal of Ecotoxicology & Environmental Monitoring. Vol.11(2001)

3. Ef{'cct of change of pH on IAA oxidase activit_v: Effect of changc of pH on IAA oxidasc activity was carried out
over the pH of 3.0 to 7.0. IAA oxidase (0.5 nrl) rvas allowed to react with 0.25 ml of I mM DCP. 0.75 ntl of a t.trtxturc
of I nrM IAA and MnCl.. H,O and 3.5 ml of 0.02 M of buffer fi-onr pH 3.0 to 7.0 at 32 + l"C for I hottr -['he
activity of thc cnzynre was estimatcd at the end of the incubation pertod.
Efl'ect of change of temperature on IAA oxidase activitv: 0.25 ml of mM DCP, 0.75 rnl of nrixture olnrM lAA.
'0.5 mM 4nCl.. H.O and 5.5 ml of 0.02 M citrate bu{fler at pH 5.5 was incubatcd with 0.5 ml of IAA oxidase irl
different tempel'arurcs. After the incubatron peilod. IAA oxidase activity was esttnratcd.
RESULTS AND DISCUSSION
Fig. 1 shorvs the determination of nT olecular weight of IAA oxidase in a sephadex
G- 1 00 column using illolecular rveight reference proteins. The molecular weight of I AA
oxidase is found to be 30.000 daltons from the senrilogarithmic graph. Fig.2 shorvs the
effbct of pH on IAA oxidase activity. From the results, it can be seen tlmt the eff-ect enzyme
has an optilnum pH of 5.5 for IAA as a substrate. From tl-re results presertted in Fi-s.3.
enzyme activity- remperature relatronship is seen. It is evident from the activitv that at pH
5.5. the optinllnll temperature fbr enzynte activity is 40"C.
K."
Fig. i Deternrrrration of rrrolccuiar rvc'ighl of IAA oxrclasc b1, gel filtration
IAAO
Activity in
units 100
5.5
Frg.l Elfcci ol'changc oipl-l on i:A orrclasc acr r1'
Jcurnal of Ecotoxicology & Environmental Monitonng Vo!.11(2001)
o.4
0.3
o.2
o.l
150
50
Molecuiar Weight
pH

4. t50 B A}.INADURAIETAL
IAAO
activity
I"AAO
Actlvlty ln
units
Temperature
oC
Fig.3 Effcct ofchange oftentperaturc on tAA oxidase activity of A cepulae'
The rnolecular weight of IAA oxidase fiom G-100 sephadex colufirn (Fig' 1) rvith
the help of low moleculaJweight kit, the MW of IAA oxidase is found to be 30'000
daitons. trAA oxidase was not stable at higher temperature optimlrm temperature for IAA
oxidase was 5 .5. The enzyme is repofiecl to be active between 5 '0 to 7 '0 pH (Krupasagar
& Seciueira 1969)"
ACKNOWLEDGEN1ENT
The author B A is grateful to Dr. s c Dhar and Dr R Puvanakrishnan' Scientists at the Department ol
Biotechnologv. Cl,,RI. cnenn"ai for Laboratory facilities and useful SLlggcstloll and UcC, Neu, Delhi for researcl,t
grani.
REFERENCES
Andrcu,s p 1964 Estrnrarron of molccular wcights ofprotcrns by Sephaclex gelfiltratrorr' Biochem' J'91. 221-713 '
;nnaciurar B. palani B. Mahalingam S and Singarav.i, Cl S9s-ifftlt of afla:toxin on RBC' WBC and haemoglobin
of' lialltr'r' ruittts ndr;eglcas Biojournat t 0: 1 65- I 72'
Annadur.ai B, pr.abhakarar.r V, N4d-.. Farrrk S a.a nrr-rttt,imaiun p f CSa Production of anrylase in Aspergillus or;'zae'
or"".r",.l,ll',|,,|il]i]l ll",'Jl;l,t;]-,, o r.o Singaravcruc leee prodLrction orar'raroxi, in conrcnrirrated storccl
I gru,,'t.. J. Ecotoricol. Environ' [tonit'9(l): 13-17'
Anrraciurai B. 1(arr-rnanidhi P and l4alralilrgan-) S t999 6"t'i(}tn',*t' of Alternttria t'epuloe in leatblight clisease of
ontotl..l" Ecobioi' I l(4): 191)-i05 i.- ,^..+r^ri.-L+...-...^,rrnn'
Annaciurar ts and Motlag D ri r99C Extlace llular cnzylrles of Alternaritt celtulue in lcaiblight diseasc of onton
Biojournal 8: 105-lt)9 .r , -r.L^ ^^r..,-r
.nnlicluratB.Coplnatho,nap,ion,RlggSStr-rc.Iicsontherolcofthccelllvalldegr.adrrlgCnzynlcsrnlcafblight
discaseofonron lillltittttccpr(l,tnn.).urr.dtyll'nrnariacepultte'
Biojournall0:l7-l-"1 iE'
.nnadurar B. ,{rul Kurraran P, Kalpana erul t<umai'Li"'JeUJ'f S't'Lan K 1999 ll'lrcrobiologrcal anal-vsrs of
cllorc drrrtlttng rvatt't'sLrppil' Bio' J' I l: l6i- I 7l'
rrnurirrrar B and i4otiag D B 1999 croivth ot ltrci'io''i" ip'lou in leafulight discasc rn onion Bio' J' I I : i6l-
i (i5
Journal of Ecotoxicology & Environmental Monitoring Vol'11(2001)

5. j
!
PI,{YSICOCHi],{IC'AL CI IAI{AC1'ERIS'IIZA'|ION OT INDOt,E A('L]1'IC A(-ID OXIDAST 1.-.I
,rrnariirrrfi L] rnd Motlag D Il 2000 [:lfcit of Various carbon solrrccs on producttot.t ol cndopolVgairctLlrollasc (]l
.iltt'rriortu ( ('pul(tL'. J' Ecotoicoi. Environ. llonit. l0 (l): 37-41
1n1r(.ir Lliti B lnd Nlotleg f) B i999 Elfcct oi tthytohortrones in norntal and int-cctcd onttln 'icr t!s l'r 1/It lit,ti r,i
t't'prrluc'.. llio. ,|. I I: 155- 16().
AnnlclLrrai et.lStlStr,li.roncndopolygalacturonaseinlc-iliblrghrciiscascofoniott(lllitrrtt.t'lr(1 Lilllr.lcaLtsct'l bt
..iltei;ntrria t.cprrluc /Ponnappa) and its irrtcraction rvtth phytohormones. Ph.D. ilresis. litrlverstty ol'
4 ldrtis.
Atora Y K. N4ehta N. ThakLrr l) P rnd Wagie D S 1986 Enz_vntc changcs associrtcd $ rth ltost PiilusltL- tntci itcttons
bctrvcen Pcarl ntillct an( Dorvny nrilderv furrgus. lSclL'ros1tortt !rtrttirtrt:rtitt- J. Phytopathol. I l6: 9r-
t ()5
Bonircr-ll arrd AianS M R 1974 Dcr-r]onstrattot.r cl indolc acctic acrd or.rdrsc rnhjbitors rn l' usnirtriittltitt)rLt hru.r.rt(1it
rniectcd roots of rapc- Z. Ptlaneuzenlir. Pllanzensclttz' 8l I i2-l i ;-
I)cnuher11 A and I(lrsuriia il iCSt, f'ompatisotr of son-ic i(lnctlc par itlllctcrs olpcroidase and IAA oxtdasc rtl thc
.or,,.s.j l,t g,-o.,",th and diticrcnttatton plaltt cc1ls. Biologi. Plautarutn 28: i()5
EviilenrcA.SurcroL,. lacoball:sNSirnciRandassoClgti6/--N-Acctvl indoic--l-acctvl-r'-L-i.vsrne.3 lllcf;tboiltc
of Iridoic-iacettcacrdil'onr !'sc'ttricttrtouttss.'rut.qu('psttyttstQtlot. Plrltochenlistr-r ?5: Il-'
Frrc F 1975 Activity oi-peroridlsc ancl IAr oidasc il1thc trssucs of r-trild$v inlcctcd bericr il.r.r.ilplte ,!rrttttirtrs i'
sp horder marchacl ). Ph1'tnpath Z 80 6'i -1>
Gabor.ianyi R. Scgi F and Balazs n tgl5 Cro",fh inhibition of virus Infcctcd plilnts. .'ltcrrtrr)ri (rl l)cr'r(lxi.iirsc
enzylt-tcs r1 colrpatrblc- anci rncontpatiblc )rost parasrtc r-elations. ictll I)htol)atltol acatl Sci' Ilrrng li
.[iI-90
lrrbcrr M p and Wrlson I- A 1972 IAA oriclasc preparatiotls li'onr srvct-t potal0 tr:bcrs. [)ltr tocltclttistr-r' I I: ](t-
16
l(rLrpasagarV anci Scclucrra L 19(r9 Auxrn destructionby,,larasnttu. Perfit(t():;Ll' Am. J. Ilot.56. -l.)()-it)^
Kuntar-"1 Bcnrrral SpSanclIlantS I()E0Dcpictronofauxlnsrnntangosccdlingsallccteclw'lilrl)Llllcil)'it)psiaL'rl:
nrango ittali'ortllatrorls. Intl. J. Exptl. Biol' l8: 28(r-2E8
L.aurenra S t97+ inclolc acettc acldc oxrdase rn rcsting ccreal gratns. Phvsiol Plant. -30 .-10l--11)6'
[-orvry O H. Ilosebrough N .i. Fan-A L O and Ranclal] R .] 196 I Protcrn nrcasut-cnlcnt 'ith tlrc l;olin pircnoi rcauctrt.
J. Bio. Chenr. 193: 165-175
,ianncs A N4 l97rt Titc Indolc acctic acid orrclase of l,upitru,s luleus: A quuntitatiYc cot]lllarison of thc actr l1 tll-
thrs enzymc in root nocitilcs and roots Acta. bot' Nerat 22: 694-7(15'
l4rzzaG.C'har.resC, I-loucheslr,l.llrcarcl JadnRynanci U lg6tiEndo..ecnousaurinsinlrcaltlri'rrrcldiscascdl:)ants
Biochem. Biophys. Acta. 167: 89-98.
[,c.el (- and (iraporn ]l 197i Control of glgrvth by', aurins and its spccilic nclrtrill inhibitor'. Plant Cell l'}lrvsiol' l5
i5l-i 5(r
fri]ct F E 1!)(t4 Ar-rxrn conlent and auxrn catabolrsir of thc sterns ol Etrpirctrbtu (1'r(1/.ll7-t-/--rnicctcd b;v i'r0rrttt'r:r
pr.sl. Fh1'tochemistry: 61 ,--{t21 -
pscnilovr -I'and
l(oieli j 1975 Enzynratrc degradation of Indolyl-3-acctlc acid u'ithtn irtatzc (Zcrt arrt.tr.s) Root'
Biochem. Phvsiol. Pllanz. 163 : 5l(r-544.
Rat p i4 i95(t Thc destruction of Indolc acetic acid ll spcctrophotonrctrtc sttrdy olthc crlzr'trrlrttc tcitclton Arch'
Biochem. Biophls. 64. l()5-21(t.
ReL^ clr R and Fcrrtra J R I 9E5
'fhc relationship bctu,ccn pcroxidasc activity and thc tcsislatlcc of totratocs
(1.1,t:1rO"rr,rry esculetttttnt) to Varricilltun dttltliaa. Phltopath' Z' 118 193-l()i '
Runkova l- V, Lis E K. Tontaszanski M and Antonszeu,ski R 1972 Ftrnctroll of Phcnolic substanccs rn thc
,Jcgradation sysrenl ollndolc-.1-acetic acid rn Strarvbcrrics. Biol. Plant. l4: 7l-8 i.
ScqucrraLandMineoLlg66Partial punticatronandmincticsoflndoleaccticrlcrdorrclasclt'otrltobitccorools
Plarrt Pltl'siol 46: 195-,l9(i.
Sri11,astavaBISandSha11,tv{ l9(rlThcbiosynthcsisollndoleaceticacidinMcluntp.:pot-rrllrrl(l)crs).Can.'1.[lot
J0: 509_5 I j
'Trlurtr C. Dcndsay J I, S and CLrpla V K 1985 Kinctic propcrtics of lAA. oxidasc fi'onr nrungbcan cotl'lcdorrs
Phl tochemistrY 21. (:75-676.
1'ayrl i1 S. Sharrnr S Nl and Agaru,al iv,l L l98l Studies on thc polyphcnols, irt'ott-ins. chlorophvlls. IAA ortdasc
ancl aptvlascofn,rrntil ancl falscsnltinfcctcdIcavcsttf'l'haorti.tstlt'es/i't.s ltttl. l'}h1'topathol. l4 l-il-
ile
Journal of Ecotoxicology & Environmental Monitoring' Vol 11(2001)