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&sdg*enybo&eg/llo,Jl
*!rct$bd*4ryd-b*
".. ... . i
MUTAGEI{IG STRAIN DEVELOPIE T OF
AEPERATLLT'S N'OER TO ENIIAI{CE
PECTI ASES PRODUCTIO POIE 'IAL
tbdul EaDrBll
INSTITUTEUOF MYCOLOGYT PLANT PATgoLoGY'
LAIIORE' PAKISTAI{
MUTAGEMC STRAIN DEVELOPMENT OF
ASPERGILLAS NIGER TO ENIIANCE PECTINASESPRODUCTION POTENTIAL
By
Abdu1 Eattla!
a T||Esxr lurumrD 30t TnE,lJLruL lmot Dtcrll ot
Doctor of PhilocoPhY
Mycology & Plant Pltholoay
supe''lsor
Prof. Dr. Rukba.aa BlJrtProlessor & Director
I dtut€ ofMycology & Plrnt P.tholog/,Untverslty of thc Purjab,
L.iore. Prl<!
IMPP
Augult 20{E
DEDICATIONTo mv Father
SABIR ,{,L[And mvWife
HAIYINA HANNAN
CERTIrICATE
$.t tno F!.eh m* th.
ol Aqug tu ,84 ro
thir lbdi! bY Mr' Atdd
Fsh wdt dtitLd lMrb$nlc
.trh.n@ Pcdds Prurhcdon
n@4 i. & odgi.tl s* of lh'
Nrho. 'nd
I's bd csicd od udd Dv diF t i4avitio l t'rc F ltry8o*
thre!8h all the dltdt4utl!/natqids rponed i! tlF nunwitl Dd cqnfy 6'it
correildddlhdtrcitv l flt0Er cei!ry th't $c mtlrial irclud'd i! $i! t[6it hlvc nol
hd u.d i. . pdl m tuU b . MrdriDl ttEdv 'ubDi6t<l
d ir th' pN.s! or
su!mi$io!. I .l$ qdt th. th6t ts td FlFd uda nv '4qsio!
ading !o
O. prerib.d fodi dd I. o!' its dtludd ror th' !w'rd ot Ph'D d!sr& t!o!$
6e otrci.l Poc.drcr of $. UtivditY
Di,
MlEology & Pl.nt ?adDlogy
D, tqlq,f
DESANATION
L .$el Ed' r.Shd lb ts+r' HY d'&G |L t' e
!!ldd h 6t |BL dd.d ' 4.a.61c Str'h D'vd'9dl ot 'rtttt{b 'trt r'
*r.d3rr.|Hhd LryomtdinbD't-'oLdn@dyt dt,trrth .t' alditeb 4e@
M**
CONTENTS
Acbowledg@edl -----_-'-Sqnmary -.-----'-------Lisl of Abbtviation -_---r. INTRODUCTION
l l.5 Wd. w|d tFatrat ---- -__-19
iii
II,I BIOIECqNOLOGICAL AIPLICATIONS OF MICROBIAL PECTINASES IE
l l.l Fruitrui..ErFaclron---__"'_--"---------------_---- 13
1.1.2 TcxtiL p@d$rs dd biosuinS ofcoro! 6b's ---'-----'_ l9
Ll,3 D.gl)ming of plart bsl 6bd '--.----_--- 19
1.1.4 R.nitrgofPlmrfibq-- 19
1.1.6 Cof.. and led fed.nlation ----------_-----_'---_---- 19
1.1.7 ?ap€l dd pulp industry " ""-""''--"-""""""'----'-- 20
1.1.8 ArnMl tird..-- -'-- 20
f.1.9 Pdificltionofpfttrlviru!.3- 20
l.l.10 oil dniction ---
-
20
l,LII Improv.mcntof Chmmticityedst'bilityof €dwird_---- 20
2. MATERIALS AND METIIODS
rsor"{TloN OF ORGANTSM ---- 222.1
2.2 PREPAMTION OF SUBSTRATE
22-1 PEp@tion of lmculurtsloc sup.nsion _------ 2l
2.3 PRELIMINARY SCREENINO OF,{' 'V,IC'X STRANS BY PLATE
METHOD ---.'--------_----2,4 QUANTITATIVE METHOD FOR SCREENNC ---_*'-
2,4. I v.gctdivc lMdm PEp8dion
24.2 Suln .gcrl Fm.trllio
24
24
24
24
25
25
25
2.a.1 Adlyticol Merho& --_2,a.l.l Polwd(ruMeturry.'
2...r.r.r sdiid cltu ofD6.16!s o&id------_-2.41.2 PatdYeAsY...,'=-----2.4,1.1 P.ctindte@ AsY
-
2.5 MINIMAL INHIBITORY CONCENTRATION OF BBNOMYL '--'-..' 26
2,6 CULTURE MPRO!'EMENT
---'
26
2.6,1 Ultoviolcl id.diarion -2.6.2 Ch@icd Mutag66is --
2,6 2 1 N_mlhvl_N_nibN_ni|@Smidirc (MNNG) stur --2,6.2.2 Ethvl nFlhvl $lphort tr'tlMt (EMS) _---"-.--------_
2.6.3 S.letion of Mut!trts --.-2-? OPTIMIZATION O! CONDTnONS ------------_--
2.?.1 fddr.don Pqiod --2,?.2 OPdn.l PH ---
_
2.?.3 Ir@ulMSi4----_-
26
26
26
21
21
2A
xa
2a
28
2A
29
29
30
2.?.5 Tw€4_E0----------2.8 MOLECUIAR STUDY --.
2.E.1 Funed Srmpld ------2.8.2 lsolariororcsonic DNA --------"-------_----------
2.Sl.l EsDdd of DNA Ct)|g!.tir2.s 2-2 Qualitv of DNA --_-----------_--_-- 30
2.8.3 Conditiotr Optimilrliot for RAPD edvs --_----_----_'--- 30
2 83 r R-A!D (PCR) !*1im
----
3l
2.8,4 As@&gelel4troPhorc$s----_-"--_-__------'----"-_---- 3r
2.8.t DabAn.rFE -'.'.'.-'.'-.-...........'-...._'-..------- 32
2.9 PROTEIN ISOLATION -- ------- 3l
2.9,1 P@ipitllion ofPbt€b -
2-9,2 Walhi.g of DidYlit M€nbmc 33
2,9.3 [email protected]'----------- 33
2.9,4 Polvervldidc G.l El@lFphoeis -----------_-'-- 33
2.IOT,\RGE SCALE PECTINASES PRODUCTION
---,.-- 34
2.lo.l ?'to.6tion of In@ulus/SPor. SldFdion --..-----_------ 34
2.lo-2 F.mat [email protected](solidsulslr.L) .--- r4
2.10,3 Hd4tinSofp4tillet'-'_-'---'-------_----_---_-- !l
3. NESULTS
3,I ISOLATION ANDSCREENINCOFPEqIINOLYTIC/I,'PtrXC1I'U'NIOER ----_--_--_
]I PECTINASES PRODUCTION IN SUBMERCED FERMENTATION --r:.1 PolygdrbMac Adnb ----------*------------lrl P€inbedivity--
36
33
38
50
52
54
51
6l
65
65
1a
8l
89
102
].3 CULTIJRE IMPROVEMENT _t3.t uln iokr Intdidim
---rJ 2 N4$rlN{ih-N{ih$gumidiF tMNNGI
-----r.rj Eln) | Mefiyl sulpho@ iEMS) TElltMt -------_--t.4 SELECTloN OF MUTANTS -_--.-._-_-----.---.r_5 oPTrMlzAt roN oF ( oNDlTIoNs ---------------
1.5 r ElEcl of F.ftndjon Pcrin @ Erzlm AcLirity ------rJ-2 ElEct of DH o Enzyo. Acriviti.3
--rjl Efhd ot lnd'nm sia d Eizyre acrivili6
rJ.. Eftcr or Y.{s Err&r d Edrre ac.iviri6
.}.8 LARCE SCALE PECIINASES PRODUCIION
3. IISCUSSION-
r.5 5 EfftdotT@30dt zyn. A.rivitis
------J.6 MOLECULAJT STUDY
-3.6.r D.ndresms wilh singlc linklgp od coGl.rion @6cicnt ---l3l Unw.ished Plir Crcu9 M.6od Atithm€tic (UPGMA)
----1,7 PROTEIN |SOLATTON ---__---
4. REFERENCES.*--5. APPENDIX-_-__
!IST OF FIGURES
Activity of PGs. !i.r 24 ho6 bv 23 slrals of'{ '/gtt -'----
39
Aclivig of PCde oner 43 hom by 23 strsire of "t8!f
-------' 39
Aciivity of PCs. aod ?2 hour bv 23 strtis of '4 '18!r ----- a0
Acwily ofPcle.sna96 hodbv 23 3!2i!s of'r' '!:9t'-- a0
Aclivity ofPt e silr24 ho6bv23 sFliE of'l tiStt- a2
Acivhy of ltlse aftcr 48 hou6 bv 23 sntins of ,l t'8!r --'_--- 42
Activity of PLe.nd72 nosby 23 straisof''l tlFr -------' 43
AcrivityofPlr$ aner96 hoEbv 213lllic of',t dAtr-__---- 41
Activity ofPEe !i4 24 hos tt 23 stnis of/"/8tt -- a5
AclivityofPE& snd48 hodbv 21sElirs of'l 'iatt- 45
Acrivity of PEe and ?2 ho6 by 23 tE its of '4 ti8tt ---- a6
Activig of PE6e lnd 96 how 5v 23 sttsi^s of A alSer '------- a6
swival c!* anduv traillmr --*---''---_-' \r
Swirtl c!ft and MNNO ti.ltncnt
----
5l
Sdiwl .e! sid EMs tratMl sl
Conporison for potvsal&turon c U/ml'nid ------'------ s3
conpdienof?ecdnlt@---'_--'--__'--_--_---'-_'----_-'_-" 5l
Conpuistr oI P@iin6c@ - --------'*'- 5t
Acrivity of 9C@ ar {ryirg fcEcnlldoa pedod! ---
J6
Activity of PLe at varyine f.trmilti@ Fiods -...--_-.- J6
Acrivig of PEse ar va.vinS fdrenrstion periods --_-_---- 56
Activity of PGNe at difr@nt pH --------"---_-"---__-_-----_ 53
Acnliy of PL@ al ditfeEnl pH _----------'--'--_----- s8
Acriviry ofPE@ at diftc@r PH J8
Aclivity of PG@ at varvinS ino.ull,n stt 60
A.dvny of PLse at vtryins inoculM siE ------'--'------ 60
Actrviiy ofPEse !1 v.rying inoulun size ----'----------- 50
marof Y..s,tcxtn t on POe dctivitv
---'-'-----
62
EfIet olYon dEfl on PLe divily -----ElId of Y.&( dtra.t on PEs a'ijvitv -_..----
Effd of rwfl8O otr Pc* adiv'tv ---------62
Fit l0b:
Fisloc:
Fig I l:Fig l la:
lig l2^l
Iig ll:Iig lla:Fis 14:
FislS:
lig l5al
Fig l6i
lig l6ar
lig l7i
Fia l8:
Fi8l8rli8l9lFig l9e
Fis 20;
Fis2ll
FiBZ2sl
FiE22bl
FiA22.l
Efi.ct of Twecn8o on Plrs. a.tivilv
--_---.----58
Hfed ofTw.enEo on PEdc eliviiy _
RAID pmdEts by Primd OF€6 ----
Dddrcsm aftd pdm6 OPCF
DddrcsrM ARer pnb{ O?C. -RAPD pNduds by prina OPC
RAPD pDdNl' by pdms OPC
Dsdogm aRspnbd OPCj
-
RAPD pmducb by pnmer OPC
De|dsgre aicr pn6q OPC'.
RAPD pFduds by ptraq OPC
Da&rstu Encr primd OPCI
RAPD pEducb by pnm€r OPC
DadDgro .R.r priEd OPCr
RAPD prcducE by prin{ OPC
D6droglu a,Lr prin.r OPCB
R PD F.d@ by prind OIC
D6dt0e@ 8nd priffi OPCtr
RIPD pridkts by pnhd OPCI
Dcndegm attd primft OPCI
Ovdll simileira dona rh. th@ wild &d th.ir nuhDrs -----SDS-PACEpFtein p{cm
-ttBe *ale divily of PCsc in SSF --_-_------tsgc sale rtivity of PLsse f SSF ---.-------'--.---L.r8c Ed. &livity of PPs in SSF
---
6E
69
69
70
70
7l
71
12
72
7X
15
1a
76
76
?8
80
80
80
list of,
--l--r=r-
table-E=-=lr
T.lttc l: D.n ts of dd Gldis z@bv A nbqist,,6
list of--Er
PI,ATES
PLic lD:
P@rin dceFdaio b.f@ 3oiti!8
Pedn deer.d.tion .Rd 3taiong
C.ral dituiod for..ti&diotr oftdery gd '_---PFpdadon of @idid suttdsioa of '4 "latr ---------'Pdir d.8t ddid by hiSh P.diEt6 FodlE3 h'foE itit!i8-P6th d.grlddio! by hiSh Peli
4nennnwunnnnmmPFiBr of au I wodd lik. to p6ar nv hMble grditud' to AILAIi SUBHANA
TAALA who cret d @ s@lv lor t caue, and bl'sed ft wfth Ptdon ro *k 8!id@€ bv
foloeing rh. liac ot Preprd Muhtdn d (PBU10, wto B tht uu' lisht ofou liv6
A b.0udfd q@lalio. bv unktoM 'A rachd s Pulpdt is 'or
!o ctat' lhr<ldc n
his oM ime., bul to {tevclop clud@ls who od cEait th!i' oM imgC' b &luuv ! ts!'
denprid about P.ot D. tukrws B!ju, mt r4'fl$r lrd dnEior' Insrit e or
MrroloCy & Plsl Plthologv, Utrimitv ol lh' Pujlb t'!oc wnrr t ford rholt ha i3
$d 6he lov6 to do 6edh .nd !h' al$ odts i! ne bch'viou of kdn B@hd l
l.m.d abt n mhd. \vhddq she fourd thll I 'tu
not PivitS ltcntion ro mv wo* $e Cot
egry, ed {h6 I did $@rhing aood lhc 4'ua€d Ec l qd nad foracl hd guiddcc'
c@p€atio|! *itdnd d{t @ft@ I pav nv lpci'l lhEls ro hd for tll th' lood lhii$ 'bc
hs done to no lhreughour nY B@n
My spditl gdtitdc !o Dr Z.ldt brrf fd hd gl!'!'&c in qtdilEntdloi
I {ould like to th&k tll lh' tachd!" EPeiallv Dr' Gh!z!l' NGit4 Dt Sdik NNU
Kbd, Dr Shalil A!tu<l Dr. Asltad Javaid ud Mr' N'd'd Str'l a I l@t nwn morc
Iion lhd ihbuehout th. €@h
My gst6l 8ndtud. lo mv tolhd w' Slbir Ali rd Molhs brth@ dtd 3G6 EtD
weF dwsys been a geat suppon 0mughout mv life n'ir suppod ed ocoDditiodl lov' i3
I wdd !l$ likc to lnsk lo mv 6idds J"6' Mrlilg Ullah "d AI wno t$tv!
Now,l f41a gql honou to &hbwl'dg' Twis dd Sci@@ b'hu s wh6'vd I
call.d thcn lor h.b tncv n*d dielpoinLd @ dd alwlvt hclp'd e
I n qdrlt ddb.d.tLd.! 6! Edr Brdd Codtr of
r|5tu0lBo i. t&3 e. tt tdd q.t. ofry do'tor&'
Sllly,.! ttirMd of .dd!!,@. t '5hbbryaiDtbn'd'd !.fr.. tuila ry Edtd dn 6'i' tntrlsridA ivd rodL r'lM
-ffitf-
LIST OF ABREVIATIONS
-aeE----=l-EE
I
tc
Pl
tMF!4L
C.Ch
C!@t
Dr
EC
HCL
KCL
tDr
M
MtsolN.OH
MiqwoL F dituic F.litrcMimolc !.r millilit!
Mich@lisMcnla cotlt&t
PACB
pH
scP
sDs
TAS
uhtUlt'lL
U/ntvL
TJK
USA
p
!
H50n
I|2tnBl2nCF
CPP
Poly Fytdi& !.1 deftPhortsi!
H$olFa iod cod€lr.iion
Sin8|e @ll PIotdn
Sodi'D dodilt dPM
Th.dootili. &tirc sdri!
U.it! Foilii@
UDirt F nillilil.f Pq ntiNb
UnitdKiry{oIn
t rrt dsataoflEio
Mutd ofdrhlll2
vl
SUMMARY
The prcsent snrdies descnbc thc isolation, screening, scleclion and ilnplov€Inent of
Aspeqi B niget stains fot qhicement ofPectinascs poduction Abollloffifrv
fito s.lllias of Aspergilt/s nige, w€re ielated ftom diffcrcnt habilsls 6d sit6 of
Pakisrsr. A multislcP scMing of all srains -4 'ig€l w6 done on the bash of
p€cb.s.s producnon. PlaE tncthod vas 4ploltd for prelimiu'rv scte 4 of
isolal.s for cvalultion of thch natuml Potential of Pcclinscs production The
stEiD! w@ cltcSorizcd s hi8h, moddtc lnd low Fctin's€s prcdrces The hiSh
ud modenl. plodu@6 w.rc *ledcd for 6nal sc@in8 by using optimiz'd
mzymc 4say mc6ods. Aner th€ final scrcening of 23 s$ins, thG strains' H12
inilicatcd the bBl cnzyDatic activig of polvgalactrronase i e 0 ?86 uidlnl-min'
It27 indicat d th. best enzFnatic activitv of pectinlvB€ i e 163 unittml_min' and
H50 show€d mdimum ozvmc activitv of pectireslcre i e 14 5 unil!/ml-min
Th6c lhde sLlins wed sub.jccted to mltatio6 bv chcmi€l ed UV imdilti@'
Aner tho nutltion those which snow.d imPdvcd Pectinasd Prcdlclto! w@
sel@tcd. The mutant of stsin Hl2 dlibi&d tMilnum acnvitv of
polyg.l&$6ns. viz. l.4E U/ml_mitr sffo dr qPo$E of 50 min und€' UV lalnp
wlilc it5 plMt showed 0 786 U/nl-nin And th' rcPcared trclrrndt! of sFsin
H2? mycelia with MNNC for 40 min, iB $uLnt show€d 29 U/nl_min of
P.ctinl)$e d colnpaEd wilh Par€nt which gav' 160 U/nl_mitr Thc a'lcclcd
mutnnt of stmin H50 by EMS crhibilcd 27.6 U/ml'nin of P'ctin'stemse while its
wild gave 14.4 U/ml_min After nulli-step scr@ning ed muttlions' thc cnzvm€
production wa! optimized bv sludving the etrect of vdvhg fmotation P€nod'
DH, iNcululn size, )tast cxtact.nl twe€n8o in subm€rged fcrnenldion The
fdimud &riviti* of Polygalacturcnase' P.ctinlvase dd pcctir6te6c *erc
rccordeit ,iier 96 ho6 of f€mdtltion. The optimal initial pH fd svnth6is of
enzymes ws 6.0 for nutants s well .s for sild strais' Th€ muimun activilv of
all sr.is ws rc@lated i, culturc filLats oblaincd ft'm submdged femcnlqtion
medium inocuhted with 6 ml vegcbtive iloculum ln oplimiation studias
mdimun aclivity of Pollgahcturonasc .td pc'ti!ly!'! wat notd at 04%
addition of )tsst dltact whil€ mdimum activitv of p€'tir6i'rale ws 'xhibil€d
at 0.6010 of sid suppl.in trt. Addltion of tween8o €nhar@d enzyttt activity up to
O,ln/o. I .
Inc. optiniatio! of dzymdic conditions, tunh.. studiG ed! c{rricd out to
.valoaic mutal.d s w.ll as vild st"ios d the bBis of tbcn gcnctic divc6itv
DNA fingdpriDting d,.t cluci&tes th. varjalioru anong $e Par€nt and lheir
nutlnrs. Less Sen.tic divasity Mong tlle Pstentll strains ot AsPe,gilhs niget
and morc g.nctic diveFity among ib nutint w!5 noticed. Th. Eslts ofUPCMA
indicares thlt similarity lcvels amonS all the slEi.s w€rc mging from 86.3-?0 E%.
Thc dzyme punficttid Ev.slcd potein of a Inoldld siz. 78 KDa @vm..
Thc eiectrophorctic paftm obtatncd nom didyz.d azyme cxtr&ts exhibibd !o
diflcMc. among lhc mutlt d stlins. lar8e scal. cnzym€ Prcduction by mulanis
of ,,{. r49e' w6 studi€d in solid stlte fmentation. The enzFe lctivilies s@
signili@ntly higlt as compared lo submdg€d f.incntdim Thc naxinal activiti.s
of PGas., Plase .nd PElse w@ [email protected] aftet 96 hqB of incubslion viz. 3.55,
35.2 ad 32.4 U&rl-dn by Hl2d, H2?n ed H50m r€s?ccdvcly.
AI fie d!t, v.rc an lyz.d by M.l,sis ofvarialc. followcd by Dutrcs,s Multipl€
R l|g€ Tcat using conputer soiiwarc COSTATE.
T!. prs.nt study concludee thst among all the i$lald, lhc st'aids isolobd non
.pple dd cirt8 hdvc morc pordrisl ofp€ctin.s6 production. Th. mblr $rain
d.v.lo!.d tlrcud W md chmicrl mulagc$ (MNNC and EMS) dhdc.d ihc
pmduction of [email protected], I'9. scde p@tiE* prcducti@ [email protected] thlt dutaDtr
cxldbitcd mor. thcn 4fold inctls ofpol'€.1&turcnsc, 2-fold ofp.ctinlye.6d3-fold of p.iiin6tcru.. ar rY
Chaptet-1
INTRODUCTION
BMyn6 s calalysl3. th.y m.l€ lhing! to work f&tcr All ltt liung org&ism tidu@
..2yn6 or bi@lldysb vbich aE b$ic nuirimts sd caeti'l sut'tdc* for divkion
gowrh ed taforo.e of thcir nomd enviti6 in living c'lL &d tktus'
Mo oft[Q.EvF.r e pDtci! in mtw Pbtein sElion k ! vit'l prcc6s in dl tungi
in ludrq y.4t drl lilddtou tungi. Sdra.d prcLis @ div.F i! 1b'ir tutctiotr
Thcy itulud. @ynB wbich plav tcv tuI. in nuttitron' ln natuc' tnicoo.8rlis have
b.d @dowcd wi$ valt potdtia!. Th.v pDduce d .dv of @ym6' which h'w b6o
€xploit d comdidly ovd llt '€d
P4dn66 ft of Srtl siglilic@ with
bdmdos pot .ti.l to oIId !o indlsEv (Datddd &d Palil 2003) Thcv e @' oflh'
lpconing .@Fe of thc omdial s@lor, spdiallv lhc jui@ ed f@d indulry
{Kuhyzp at at, 2ool) dd in p.p* and pulp i.d$ltv (B6e ., 4r' 2001 ed viikdi tr zt
200D
Pati|@r E a gNp of cMyne Pbduc.d bv v{i.tv of nicoorglnis6 $ar @blvz'
UE bEakdoM glt@sidic bonds ofthe long clBiG ofgd&turcnic a.id Gidls ofp€tic
subst&6. wltich e tttc nnctMl polts&chdid4 of plst cellt Mljor P€iiMB ePolygalactu@c, P61inltB., tnd Pedn 6t€@ @ecagdio a aI. 2002)
Thc bsi. stl@tw ol D..tin is @ l-4li*.d Sd4nsni. &rd eitl uplo 95% of i!3
cdtbxyl greuF 6tdifi.d wiih nclhdol. P4tis @ Peht s ltructural
pdys&qisd$ in thc niddl. lan llt ed tn6 pnn.ry 6ll w.lk of hr8!6r Pldtl Thde pan of oB di.l .ithd s additiv6 in $n fod prducis or a @Gdru.ds of Ew
natfiials !!.{t in thc f@d Dtod@ts Thei. phFic@hdicll pop.ni6 wnhin rhe c.ll wdl
mtrtu or.ftd qr&lioD, e thercfoE ofprib. inpottae both Aon ! fucliodl &d a
nut ilioml toinl ol vicw. Indukial p4lins @ crtmcl.d tom by-podu.ls of thc lililjuie industy such s ,9plc pon@ d ciE!! p€k Thev e qt .1cd i! -idicconditions dd ch@ically modified kt givc high M.thoxv (HM) p6tirs aid low
Mcthoxy (LM) P.ctins (smc my sls be midaitd) Th' pht5io'h€micat poP'ni6 of
rhe two Min typ€ ofD€lins, 4 scu 6 th.t Cclhg Prepcnics" <tlfd widclv Th' HM
p.cri6 fm 8eb h &idio @nditions dd h the pBence of $gc whcna! LM p@lis
ne.d dlciu for gelation. The.tpliqtion lras of th* gct5 F Ninlv ir th' food
indusiticd j@. nm.hd$ md jGlliq (l€d'Fe@'!' 2001)'
Citrus indudriat to.fm.tion giv6 rtuc ro bsid6 th. m.in Prcducls jui6 ttrd
.sstid oils. sclrl estc! *!ot dicpdd oaug Dov prcbl@ Tlc Pmblm 6kirg
6om wste odlp pc.k drtp6d e rh. b6r 5n di.d be!@ of lhc CG'l v'elv
D6dwtio which hs l.d to difrlMt but not qlstiva pbpotd! for di3pcak by singL
c€rr por.in (scP) pDdEuotr ([email protected] aL, $a9 d caL a at 1992)'
Pcdt|ls art clsif.d ..cordi!8 to lhcir Poirt of .tt .t on P@tin nol*ulc Th' m'thyl
6ter i! spliled by P4lincst @ ard thc Elttosdic boids of p.ctin chdn s 3Plitcd bv
hldFlys,s wih cndopolygala.tuD@, or ooPoltrd&thn4d Alolhd m'0tod or
splifting is nur.limiMtion by m*! of bst rial odop@t t.lr's. d ftDgtl 'xadd
?..tinolltic dtsd @ .le poduccd bv pllnt patlogdic tudg ed brcldia, md ctu'
laf 3pol, son ret ,!.t wi[. On th. olha be4 rbcv e ll!'tul ild!'lrid 6ryn6 fo!
at@tiol' cldifidtion ed liqu.fetion of frlil jui@3 Ih.v e ds 6ed in fabri'
irdusry 1o .ct plel 6b.6 $ch s fld, lct|P lad jutc sd in th' pald itling induthv io
solv. rct€niion Dohlm in E@hdiol pulP! Gohru, 2001)
r@ritlls @ bc Pr.dNcd alh6 by $bndeld (ASrild tld lruitio4 1987 ed Aguilt(
e,at, l9l)oreUdstlt (A@unA ./ aL. 1994, ed 1995, RousN '/
dl,l99l' SiGs'@
an.l S.d 19s9, S.lit .r al, | 93 Dd 196) l@6l!d@ SubnaS'd fmdirtion (SnF)
sy6tctu havc b*n u.d dt€$ivclv for lhc srudv of svltldi' of mimbial n'tabolitq
and for lhe roduction of higtupric.d mlcri.ls (Beios t' af, 1990F!d Soltu et ar'
1993). Solid-sl.te fm.ntation (SSF) hs h*n w.d for th. Prcducrion of Pordd
(Mitchcn d ar, l98S) @yG (BattaCtu ?, dl. l99l ard Ntuio a zl 1979) sd
chmol ((s8i ed Cmq 1985). Snrdid @mning Ggulrbrv '!Ft5 of peiil],g
po<leti@ by nold! [N. t@ cdi.d oul i! SniF .y'ld. Thc dvn6 @ inducd bv
D6th or by $mc ofib ddivlliv€ (Maldontdo.t 41 1989 $d Pdlcv tnd P!€! l97l)
bul pF<tuii@ q be controllqd by cltalolic rcpBior Go$.tv dd Kellv'19E3' Ia!'
sd Vd l9E6 dld Msldontdo et dl 1989).
Th. oolDolylic 4jmd hlw be.i u.<l in ollid pres eh@ 'limiEnon
ofp6ti!
is 6sdli.l s in win. nalin& @ffe ed ta pr'cctling pLnts t'd n..drli@ of
vcgelab!. tisu6, Othd lppliqlions includc r.movrl ofcitus &uit P'cls tiil irct!'sing
ilc 6]ms of eo. filits (Mrrtis ?l dl, 2002) R*6t tpplicalioN he sndScd rsrr
s lhe trqllndt ed dcgu@in8 of Et'rdl fibG (8ltldnD a al' 2001)'
lor irdultrid ue, p€ri@ cs b. prcde.d cing s6l 'gnculnr't
patin @ntlinirg
{.nd $ch s .ppl. po@, .mbqrv sd .Ettlarv !oo!'c' H Pdp' 6116 t'iP'
@coa. lmon pulp but lh. Dain !ol@ m.int citlu! P"ls (B!rcvic &d D.Mci|L 1993;
C6tilho a al, 1999i G.rd ad Hos' 1992).
'Ite abilily to 3y h.tiz. rh€ pcclin$d b wid. 3pE!d tmong alt o micmbial srorps but
tuala'(..t kPqailt8,icq M.hod.tu hadifuud &tizals) trc PFfdr'd b'cN
s nuci s 94,4 of tnG aE]'n 6 b. dcr.tcd ilb cdtuE mcdi||n @lridim tr dr'
2on\. Atperyi\s nteq it ^
tungus that ce b' 810$1| ddv wh@ on ch'sp n'drd
Tb*foq p6lill,s podlcld ton tbis nDtB @ t' ofgrar iDPotiatu'
kpdgllE ,igq ii a fltsndtos 4@mvc€ie nsgu rhat is ubiquitout itr tlc
ovitumol. -r- tigt is mosi eidclv lmn fd it! bl' 6 ! ciric &id ed indrebal
ozym.r ptu rucq (M!grNn 4d I's\.,2c{/). 'lsPcr8tll6 "i8* ir I @6non
'!@Mor$c micdbial comuitid foud b $it Ttrs orSrnim i5 t &il saprebc vii! wid'
@y of hydrelytic dd oridttive aEldd involttd i' baldom of dalr
poly!&chri.le!. A vdiety of th6c @zvn6 fom ,{ ttgT t$ inponot i! biotdhmlory
dd irdstry. ,rry?43/li e u5.d bv indu.lry for rh' pmducti@ of pdnuv m'Lbolitd
dd dzym6 (orsani. &id5 viumiB, fttlv eiL td mi@ didd Mrinlv ts 39€id 'lniset Nn a. oDaa., fJr.il s dtn. poducN lotdboll, l94r qtblc '{ lgt i!
ruinly qptoilcd ir thc aE of food (b*ctv, starh indulttv) &d b'vde6 (wi!€ ed
juic., bwdy dndlinA indurr). ,{ d44. i. apli.d h f'ddllrid of oddLl $tct'reh e .oy! s.. .nd wis (a,doo et 4t t994J
Tutob.tlo dtd Miu (l%I) sludi.d rlrc .fier or suqlc' d ln6 h}nelFis of D'ri' tld
found thrl 0E n.dium @nbining swlos. s $€ !ot' csbon sourcc has ladocv lo
uod@ lidt d hldrcbsis wDiL lh. h.di@ dt itiDg Peth sbos @mpld'
hydrol,sis. Tlt u. of nixte of !ucr$. ht! 'fi'ct
of ditlcrcnt cubohvd6lc other lhd
.ucl@ t ! $dc ditl.M4 in sha!. fl6k but thlv vitusllv ditaPPcgr'd in tnnlxt
fcrm.nr.B.It w.!.!o r@n d $.1fi.rlldidon of lu€Fn' Po*dd b n'dit itd!{!'d
dlc }rcld of P..ric oEvnd ftln Pdcttltur nofo,nd ud tinils cfi"t w$ obt'iv'd in
Childlal. (1981) Slulti.d l!r8. $.lc Pr.du'tior of p'ciolvlio @FB bv did nttc
f.mdt tion, Studi* |wdcd Url tnc prcdrctron of arzvo' bv kP"a lB d'tutdta
CFTN 1048 on whd bru n.rtilm ws rdimun 'r
2l h of fdmor'rio' 'l 3C C *ilh
o etivity of lO49 U/cm.
Mddd.do .t ar, (1936) Pode d pad'apbr *pq N W urn8 vuou ptsrc'Ld
ldo. p€l D thc cltd &!@ it|lrad of Peln & s found thn tb6 pidutdd of
pdySrlr.iMe w do!| th. .d! .!d U'! of Fti!'dru sbdsli'lv 6i$6
*h6 os.!h.d lish l.llon Fl *s u!.d in5t"d ofPeti!'
rlios ,t .r. (t9S9) inv.diS{ed adogolvS'btnsore podu'rioa bv AtPadnB tP'
CH-Y-lO4l sing ut!.lcd lqnon P@l u th' olc crton toute Rc$ll3 lhosld lnr| lh'
&rivity of ddoFlyg.l&i'Mt c a$ moE '!
lqc tho lbll 2tC Ufiaicd ld@ po'l
Dd.d io bc I bdra c.tbou oue lhs citu! Pelin fd '!dotol'8dr'ttro!4
ptrdsdon. A lo fold i|tlae in dxtoDoll4drruo|@ *s obw'd tv lFdiryi!8 thc
ncdiu ed der..rilg th. ncdid PH to 2 8
A.iby llld Pds\ (l9o) dcv.loP.d . pc!$ for th' PtldEtion of polyt'lrciMl|c bv
,,1. ,ig./ VTI-D-7050 .xl i!. nu[Dl VTI-DE6267 Hith lcvcl of Podrcdo of lhit
.nzyrc ws oboin d in labodory f.tna |rrG on bG'1 dli't {$tt m'drlln' 'Il!' b'd
*rsL *s liomulrt d b ! oncqfilrion of3oty'n'&d attdod b n'diw @nnhint
otb6lutiiord. CuttGwsFfom.d !tlctw'cnPH46ud 5O*ilh|git'tidof 200
an ed &rstion of l0 vhin Pilor glst 3tudi* w@ a&o tdfoF d iD 200 I fahmta
viih e lddion Bt of?t ytnin but the tnrdi6 thowcd vtry lw i'ld'
P6cin at ol, (l9l) EPon d rh|l" vna Phonopss h'lianth4 it gom b subndg'd
clltw, ii cxcr.14 4te.llulr petine. ?oduction of @tltitutiv' dopcclile by t!i'
tunrl! gFM 6 d@4 ws dtlblish'd, whilc lht ddoP*tins ed Peti! dtd'r'
&rivili* @!tl bc d.ldtcd onlv in th. pF64 of tdiin in th! n'diun Tdpdnls
dtihun of endo lttd do r@dn66 w.rc obtu'd !i SOPC' pE dc9'odd@ or c dc
dr.&clllld ddepeline etivity show'd two o&dM al PH 4 J ad 5 ? Mdirnm
etivity for qGp4ti!$e ws obFded d PE5'5 (!t 5CpC) $d for p'clin Gsl@t d pH
5.0 (20'c).
stutzdbdgq, (1992) Eporld lhct pdti!.$3 !4oul! for lo % of thc global indudriar
@ym.s pFducql,Hqevd, in lpile of lhc obvios lttvdtlges in usirB @Fca for
[email protected]| pd6iry of foods' @mdic fdo6 tub s sraDilitv of ih' cnzvme
m th. n.in dddnts in th.t s. stabL .nzF€s n 'dvsttgou
for p@6''8 ro bt
@st cll*tiv. dd for bcing .4wlabl..
M,!d@no d al, (193) u.d ! ttrin of '4Pt4il6 ttlqa fot lh' s'd[6it of
p@tine' Dding lh. prcdmtion of th€ €EF n'thel es libmt'd inb tn' n'diufr
du. ro cls{8c of pcrir hol*ul. {hich i! rh' crbon &w' lt w! tlp'ncd thtl
n thmol is @cm.d bv ttc o€oisn b$ ntilhd thc stitt6is nor thc etivity of
Ddlitr4td@ dd polygat&tu6n@ ws alT'cl{t by EcthNl ll !i! pbpo6'd thrt th'
n4hdis inrclv.d in l!. utili4lid of ncthatul is sinild to thd fot odltylotlphic
w6s,t al, (193) snrdi.d pdtitrs6 ed scLDtim preduchon bv '{ 14tu kobt€d
tls foods in a p*ti|l,s dltw plde.ssy ed cz 3 % N'NOj mldim plalc lgv
Our of 82 i$hr.s, tt% prcds.<t breM'bl&k $ldlia th' toEseoic tFlils showing 'tcndcncy to hlvc sall€r scla.li& No stnint pbduc€d bo$ alunddl smtll scl@tit dd
a lrg. q@tiiy of .Odori!. Tn@ *!5 m lil6 coGlrrion bct*d tbe dount of
!flatoxio Drdu..d od rh. trubd of ol@da t'v'b of petil@ PDdrcld by tbc
toxig.nic sFdm w€t€ hids ths lhop of the lon'loxig'nic atrais' sd ili! wd more
ohvioG in elmlium_pFducinS tltliG lhd in the 'on_el@riM-pbducing
sfiiB Ir is
su$ested ttrl in this -4 14ru populdion (*ith t low toiieloic atnin ratc) toiiSdc
shaiM hay Equirc hi8!d lacls of p€dinse lo mp'tc wirh thc non-toxiSenic lf3jns
{h@ id-l.d food5turi
Bch.r. e,al, (1993) tuolat d | sElinof-,,iga lDD onion trdprcdw'd Lrg'Mout
of polygak tNne Sodiuh lolrlet lG ws u'cd s main tubsb'le in dcdiM in
.ddirion to diol! tlt dd 8l@s Two .rotolvgal&r@e fo@ (robralel@ I
ed polygaduond. ID od onc ddopolvgdlctubn4' fom (poltsala'tuF@ IID
w@ premt in the n diun. Atl ttt e PoltAda'tuh@ foms @ oplicallv !'tiv€ at pH
5.0 dd pEfcn d edi@ p..t!rc $ a $bstr lc ovq p@tin
Solis-P@i6 ., u t. (193) studicd the pD<luc$on ofPdtinr*s (cndGP &d a@Pp) bv "l
r,ger CIL st 350C Mlh inidot pn 4 5 (a) duing subods'd cu[@ in shdc fldks on
!t6li4cd liquid medrm btt d on 3% perin wilh or wifiout 3'l5 su6'!' duc@ d
sds.tNnic !.id ed O) d!d!8 elid sr't' 00q' misl@) qrlte on nailiz'd r'dim
iictldins ?7% (on DM) $8Nd€ bag$!' 6 $pPon b&thd wi$ 3 5' 5'5 or 10'6
p@tD or 5.5ol. Pdtin +3.5, ? or lO% scrosc' 8lM* or gtl&tlMic @id' in 150 nl
st r a4t d d- 40o bl^ F I DM Thc 6ulrs sto*'d tul (s) &livitid of qo I ttd
prniculdly 4do P *d. hoth d@ts'd bv ll|a Pr6.M of dud* d 8lq@' i O)
acivitd of both €ndo P sd dep incrc'lcd with in@M in irutid concoFaton or
in6e ca6o. s@.. For O) in @npdi$n wilh ('): 6dl &liviri6 wd 3x hishd ro'
dldcp€lill.sDd llx hiShd fd dt p6linc'5' oEdl pllduclwitv (U/h F g d dl)
po$ible es lE 8 x tigha for ado P tnd 49 x ltighd for dG! Difidq Gglrlatory
ndheim ep?tv in dir.rcnt f'm4ttrion sj5lffi-
Pbitip d{ dl,, O9!) [email protected] ltc nold dni$ fd th' pr'doction ofpedl|lc bv $lid
st t! fmltttltion of @tTGc Pulp 248 ltdint wft isolat'd ''
Mdi@'3 cofi@ gwng
ads, anong whrch wild srldn s oI Atpeql\B niSer sitna'A ^
Peat rtluc of pdtintss
of 138 U/8 dtv p!)p in ?2 h P'clDd6 Pto<twins Dut'nr 'trin ws ielAtcd in m'dim
@.hinins 2{dxvslMtc.This murant strjn *s Ftdu"d 228 U/s ofdrv Ptrlp
g@ ,l ar, (199a) sed c modifi'd abnolphcrc of loEq Cq' 5% O'1 rnd 85% N2 to
dehinc cffc$ ot [email protected] !'tivitia of veg€t'blc apoiltge blaldi! C\|ltlB of
Etu1.4 aftko@4 Ps.iwi@ Jtb@6 ttrd XadhMotu 'Mpavit gbw rr
2fC b liquid m€.liw, wcE e)€d for polygal&tene ttd P€ttttbst &livitid'
Etui,ia dtd PwtLnon$.xhibiled hiShd Polvgal@tsnlsc ed [email protected] tcrivitiA
thd ,(@l,,@o@ {nds groE uda nodr6'd dst$9hcrc or air' tt r'6c crs's'
b&tqia €rom udd lir cxnibited nigha pddntG !'tivitv lr g@<al nodifiGd
atrnocph@ did not sigilic$tlv rcd@ed p*tillag etiviica in 6v of6' b&tdir l6tcd
[email protected] d 4l (1994) i6t'd 'llvlac glJ4ol' etbitol dd 3lv6ol a d'Pl!$B
ofw.td !.tidy in lh. study ofrhe ctTet of {tid &tititv on p'dina!" Ptldutti@ bv
Atperytlg nicq CrL, Elhvl'ne elltot d'9Es€d Perines prdNlion wirhout
sDoftig sowth or saongly afl4Ling Pc'tinsc! Poducrion in p'ri dish culful6 This
".il*-,""-" *. *""* *c influsc' orwl*-erivirv on exopatins prcdsLion
i""* ** "* f.m'nrtron ortucos'Jparit/trEdlahs mcdrm {upponcd
"" *t "-"
o"t** o'* t*tines Prcduclion d@rcBed st low wrtd a'tivitv val!'s'
0", *, -,tn, *Ut exisied !t vdcl'lclivily ('w) vddd s low d 090' indeld
U""."*"a tn", -t"t U"t 098 to 090 indcsed rhc aD6ifi' a'tivitv bv upto 4 5
fold. Exr; *Iuld &clnllalion of r'deing sroup6 @cm'd sss6ti!8 ih4t low wars
adivity lm'ted the rbililv of mcnbd* o lrNsPon 3ug!s
Coelho, (1995) obrain€d m cxt@lluld PolvSlla'luod* fon lh' dllsB of solid-
,t t" "'riu* .t,l,p"ag'rtu" iis€t 3T5B8 ThG @vn' wd Duifi€d bv sdtins{ut
fr&tion.lion, tli.lJ6is sd scl filttdioo 'hftn4ogr+bv
nG dolell& wciSlt
detmincd fd tnrs pobldetdns' w$ 34700 D'tont
Crvalino. (!995) studicd $lid{tltc f'molllion ol'irPt'stla/@'ldId NRRF4I dtdd
dJ* gdi.**0"- O'* bottld @ntliniog 5 a of*hctt b6 rtd ? 5 @r of 02
0,3,0,4 ed 05 M ltcl {6t 'utellv'd
dd i!@l'i'd silh r stoF $'pasd
dorcorialelv drturcd .rd incubdcd al lfc Toltl pctjlns.. e$vitv w dpcs'{ s
L, ", ''*t*"
*'" * gr- of wd sublt{G (vu/d ws 'tTdcd
bv oc itritial
-iAity "fO. c"ft".", It *'s tf'om lhst thc p6rin86 etivitv ild'ts ei|h indss in
iii -,ao "*" *n""' t*sd 8'owrt w$ rct ob*ntd at hield &iditv (0 5 M
t"tl "-'.* -U,t,t"' w/d wls Eeh'd rn416 h i! o 4 M ttcl @nttinila
"'#. *t* o, dd ol M llcl' lotd poctills rclivitv ws 6uihm 6r l0 h
ittt*rtl *" -"*t'u- of PEtin'!6 ws tbo tfrdtcd bv lhc oltw &iditv'
cun* -itry o "'*ot
ot*"m! to lh' PFnncslcdt a'tivilv dd Fopotlonal for
bolh Poltmdhvlealet!rcntr' IvEc ed polyg'letuole a'uutv
Nair dt al (195) rclodcd pdlitrs6 l}{thBh during cultoe of 6 sD'cid of'{rPry?B
", *" it,t, * i U - t
'cdra dir'dns
'itha in thc idcntitv or con'atation or rh!
"*" **"t t"oont-ttu Nilh cod powdct (lo kg/mr)' oul of thc 6 srni6 ued'
-fi. "*, t"* utu t"'* ro sv{thclizc ' whot' unit of pcctinls iom ev ofthe
-"i"te,*u. "*,t*" tt*6is ws $u6llv hi8h"t for '4 'rsT
NCIM 548' which
O**"4 t., U **t," -*oatc @ddin'd 'nh'r
gt!'d' o' lO $corc tt 40 k8lti or
t.ra u it tt"-" " 20 Wtt or invcrt sugar st 40 tg/mr' oplinun tii6 v@ 8Gl0o
t!,{.r.rldB NclM 505 8av. 13 U in 36 h tom thc invcn ncdiun but littlc tom thc
othd n€di. ,,1- /@rrir6 NICM 5l O ws the onlv sl|ain $dicd lhat r€$pondcd b€sl (l 06
u in 60 n) io ihc sle le$s d.dim @rddng P*tin (20 tslmr).
shivatubd ed N&4 ( 1995) ud spt4 tom hiosd dis6t c, wae! pords' |linal
hou* cmlot' ed food pr.€itg w!st* in diclm@t stsrc6 !4kio8 ar@bic
brr6i! pDdu.ilg pedn.g. Ahong th. ,16 Mbtc coenit ddclopcd noE vdios
sm9l6, four slbw.d high pdtinlg &tivity unda sblic @bic @tditions,
lnv€stisadon of fmdtdion vdiablG [email protected] ln. opdnm coidilioB fo. petilDs&tivily wd€ pH 7-0, 45oC dd 72 houB of growrh witn 0.5% petin in the collivatid
mcdiuh. .A 1,4 to 1,6 fold incils in lhe p6tin&s &tivity wa &hiwld uoder the@ndidoG. The nain!ft yield of @ynes (62.?2 U/nl of Fcri.es 4.74 U/ml ofpoly8dactuD!6., 1 l3-30 U/Brl ofp@tinlya., 2.10 U/ml ofp.ctin Bt 6.1 0.75 U/bl oftolal @llub& ed 9-27 U/nl of iyl'l@ w@ @rd.d wilh lhc @Bodia C.S2
d.v€lopcd 6od dasposld pldt spl6 @lldcd nom . po.d
F.tfudd-Sdm ., dr, (196) Fpon d th,r nr obtrin petitud tud hr?crplodeing
rinb., P. e&auw ws hurlr.d ei|h W tDd MNNG (N-ocilyl N-ritu N-Dir.o&
8widitu} Th. lcl*tcd mut rts e@ ..rttz.d fo. petiellic qvrnc Eld@rion &d.onpdrd wilh thc pGnral lin . Mut nk wirh upio . 2-fold inG!. u petintrs. dpolygd&t@rue a.rivjry w@ obrlin d. Mul:nr Pa62 D|lotcd a 2 fold i@ itactivily olbolh.Mymcs,
Marlns er /1, (1t97) studicd lhc crystnl sttucurcs of parirlyroc. FDm rwo strjins ofkpetgill8 ,ie.t, N440 dtn 4M- 14?, the @livity va! d.tcmircd a1 pH 6.5 Md pH 8,t
'rsp6tiv.ly. thc struc$E wGrc dc|mincd by . conbidliotr of mol@ute r.pr!.dd!
aultiplc iemorphou..pL.@dt.!d irt rcr,5ral avc6ging. hrdclyalc A fotts into a
p.r.llel b.ra hclix ed shG I@y of rh. srrucruEl fealE of p@rinlys., d6!nc &m@ iho I?96 r€quacc iddtity ana pan wi$ strucruFbs.d digrhclr,'nre shd.d
sttuctMl f€atc include mi@ &id st .ts ed th. a5!@eiE ladda. Ho9q.., rh.dillcMc6 in rhe sublr.tcbi.dine clctts of the rM aEym6 e sEjting, Inp*tinlye A the cbn is domiDatcd by enatic BiduB snd ir dvctop€d by n gativ.
cbaosbric poLnrid. In. perdelye llb ctefr ir rich in ch&g.d iesiduB ed cont.in!e clorSar.d ribbon of psiriv€ poi.Driat whs C.r' i! houd. llE mojor dififficc
b.lvcd 0E t*o pcclinlysc A structuB Aon th' two s!!i6 is in the @trfomanon of
lhc toop fom.d by t6idu.s 182'18? ft6e obs'N€d difcrcnc's !rc duc !o 0F di['rc'l
plt valu$ of cryslallizatiot Th' substrstcbinding cteis sd c'tdvtc n&hinqy of
p@in o.t pcr.tc biscs h!rc div.rSpd sisnifi@rlv sP-ificitv it dicttt'd bv bot! th'
ndw of lh. PolciFcsbohtdsr. int'n'tion ed )ong-mge 'l6tt!61!lic
fo@t ThFc
@ldtial calatvlic ci.lu6 hav. be! iddtifcd in p6tis lye'' two oflb's' ar @snon
ro petarc lyig rerin ryc .e. dG rct bind Cl'bui s e$bnc sidlt is founil in e
a;i'ulat posidon to th. cJ'ion in pctat' lts'' lussdling a simibr tulc in crl'bti'
T1r. &livily ofPetinlts A i! pH d'p'nddt with e optibu envitv 't
pH 5-5 llrc
&tivity drcps abov. PH ?.0 du. to a @nfotm'lioMl 'hdgc
at the binding cbn' lr'ggald
by the ploiimity of two bE.d lspatutc 6idu6-
Tbalu d rl., (lao?) rcpod.d thc fMl Md vcgdtbl' indunnG ur €dvm6 'or
num@B mit oPa!1iotu. On oflhc non imPoddt CrolF of'@vde u"d in lh' fiuit
dd vegchu.-po@sing iidu3tv is p€di'dd to incts' lh' jui" iclds 4d c)drv
juic6. Thce @,md ad on p6dn, a cls of 'dpld
polysaabdidc fodd in th' @ll
w.lls of highd Pld\ whd lncv tundio' s a hvdntin8 !c@l md cd@tin8 marcnd
for tb. c.llulosc n€twork
Pdici. ., al. O9?) N.d a 3.phs pMitionins stlrd consisrins of 56% (w/D NrU"
so. in srd/lo./a ry 4 lF-but ml to iFbr' deP4brG no6 P rq@dd' Th' low
@livity ofth. 66ttucive .xo-p4til@ podu@d h srbndged cultuG w6 breught 10 3
dcEtabl. activitv ed wd ft.n puilicd I-fold
Acune-Arguelld er al (1997) noted thd $c 'xh{ellultt
p'ctin$6 ld
pr.il@d bv kPztgllld tigq CH. bv slbFrSFd ed slid st!t' fmdtation dd
th.n physiocndical ed tinclic popdti€t tae studied The high6r Prcductivitv ol
ddGard dcDclilug 4d pdlinlvr* w4 obl'in€d wilh slid sbr' [email protected]
Thc tinctic !d Physi@hmicd phP.ni6 of th'$ @vn6 w'E influcn"d bv th'
lt?€ of cullu.c n thod us.d All rcliviti€s lrd vqy di|I.@l in tcms of pH dd
lc',o.6hft oFiD., stlbilitv Ps dlfcqt fd llc subst2lc (lstr vslu6) ln slid
$de fcmotltior\ aU petin6B dctivitics wcrc noc alablc !t qtEhc pH &d
lmp€6t@ v.lu6 but thc tn \tlt6 of€ndGpelil@ dd petdd'i* {6t [t8Fd
witn r..De1 io th@ &tivili6 obLincd bv tltc subndged culte t'cbniquc Th'
p6tnlya$ &tiviv ohancd bv rhe $bnd8'd cdnrc lcjniqud shoe'd 3owatc
,*ot* O* t" GEvm. ob|!in'd bv $lid $at' fffiiltion did lot
clabolho6i!, ucins sdium dod@vt lulphddPolv&ryldnid' with wvmlhc
dtr..ts obLin d ,for both cultu€ ndhods' 'hovcd
lh' .me nunb'r of grot'in
bcflls but m. diffcituls qtre fodd in thcir cle'topbotltic pGitiolr Th' r'sults
obrai.d in this wo* !ug86l thal dtc 'ul|!c
n'tb.d (sbn''ged or slid sldc) my
b. Fspoldbl. forinducingchds6 in$!j ofth' petinobric aEvn6 produtdbv
Chiloti e, ,r, (1c97) sMicd thd eiolaEd tott's hypMtvli Fmwt 8d ap?lc tuit!
w@ i@ at d with 3 rr?.s or i@dm ol a 'iP@ (ugcdillLd @Ddia
gdiEte{t @ddis $d DtsliM) All iF obn rv!6 @n&d w!td-!o!k'd l6id in
t put dso cxccpt ror ugemlnat'd @ di! *trch s!rc n€ b nebtic .@ tn
ollreo *arings lnf*ra tissues wae satnpl'd d vdios inldv'b ln.r ino'ulation ed
**ya r". w"ti"tv* (PL) dd polvgtl'c$sMe OG) @tivitv And is@l6Lic
focsing ihe pa{tu of lh.n i$dzv!6 wcE obw€d md PL &tivitv ws not foord in
fic ne;dc ..!.! itrds.d in elten bv un8'mi@1e<t '@idi!' *hcr. slv!@ltr L rh'
pnncipal eyhd phvroalqit' es @td'tl c@nitltive PL trd PO tu@Mlm* wd
*"-t, "t
*. " "t*t debt'd h c@paliblc ttrd i4omp'tiblc intaltliod
durins ilc €dly tisg.. of inf6tion'
wei.hd a at, 0998) dared nd ' Petinlt@ (PLEc422l0) wd pDduc'd bv
Plri6 sPh,itt% in inf@t d @cMbd &!itr rbc dE)tnc ws pai6'd io Fr
hoDoeldeily bv d!tuniu slPhd' ittli@tidl CM'!"lr.,w @luDn
chrcnabgnthv, sd prcp@ti* is'letrrc f@uinr; lt€ G"}m hld ' norduld Da
of 23KDasdctmindvSDS-PAGE Thc pli of PL{d 80 whi'hwd 'ls lh'
opdnu pH fot .nzvm. &tivitv, lhe @ym' wd slabb ar 4-50'C' but bcvond 50eC il3
&rivity ddr4lcd npidlv The p@lintv'$ d'8ndcd high nc$lyl qtdi6'd p@tin by
o-*U."""" i! e .nd+ttldci tld ws Erirura&d bv lomc divaldt '3tioc
iFludils c.", MC' ed sl' Addirio! of zJ) bbftitld lhc astDc &tivitv!v 36%'
rt" ';a
pi ."*a '.piA
t@'lion of potalo sd dcmba dis Ttti' is lh' 6Er
rcpon of a !L Prcduced bv ryl,B' tpl4dt" t pldt pa$osdic frgu {hicb c@s6
!.edling danping.ffrnd 6uil rct on cucMo'r'
t0
Mddotrldo dd S&isg de Saad (l9E) sMicd thc p'odwtion of P@rnetc@ drd
polygde$o'@ bv AlPe?illw 'ig n sbDdged md elid_s|tc fcm@btion
s)€cht, Wiltt p4tin s a $1. cdton tow') p'cth61€@ 4d poltllleluolw
Dodudion wde iou, dd sx rinca hisher rc$*riv€lv in a sold 3hte svstan than in !
subm.iecd fcm.nrdid .)51on aftt r.quir'd a shon€r dn€ for Gdvmc pbductior ft'
addiiion of glucos incresed p@tin61cN aid Pol)€tbttuo'se poduction in tt' slid
.t!t sysld but io bnaB rl f..ndt tion lht PFducti@ *6 ndkcdlv ithibit'd A
@mpdisn of@zyn. ptu<tuclviid show'd lh!! to$ dddni!€d for D'clit"t4e aid
polyrrt&luo@ with p€litr s . csrton d!t' wa! lhc &d 6rc tiDB hislt.r by
ciry thc solid ltd€ nthd ilm tbc $bhdtcd fcddt'lon s$ten 'Iltc Flducliviti6 of
th. two dzlr6 w@ df4l€.! bv 8lu@e in bodr fandtrdon svslau- Thc mdbnn6
6a cclh fiom lhc solid srat fmcni.tior sltowcd itwsed l4els ofcl8:l' Cl6:0 4d
Cl8:0 fatty eids. DiffeM@c in lll. rcgulalion of'nzvnc svnth'sisbv '4sP*gi a nigq
d.Ddd.d on the lm..lalior stst n, fsvomnS th' elid sLtc over $c aubncrged
fandLtid &r PetitDs Poduc[on
Ddn e, ar, (199) cdi€d out studrc o rh' pFducnoo of c![da!6' patre dd
potcitllg by lh. @lomrcotdi?'t '{tibt'
Duhrld Ca"'zr"lls 'i&ts Two lrlrid
ortr'is nogut (aon u mirou dd '
d*ftluou fo6t) Ploltr'd tl@ 'idosluc'D*
elivity the thc i$lde tom deiduou toBt, but thc isdlal6 lnd sinilu doBle$c
erivily. TIE surfeclant lw4ri-80 g.n.dllv rcduocd @ltlle &tivitv ofboth i&lal6 bul_
!n nod csa th. .fTd w4 nol siSnificmt ThF Pelitu' 'dvm'r pFduc'd *@
pdtat ltsc, polvg?n&t@nd' sd p'clitrttsa Th' addition of calcim incrc'!'d
o6t.tc lyc. . l_pdtin 1v!* i! bolh ieht6, but onlv in tht c@ of 0tc l'tla 'E
m'
nlE lhc @nif@6 fo6t iel.t {s 'f@t'd
si8!ficettv Borh i$lal6 podu"d "id
dd ndL.t oFlci|l.g anrt md oflh@ oEvn6 wd ptrducld eho dF fun8i wF
goM on c.sil s rhc $le nit$gd e!@ lhs d v'!tl od&r Th* *s no nEprc
Elstio$hit b.twd biotts po<tlclion od cEFc etivitv for lh' @vnc dudicd
[email protected] 4/., 0999) *dur.d p*lDlts' rPLl indEtid bv orSeic Dd indg&ic
@mDonot! of )tdt eiMr {vEt in P''t'iltud srbturddr' cultsd in a nitr@l
ncdium @ dnins slme, bv ddcmining ?L etivitv ed n'@tial gowth (nvcdial
dry e.ighl} Tll towdt YE @Mlr.lion th't premicd sicnificel PL induciion *nnoul
&ting s . qrbon sle lor th. tbSs corcsp'ndcd to 0-m?5% Neithq cdcin'd YE
ll
@ a nuliior $lulion @sldining micon ti6rt indlc€rl PL poduclion' indioli'E $at
6e ind!6 war d o4rnic compond' Vitamir& lhGgholipirb @nPoDcnt' rd@
U*. -a ni .g.no* t*r" ,u@ l€sted in Pl!@ ofYE and Pr'mot'd no signiliomt ?L
tu*,*. o ti tu** *.*utd wd fouid to b! ehhl' in tc nlcl@tid' ftlcnd
obr.in d duing *ta.rion of YE- Th' indud wG stoM lo b' I rhdo sobl' lollr
$bsiar@ {tirltdblc at 2ooo D'!to!" hvrltotv'$lc bv ficl ' dd
"rivtrcd bv boilina for
up to 60 min. Cy.lic AMP (oAMP) qo8@u!lv t{tde<t lo t[' culnrc oediun 't
5 tld 10
nM w6 c.ptble ofinduci.S PL in P g'r'oto3t{t gbM on 3ucos' sugg6lin8 lh8t d
l.!$ orc @mPould otv h. Plt@t in YE &dng in a @pdaliv' flshion for $e
nri.{en.n@ ol high l.vcls ol aAM? into rhc ccll PL etivity ald th' lewl of cAMl
n$id. thc, fugal cells incEacd lid rhc addilid oI YE o th€ cdtuc nc6rn'
suggddng U. pani.ipation of lhrs ness8d in th$ @vncl 3v na'
Beg a at, {2ooo) EPon d thd xvlll@ ed Pedl@ pmdu"i6 h1 Str'Pton"4 sP
OOtn-3, "* stinulatcd bv DLnorlcucinc' L-l@irc' DL-iolocinq L-It5l!e
;dohldochlo.ide and DLb't!'ph'nvlataninc bv up to l t2_md 2 ?8'Iold' rctP'ctivelv'
wh.@ th. @nbin.don of DLnorlacin'' Llacin' dd Dl--i$lacin'
syn{gistiolly slimdar.d lh. xvlu@ Md p€tiM pilductid hv up ro 6 72 'rd
5 62'
rotO.
-rc''orletv. Glvcinc, Dl_nomtlisc' Dln€lhioninc' &d DLasptnic &id
show.d no sisniti.sr srimulalory'fTat on 'nzvne
prcddclion
vshy4 dt ,/ (2000) ob6d'd lhal s $il 'slar' sa'tttB rPP DT? lo ptudu@
signifidi dounl of 4 cire€lhl4 p'ctr@ sbc'quollv chMtcian a
p;$rlysc (Ec a.2.2lO) Bv oplinizinS grcwih conlitio$ 'oti{s
lp' DT? poduc€d
highd dount of petinlF* (53 ulr'mD tiis th'l hd b'6 rcponed in lh' lit'ratlE
uL gcr frrrarior oa ior qch6e! chdntlosraphn ihit dtD' s puiicd dd
"*i " t* .t*t- .*s of to6KD& Th' p66cd @Fc abibit'd Ddim'r
divity .r ! mpdlw of 6O'C $d pH 8 O Th' Ps'n" o' tOOnM concdulriofl ot
CaCL sMl D.rcapbcthldol 5i9i66tlv G'nec€<t Fctjn!$ &tivitv of lh' pui6ed
R€id and Ricdd, (2000) rcponfn that tr'dncnt wi$ the c'zvnc petinalB lowc6 tb'
caliotric dc'nald of lncmo'mcchoictl pulp (TMP) bl*hcd vitt tlkalin' p'roxid' Ilis
disvcrv wd dt@{t d lo bl€'l TMP ptu'|lction indBldtllv hi!' sh4n 0'!r
t2
conneoial eMtBc pr.pat tioss 'an
tlal putp wiihin IJni'|. !t !h€ t@p@tu€ tnd pE
valu6 prev.l€nl in tap€i milh Aboul half of tlG dtionic d"ndd in tnc bl@h'd p'Ip
d b. ddiroy.d bv p*lir66 DFmic d6idg' 'xPdimols
snq thtl lhe rdlE'
r-*- t*t* * **"'ncs olscvdl catidic pol'mas to in'@ Eicndon of
fin6 sd 6lld p.nicl4 It doe mt iffise r6t'ntion in tE fi6@' of El6ti@ aidt or
wnn noNdc Poltn6, &d tto6 not ddag' thc stln8lh PrcPcni6 of thc !ulp'
PEti@6 @uld b. @ilv in@oolaLd hto p!!d t@line dock Ptca6otion svstds lo
low.r rh. chaad of catio c rtdiion ail5 naded id ihishd @nt'jling p'rcxd'
blc&h.d nahdicat PulP
Mohdb ., a/, (2OOO, apon.d th.l Pi./"? P'nu wN cz'9!5tc of grwing on muSo
w.st." producing PadnltB€, (EC 42.210) @d b'lr 8il&iosid6c' (BC 32123)
6zFes. TIE 2 .Mvnd wR $e*ivclv pdifi'd bv pdipidi@ w'rh ffiorum
lulpharc fouow.{t by chrcd.ioenplry on s@hadq C'l20 The Puifistiot Poq'dw
prvided 1846 dd 929 fold prfiotion with 20.6 ed 24% vi'ld '@Ycry
of plclitd6
&d lel!.6 r.srdtively. Thc Km valu€ ofpatines *s O.3l% for petit 'l
pH 4 5 and
liisi for l&ts6 wd 0.166% for lallosc tt pH 7.o Th. puifi€d @F6 e srtbb rpto
5ooc for 60 &d 45 nin, 6p.divelv, with 20 dd 3tol" tos of 'ctivitv
G't ati@tion @
S@hadd G-200 iidi@led thit $c Molaula! w.iSlts of tbc purili'd pdtins w9orlo3 Da lrd lat.e w6 ll5r lO3 De Th€v had the Polmtial for lrc.litrg bdSo
ps(6 vitn 2 PE?ltld c@Fe dultcd in d i.cre @lor of inldsiiv' lolal
carbohldEi. @n&nt ed jui@ yi.ld ldlinc nilk vith puncd bta!. d$ dDwed D
i.c@ in $c robl carbohydrr. dd tldwing lu8q Pnduc.d
T.neitu a al., (2ooo) rcloncd rhc cff@t of difcmt qbon euc6 or ine
eetin6ilru, ado dd dopob8.lr.tft.4 alivitie frcm '4 jaPdiN 586 wa
d.tu.r.d in liquid n di. $ppldmtcd wilh difrGmr subsb.rc @ncoFatioB Thc
cdtue m.diud {6 i.ocul.l.d *itl J l0' tPordnl dd haintiilcd u ld agitatio!
(l4oi!m), a1 3cc, duinS 122 hoE. IE @tD. a.turli@ w @i.d out hos and
tiltntior Th. code cxltet frn A, japoni$ 586 irdiel.d |h.r lh. bsr @vnc
trriviti6 w€r. aforded in ilc pBencc of 0.5% (D4tin6ttu), 0 2% p(jnn ad 0 2%
slycml (e opolygal&tuo@e), ed 0.5ol. petin lschcd to 0j% elu@c
(dopolygalelureD6e)- Cdbon ews @ncotration, islaLd d tssociatc4
iigtrifi@tly afldB l!. pectin6td4. ddo ed dopoljB.Lcfrftre etivitid P.ctin
t3
glwose dd su.cha$se, when addld to thc culrure m'dim in hiSh colmtltic'€xhibitcd a @Fsion .fl4l on all the dnltzdl 6zvd.'
B.e d at., l2WJt i{lotd Str.Ptorr@ lP' QG_t13' {hich prcduc6 a @llul6t tuc
thmitablc xyb|N (96 ru/mD &d . petrn@ (gobrddmn6'j (46 ru/ml) on
Hdiloshi m.diun $ppl€not d viih M, whear bM Cdbon 3o!M thd Evoutld
xyldGe !rcduclion wcE ncc br& (s2 lu/nt) and binh wood xy'e (81 IU tr '1)'
leriss6 po<tuction vs allo ninul.t d bv tetin ald @t!on e'd cak' (34 ru/dl
dcn), Th. p.nidly plnfi.il xyls4. ard p.clirs6 wd @dsdlv etirc lr 60"C Borh
cnzrm.. w@ l0oe/6 flablc at 5dc for noE ths 24 l! IE hdt liv6 of xvloac ed
p.cti@. !t 70, 75 .nd 80oC w@ 90, ?5 dd 9 nin &d 90, 53 dd 7 nitl, Gsp4lirclv
Th. optidM pH vd!6 for xvlde ud petine ld 8-6 t'd 3 0' 6p*tiv'ly' at
60'c rylde $d peti|e w@ ttdl. or{ a bbad pH ma' betwq 5 4 @d 9 4 4d
2.0 !o 9.0 c?e|lvcly rctli ng moF rhd 85% ol $cn &$viLv crr' $imut 'd
lhc
@tivity of bolh ffics lPio ?% whercas CdL, COr', CtI iodolc'tio &id lnd
iododrai& iinibit d xyralw uplo 35% 4d PecliIres uplo 63% at lmM, Hg?+
ifibitcd both 6zyn€ @nplclelY,
Blddino .r ar, @ool) invdigaLd il& pNibilitv of prcdlci.8 pdril1,g bv x,'op6
stobnilet NA ttpegilts M@:. Bing cdlll de n,l4.is 3 substlre Thc *tlole
what nour etcd s a good Nlri61 ol@ for thc cullivlrion of thc Djc@rgettu'
ErG&d cidcpob8slet@llls (PO) w@ ProdNrd in subncicpd culturt' In lhis
FsDe! it ws pGiblc 10 obtai! polySd&lNn * &tiviti4 al d @ptabl. vi.ld' in
@npdisn *i& t 1,?ical dcfDcd mcdium dstibed in thc litcdurc for Fcri.,.6
uuluction tn. synlh6is of both @F€t @cdcd i! bolh slr.iN in th. .b3cnc. of
r@rin. denotumtio. th. @mtilotive natur. of th.e dyhcs; n v.rih.l4, prcducl'otr
w6 incrclscd by thc addnion of.3nalt smountofp6lin ro th. floui
Hifsdsf ., ur., (2OOD not d ln.r Pdeilli@ gribrBdn 6lrw.A in lh. Pl!s@ of
.uclw snd ylst cxln l podlc.d p@1in lyec (Ec 4,2 2. ro)el) in the ab@c. ol irnaturalildudodpeti! tlit 6bsuws.ultur.din!fmaloiats @rior .of 0.5
tnin for the n6t 25h Dd l O l/nin fot |h. llmindq ol1h. culte pdiod. ttrd .l .stiFing sl€ of 2OO d/mn for thc dtiF clte pdiod. FuStl 3poG w@ iEd.cddir4iy i.lo th. f.@cnld al a linsl @ndtnlion ofsxld spoEr'd $pdmdt.d vith
t4
!owd.r!d ddrvdrlkd susar sd juici, Producing PL without addcd Jt1n cxtraa
Msin.l PL &tivitv (0.06? ru/mD {s obtti!'d 'fid 65h in bo$ P@sibl' b
'anv olr
thd cycl6 of iipaLd balch cuhE Tb' @c Dodrs {s u!'d for F|@d a for llc
singl. b.Lh @lt!& Th. initisl cy'L vs 53lr tft'r *hich appoxinltclv o l03 lu/il of
PL wB obhne4 and lhis pdio4 tlc tudim w6 m'*'d cycl6' etich bn'd
appFxihat ly 2On divity ofPL ohain'd in th' s4ond qtlc wd lpprcxin'tely 0118
Iu,/rnl ud ir lhe thirq alpoxiD.t€lv 0 109 IU/FI'
Mrde. .r.1, (2001) F!.nqt ltc s\nv ot P'$gtN sq@h@a !o srow sd Pnduc'
@lirs6 in d aquou two Phe mc{tiM cdnp6ed ofpolt€lhvldc eltlol ed 'dd'
ddt rn Fusd grc{ah ws rshict'd to th' hoiom Ph@ l4eng th' lop phd' ccll ft"
Anou$ ofg.duc.d biorc &d @do 6d aePednsc activiliG w@; slpai6r or
.{u.t lo tho* obtain d in hodoS@u n'did Thc pdtirid co'lEci'nt frT lh' cndo-
p61in !. was I 52 follow.d bv a top ph'& i'lit of ?0 85% Allhoush th' phtl. 3vstit
@mootilion 6vo6 pl'lilion of ! gcater Pd of do-pohnt!' elivity !o fic botion
Dhsc (K-. qd 06 dd icld ir top Ph@ 48 56%) th' putilioo'd 'ctivitv
in ll'c toP
ph& *s .qu.! ro $.! preduc€d i! [omgm$ cuftivanon
Srkrtm ,, ,r, @0ol) !tudi.d the cftdt ofadoPhltrc b&tdid lttivitv @ t!' qudilv of
coflc. bdaag.. A suflcy of the nicrcorSrniss in 'offe chcnid w3 pcrfomcd
b.rioF hd6tinc; dd lh.ir srewth on rh' ruin nuEion svlihbl' ia e^r" (Weo
drdhi@\.v. c|t@i vtu to ')tqi6 *cF dctabin'd io vito Mey adoPh'tc
b&tdis w@ Gola&d fron surlb4<'dliz'd 'oftc chai4 Onc of th' p@timlvlc
strsiM wd phrsiologicallv ed PhmlDicallv chalatdiz'd lnd vN Gnlltrv'lv
idcntili.d by paltd 165 IDNA lcqu'n'ing 6 Padibocil6 \n'AyiN 7ni3
cndopht4ic n6in pbduccd .n qt'&'llulf pelin4 slh tdihd r'livilv al 4dC sd
otl ?,9. dd B thdot|blc Dp ro 45dc EDTA lnd mctd i@ hld lirde Gfi€t on Petr
lt$. a.livity. K* dd V* wlo* *@ 46 nglit trd 940 1G8 oovnit^tr|'
rct!.cdvelv Pdtinlvsc h.v€ h€d founl in fungi bul @lv in b"144 dld thk islat' i!
a DFnisilg tool for rcguldi@ stutli6 of th6c @tdd'
SosB ., dr,. (2OOl) iel.Ld fv' 'u'!ls
tElis non &'ring v'gd'hL mrl6irL
culrivd€d on whd bE 4d ddo_polv8'lrt@na5's' qcPot'3d&$mld 'd
petir
llsc &liviti6 in lhe crudc ozvnalic 6olution ww ddcinin'd Hi3h6l actrvrly wsl
t5
obst d for .ll @zvm.s who fcmolalio! w Mi'd our ar 2E!c' tne hi8h6l &livilv
ruh€s wc obllin d an4 120 h orcullivation for qcPC tnd rnd 48 h for 6dGPG ed
PL. Th. u!. offi€ enzvndic aolution for tcatndt of fruii! lnd v'get'lble mdh tlloitLd
a biSlr jucc .xt24lio! dd a pulP wilh good P6irg ch"futcd6li6
Manis at ,1, (2002) ctni.<t out pedn lv's (PL) 'nd
lolv gddurq@ (Pc)
produdion by II@o,r@ a,tu,,utu bv n.s of $lid tt e fmattdon uilg
odg. bagec; sug& ce bag$@ dd wh4t brd 6 a @rh6n sl@s ?G Nd PL htd
opdmun elivity at pU 5O o.t lO 5ll 6p*rislt Mdimtl lttivitv of thc @vic
w@ ddmiE !t 650c, PG *s 3tallc in thc lci'lic to ndrld 9tI rucp 'trd
tt 6dc for
th, whcrcs PL ws stabt€ at b$ic ptr md at 6ooc for 5b
Tlieb d dl, (2002) cler.d |lr . wild rtF Pa'ctltiw eimrt for ! Poginmc ot
sE!.tic iEPtovffi! Md . sod' Fuld o' D[ou &id nu!ts66it' I
htT€rp@litulttic dulst {s s.t{tq{t Il 3"ntql tlour lillv lim6 ll@ of ddo ed
dop4lin$.s eliviti.i lbs th€ wild t,?' on' MGt inl@stinglv' thi! Petin46 on
eju@e dd slv@l flown B tqt€$c of Peti@lvtic! g@e lL det not htFr'
prcducefta htndvtic cEvn.s $ch s elllle IEF ctoltlk @ftr'd th* t6!ilt3
dd showcd thsl *v.dl pednM wft ovcr qpcstd in lh' nut'lt
S@mv! a al. (2002) cei.d oot snldi4 on i$brid trd pFPdtid of p@ti@t nom
tn Fmgu lsPegilts jaPoaiw Usbg sionqchdgc cht!6t!ogr4!v on difidt
crdi@ dd phdvls.ph.rosc hldrcPhobit chbmriolnphy' nvepeloMic aEld6
*d isllt.d from lhc tiquid cultue of t dolst sllan o/ '4!Pttg 16 joponkat: n\o
.ndGpdygddurelae (l od n, 38 dd 65 kD' p/ 5'6 ed 3 3)' pdtinlr* (50l(D d
3.8), .nd t{o Petin6to$6 (I lnd It) wi|h 3itnild mledd slidhls (46 ed 4? tD) ad
rh€ san / (3.8). ft. pcclindtqes appMtlv nPcdt two irofom of dE tdc
dlna Alt puificd cnzvn€s *crc honosdous adording 10 SDS-IACB dd
polr&ryl.Ddc C.I-IEF, .rc.pt for odcpot,sala'tsult tr thal srve two bdds on
i$ldt ic a@usins, but onc b.td d .t*trcphd6ia-All 6uJmd tad nuimtl elivity
in m did D.diu (.r pH 4 G5.5). Thc pdrin lv!* ed patin6t'|@ w4 3rablc !t 4G
5o'C. Thc lhm4t slabitity of bolh €ndo-PolvgdlctN@a! ws hu'h !o{s ('nd 3 h o'
[email protected] 3O'C, odo_Poltgabcn&ns I ald II t@t 40 lnd l0% of th' &lvilt
Gp@riwty). Tb. sriv'tv oa odFpdvCer@|'sg t !d n to*{d! polvSdlctlfuic
t6
a.id stongly d.Pen.tcd on NICI @nccfiarion (oplim'l conqcntralion oflh' sall w6
O.l-0.2 M)i tbe dEyn6 vcE also pa.tidlv Puificd cnzvn' ws ilobdcd vilh Dlcrdid
DNA !d lhe @mpld w6 shitlcd to . hishq not'clld t|s Alabtt ofclet'odurql
pet.is tln thc Pret in DNA @mpld daled tb't on€ of thc el4troelutcd prer'in
beds we PL, tutihodi* agaiNl the ovcr dpr6$d PL ww tlso prcpsEd Dd ptnitlly
Dccr€gorio d dr, (2002) rcpod.d s'ncle ccll pretan (SCP) &d dd' Petimltrrc
dzlm6 pFdlcdon f.om citn. pulp.. SCP od dzvm6 wd pFduccd bv slurrv $!rc
ilsk cufrivtrion of,,GP"tstrl6 r Isu $d Tithodet$ vfllie on PulN tom lnon jum
clelicalion. Prcduclion s wetl s code p6tine aclivitv ws @t tf@l'd bv lh' niSh
dry FltGr conldt ofth. Pulp3. Boih O. Po&in @Dn iD th' 6idE dd th' @Fc
&tivity in rhc s!p.h.ta,t *@ hisha ia ?'' vtlL ihd it -,Jri€t onturc' Tbc cod6
p6!i|e of t '':rlda whoe ,pei6c diviiv wd ri ld t6 rh foud lor ! @mdiil
@dlr.led prcpdtio', 6dd b. ulitz.il in 0E s' citlus PM6sing t4rory 4 *eu a
in ooE f&tori6 whicb w Lrgc @ods of P@drclltic crudc Pr.platlo6' for ddp''
lo enirtue deflratio. plel p€rfomancc,
Dhillon., zr, @004) rcpon€d $ct ftlil pto'din8 induldcf ptod!*d a ld8! aou of
vd€ 6ardid, which pos6 co6id.6blc dBPotd PFblds and ulfmt'lv l4rl5 [o
Douutio. h th. PMdsing ofcit!. frlits, ' hr8' poDortion ofttc prcdu@ 8oe eat'
i. thc fom ofp@I, Pulp !t t 5.Gd Dti.d citns pFl is 'ich
in c$lohtdnle" !re1cin and
Ddtin, Pdti! dt s thc indu6 for poduction ofp@tinol,ric 'nttd* bv niNbhl
sysrds. Ths lbcv u!.d dti.d citos p€l s $bst:l' for ftc addw$on ol
odopolygtl!.tllme bv,{ t,igs Mdibm 6zvEc &tivity w obt'in'd wiih l5%
slhs1aG wnich aiv6 a $ni'elid m6s, who incubaled d 3ec 'or
120 houB
Bd .r.1, (2004) qoncd p€li@6 6 6 elicilor of Pldl die6c ealancc ed es
Doduc.d by slid si.te f.mmilti 6 with Aspetgill8 nigq- StAs b"t putP w$ ustd 6
carhon 6ouoc dd rh€ w$tc warq tDn monosdim Slutdld' prcdudion {s utd u
nilrosd @d *at r $us. Thc lmdLlrotr t@pdatut ws 3OoC ed ih' Elativc
h@idity ot.n {d ?5'90%. Th. nuior prducdo! ofpeliM' M rcacn'd and 96
hs cultivalior ln crudG P€.tiM dlr&ted ii'n tlE fmulat'd @ldials 6!ld clicit
dis.e Etui!@ in cuc@bd sd bmaio s'€dlings
t7
P.til and Day.nrnd (2006) 6ied oul th'ir stldica or poduclion of p'clrnaes tn
subdaScd ed mlid str. fmdt!$on ftoE di*ts! tuflovd h"d dplot$g
/!,petgi&B nrgr.TlarrtnPr.ntial lt'tiN of'{ '84 DMF 2? for $bttrds'd dd DMF
45 for elid stde N@ isolsred bv moltltt@ !'rqing kchniq!' b$'d on c@ftcid of
p.ciolrsls ed capdilv of Peli|trs ptrdu'ton Th' incd'd ldcl of p@tiBsd
Dodlctio. w6 @ord.d al pH 5 0 Md lFPcom J40C in subD<g€d dd olid naE
lcmenuLion rrc oprinu ineuld sizc ws lxld ml' lor submdS'd &d lx lo'g' lor
solid .latc f@diation Fiw hmdr.d nicondd panicl' $tr &d 65% midue @nr'nt
of lh. subsl,*c wce optimd for lh. mdinun pod!'tion of P4til@s in solid sLt'
@ndition. Undq optimM @ndrtions' naimm plodudion of 'xo'Pe'ti@'
wa 34 2
'/g in Ssfed .ndop.ldnse wD 12.6 u/sn in SnF'
Di .t 4t l2}u7) purif.d dd ch@lcriz'd p@tinolltic cdtme poduccd bv
.lsperyils nigl MIIJG $-Fi!. petinoMc @tDc t@ pui6'{t uin8 3 @mbi'atron
of clmd.togrthic tctniquG Thc pDP€ni6 of bomo8cnos dvm6 wft sslvz'n
Th. pdiicd .nzyna wd dsificd wilh t6p*r ro th.ir biochhical pbp'nics and
.lbstBt rpdificity. Itct biocheDiat Prcpetti6 sd 3ubltiat B!4ificitv anon8 ilt@
rrclcitrs, orc pc ed tolySalactuFN &rivitv, eotha.pp.{.d to t r pdin
mcthyldtc@ dd th@ w@ idotificd d p@l!t bt. Th...Paitv of fsSll 'l 'tltttopmdu4pdt elys with opdnm PlI in &idic doFain ea Fpon d fdth. firn tinc
1.1 BIOTECHNOLOGICAL APPLICATIONS OFMICROBIAL PECTINASESOvsr th€ ra$, pelin.s h.K bc.n u!€d it svcld convdtiond ilduslrbl
pn6s, $.h 6 l.xlil., pl&1 Rb€r pr@6fi& i.., @f.e oil dFa.rio4 b..r!D@t of
itrdudrial rctMtd, @ntainina petidcios dlldiaL c&, '[!cv have als b.@ r.poncd
!o wolt on pui6..tior or vitlg (Sal@ ed htsinshc '
1991) ed in tuating orpaPcr
( Reid old Richr.d,2OO4 dd Viikdi dr dl2001) Thay de )tl to b. @mdidizcd
.l.l Fruit Julce ExtrrctlotThc ta€61 industtitl applicltio. of peti|]ls is in liuit .iuic. ext6ctioo Dd
cldiUsrior P@tiN cont ibuL to fruit jlicc visqi9 ed turbiditv A nixtw of
periM6 dd dylsd is u*d to cldify fruit juic6. It der6* f Ination tm. uP lo
SorA (Bl!@ et eI, 199). Trcaltndt of 6!it pulps vith Potin.s tlxo showcd eincrcse in fruit jnicc votume aon b.nda srap€ sd +plB (KN et ol 2sJ1)
l8
Pcclrnd€s in ombi.aliotr with othd mz)rn's' vi? '
celtul'sca' !r'bin'!6 trd xvlans'
h.v. bs u!.d lo 'Fl!e lh. pr*D8 efEci4v of the Euil! lor lEc' extFction
(Gsiling dd Guib.rl, 2O0o) veuM itfuon ofp'ctinsd hd '
@tlnftial aDplicdion
b $nd tle p@t of citi! nuiis for mo{t Tli tehniquc tuv dplad '!
futw lo
Epls4 h.rd cultiry for th€ Prcduction of cm'd scgndt!- biBi@ of EE stot'
Dach6 *ith petiMclhylsl@c sd catciui r6ults in four tim6 6nd iuil5 This
n.y be appli.d ro pictl. pt!).4ing whac cxcsiw $lleniDg nav oc'ur durng
f.mdt tior dd stoBsc (Bat6.rd wickd, 1996)
1.1.2 Textile processing and bioscouring ofcottod frben
Palitlac hN. bd u!.d i! @njMtid wi$ dylass, lip6d, @llulas€t 4d
bdi@llulls to rdove sizinB rSo|l 0om otton in . sfc ed @fn6dy l]lmcr'
Eplrcins Fxic .aurii. soda uu.d ror thc purDcc dlid Glondal at a/ , 2000).
Biostuing is a novcl p@6s for @od of non@lldotic inpuid6 noi th.
fbs wit! speiEc @116. PetinM hav. b4n ued for th's purpo$ without &yn.glrive sid. €|IEr otr ellule d.gndaion (H@!dd .r ./ , 2000 ).
1.1.3 Degurnmitrg ofplant b.st libe$Bsl fib.6 @ ilc $ft lib* fom.d in grclps oubid. th. xylm, phlom or
paictcl., a& Reic ed M hdp. Tt. 6bd @ad! 8u, which nut b. movdb.foE ns e for Iatilc n.li.8 (HMd.l .r dt . 2000 ). Iltc chdicsl dcsuiiinstrcalndt il polluling, toxic ed mn biod.gldabl.. Bior@hnoloSiol dc8ulning uirSpolinds in conbinarior wilh xyldes !rcmts e eofiiddly .d @nohic.lt€intlive lo 0tc .!ov. prcbld (K.poor d al 2001).
l l,4 Retting ofphnt fiberP-li@6 hlv€ t d ued in Euilg of flrx !o s.9{rle thc fb6 ed elimiMto
peti$(HMdd "r
,, , 2000 ).
1.1.5 Waste wrfer treaimentVcg.{sblc f@d pEssing industris rcl€rce p@tio @ntairitrg weiw!1.6 eith
peliml,lic 6zyn6 fa.ilib16 @ov.l of p4dtu@u natdid od ddd ir suir&lc
for ddd!.sirion by !.tir6rd 3bd8c tEt!6r ([email protected] ., a/ , 2{n0 ),
1.1,6 Coffee and lea fermentstloD
P{tlrls Eshlat a@laare tca fddtlrio sd rle d61roy! lb. foufomirg ploFny of iDllad re Fwdd by d€.iloyiog peria (Cu, 198t. rt.y e .t$
l9
ts.d i! @II@ fomotltid to r.novc nucitagimu cott ton @lr€c bcans'
1,1.7 Prper and pulp ittdustry
Duiins paFmakb& PetitN @ d.Polvn ris! p.cliF 'nd
dubs€qumtlv lowd
th. ..rioric .lddtd ofp!.tin slutioE sd rhe fitftrc from pctoxid. blc.thiry Gdd tdPich.rd . 2004 ed viikdi d aa 2001 ).
1.1.8 Atrimal f€€d
Pddns e uscd in tn. @ztda co.ldail, ued lor lhe prcduclion of.Ddal
f..d!. ftis rcduG thc fEd vis6iry, which incllls atadptio! of nuttidls libqdd
ruridra eilnd by hydrcltsi3 of rcn-biod.Snd.bl. trbd d by libdatiag nuFrdtr
bfdkcd by ile 6b.6, ard rcducG rhc mount of fa@s (lloondtl et al ,20/.n J,
1.1.9 Purificatiod of plant viruses
In cac wher€ dD virus pdiclc t Estrict d ro phlod, .|}dine peli'l3s ed.cllula6 cd bo u..d lo lib.dle fi. viB noh dc t'Bue lo givc vcry purc pr.pdtions
of nte vitu (Ssl@ sd Jlyailshc, 199)
1.1,10 Oil extractionCitNs oil! 3ucfi a l6on oil cr b. cxc&r.d wilh p@ri'l.6. Th.y d6lroy the
dulsi&ir8 pbpdtia ofletia whicb ircrf@ vith the @lldtion of oilr ftom .itosp€cl qtr&E (S@t! 1978)
l.l.1l Improvement of Chrodraticity rDd Stabllity of redwloes
ldtinolyric azyn6 !dd.d !o n&earcd Aljr bcfo6 thc ldditim ofwin! )€.5rin the p,l)cs of prcdlcinS rcd wine reulied in inprov.d visal che.tdisriq (@lou
&d lurtidiry) a conpdld to the cnmafic ch.@rais1i6, *hich @ @BiddEd b€n rlha. lh. conttol win6. In* viB also show.d gr@rd slabilny a @mpaied to the
6.110l (R villa ed Garz.l€z$!4 2003).
K.epine in vi* dre widc spE d itrdGrial appli€tion ofrhis eicrebial @F. prejetwa d6isn.d ro pbdu@ p@ri@ by ,.tp.rs,?6 rtd' &otu slbmcrged fedeolationwitlr m.jor obj.ctive to develop gmdm for bcn r perird6 poducli@ and ttE orhdobjdtiye of lnis rfudy v@
L Sq.e iagof,lsp.tgills nieq srliB for 6c podu.iion ofp@riM2. Optmiz.lion of culrliot @nditioB for sulhdg.d femorldon of citru
p6l Ior palill.s prcdu.tion
20
3. rywe.rtofrtuhlbdgd.b.tldtd4rdtloedddffiofrffar&
+ Stsilicddotodot &cdd6d ridd d tsnto d4. sEh.ceEdrll.!(blidof@tc
5. eoldid rd !!.if.& of.q,a
2t
MATERIALS AND METHODS
ISOLATION OF ORGANISM
Sanpl.s froft dificrc.t habiraB ($il, c@l water, fruir debri6, rouen apples, roieD
br.ads, peanut, naize sce&, <lmin walo, ron€n cirus Dd airl weE uoltecrd rn
serili2ed pol'lnh. bags or diBrly on PDA plat6 fron difr@r t@alrries of plkNhr
i.e. Lahore, Sahiwal, Ok@ Md Faisatabad. All 1he samptes weE noisrened sinply wrrtr
stqili4d dis(lled wab. kpcrSilli @ chml.rized by o rrprat morphotosical
rtucorc. A @nidiophorc which includ6 rhe sbtt ed t[e dnidial h€ad, eiss f.om
spsialized thick *all nycelial srncturc qlted foot-ell. ConidiophoEs &c non+eplale,enlegcd towardr lhe apex .nd t minate in a globos., colortess ro ye|ownh sweltrng (thev6rcl.t Th€ v6icl€ bcd ov.r thc wbol. surf@ tol5lm tong, uualy bb*n.metulae, which rhcnsel!$ bd phillids. Conidia d€ poduc.d successlcly fiom the
nps or the phialidcs, thus foftin8 uhb@ched dry chajns. Conidia de sn@th orommmted, hoE or lcs glob@ {d 4-j!n D di@cter bul @cdionally smaltcr
The tunsul cuhB Ncrc puifiei flm b.tdi.t cofillMb oy us.B llrmgl olchlorostrepiomych in pctdplat medih. A the isolatcs of iungr were idenlificd bynicoscopic exanin.rion mdd coDpoud nicros@p. after onions 4 a/. (t986).hd€penddt coloni6 of.&n id.nrified isotatB w@ pictcd up dd trahsfeftd to C2epak
Dox aSar slmts for culrue nainrennc., Apprcxinarely 4nl of hediun *as pouql ftothe individual stqiljzed cutrur tub.s, Ancr solidii/ing in slandng posnron .( toomtenFBtue, fi. ncdium of a.b rd tubc w6 inmLrcd by rEns&ring cond,a ofAspdgtll6 niger wfh rhe t.etp of srcdlized in@utatilg needle ed were incubarcd at
25120C for t dat{. After grc*lh rhe tuogat cu!tur.s w6 srorcd ar 41loc in refiigdatorio ue firtld for scraine r6t in rm ofrn n Fcdn utitizarion rbiliry.
Chapkr 2
2,1
22
2.2 PREPARATION OF SUBSTRATE
cirrur pel ws oll€cr.d ton lh. fiuit juice vflloB of th' ld'l nr*!t of citv IdoF
ciuu! Del ws dned at ?dc lor lire divs in ovfl ed Eround lo powda sirg e.letri.at 8nndd, Tte powdd subdr.rc *s ldctionarcd throuSt sicvc! of M$ft No 20
lbe li€ved powild of cittus p.el w4 slotcd at rood lcopdtuc i! .n tidi j& od uds a &u@ ofp€tin in turhd studi6.
2.2,1 Preparrtiotr of Inoculun/SporeSusp€nsion
In ordd !o produ.e copiols number of spots for in@ulus, f6h cilr6 Pcels wqc
choppcd itrto sall pi6.s. Abolt 20 grd ed al@llved in . 100 ml flak fot 30 no. A
l@ptull of ntlelM &on Czepak Dox ncdium platd sls added to th. fl6k dd w.l
shaken to ensur€ cqual disLiburion of ino.ulum- And ircu!.lion .t 30 t2'C fot 8 -10
dats, rhe infatcd citr6 pals wcre agilatcd wiih dislillcd wats 10 giv€ .Glliirg spoE
susoosion of ld soo6/nl.
2.3 PRELIMINARY SCREENING OF A. NTGERSTRAINS BY PLATE METHOD (QUALITATIVEMETHOD)
ln pbt s {ft pE?aEd co.taining Cz?{I Dox m.di@ supplemdred wirh 2% Fltin6 r @bon $N. imtead of glu@F. Tlrrce wells w@ m.d. in @h p.t iptal. with
sl.rilized @rt hoEr (l3mm) in tlhind follow chDbq. Corjdid sup.tuioD of,4, ,i9.twB adjued by h@'romerd @ lxld spoc p€r mt. Tnis @nidial $rydion w$pouFd into *h w.U @ 5qrl &d pt.i6 9@ ircubar€d .r 250C. A0d 4 dlys orincubdion pdiod, pll&s w@ llood€d wirh 0,0t% rurhoim rd $tuiio! for one hou,Plard w@ wslrcd Mlh t!! w.ra r peli! d.SEddid *D @idd1 by clod hemmd th. funeal .olony (Coty e, at, t95), Tt dima4 of @t@i6 &d cl* atr6eft m.suEd. Th. rcl.liv. d{ am w€E d.t sbcd cing ihe followirg cqutioni
Dimdnof cl* an.(mm)Dimeid of f6sal @toDy (m)
Th. sfains cxhibiting lhc latgc El.rivc cla @c w@ scl@rd for ftrtha e|6in&
2f
2.4 QUANTITATIVE METHOD FOR SCREENING
The cultuc Ftdld and qulit tivc sl@ing, w@ fulho @nfm.d bv queliiitivr
mcrhods, in ths of polygal&tubnde, petinlye sd p6!i!csr.e ujl! pDdrction
by $ina $bm.rgcd femdrdion in 250m1 Eelm.tq @nicd fl4k @nt ininc Czcral
Dox bre1h [email protected] wirh 2% cit us petin $ . solc cribon ele,
2.4.1 tive Inoculum Preprrrtlon
fifry hillilic of lho bmrh (d.prt dox) wirlr 2% dru petin rnd i.iriat pH !dju.r.d !o
2.4.2 Submerg€d Fermert tloo
6.0, wa Ee|G.!d inro ! 250!D! @nical fla* fottow.d by th. rddirion of 15 gl.r5 b..d!(2.0mn dia). ftc nsk pfr plussed dd 5rditiz.d. tou nl ot 6nidi.t sp6ion! ( tO6
conidiar'dl) w.c irrdsfdEd lo inc 0st, which v6 thd| ircubdcd al 25 i2 0C on a
Didy snata al 150 rpm lgiiation fo.?2 hB a.---'
)
ldararion m.diM contli .8 in A/L :KH, por, 4;FeSo. 7H, q O,a Na, tlpo.0.2ic&1r,0.01; (N&) So.,2; md (in ngll):Mnso. _7 HrO,70; Hlpol' l0.dCihu
,/ P{nn Qo bntL) w .ajEtcd io e inirid pH 5.0-6!tomdo- ald stsd, t98).Twary liv. nilliliGr oa fmcnralioi m.dim v6 r,t€n itr a 25ohl plugSed @niql,lqt. AAd sGritizrion dd @oling at rcob i.mpeEruq it @ irc@larcd with 2.jnl ofconidi.l inoculum ..d incublrcd on Drlry shskq a( 2J0C {i$ Isorpn fo. % h6. &.hisolar. had four fmfit!1ion ns*s, spl* w@ Irt@ an( 24 hB irrldrls duing 96hs, filcrd thDugh Wtuine No I fitd papei lrd w4 pe6$d iMcdjlrely or srorcd
2.43 Atrelytlcrl Methods
2.4J.1 Pollgrlacturonrre A!!.ys
PolygdlctuDns. &tiviry i! c.I 6E !up.6.Lrr of *|acd,i. ,isrr wF !g'rd byquetifyilg r.ducing 5u!6 by BinC DNS (3,5- diri[lr.ticylc &id) eeilod (Miltd,1959), BbS D-gl&tuenic &jd 6 a rcfeMcc. th. @ction h,rtur. conraninc Iml of
24
culture filtrate, I nl of 0.9% pol)E.lacn@nic acid in 0. I n .cebr. bulId wnh pH 4.5 and
*as incubatcd at 45!C for 30 mi.. (on€ uite of polyg.hcnm@ w6 d.lihed $ lne
adou ofenzlde that libdrA one Fmol€ ofFlacruonic 4d p.r mmut€).
2.43.r.1 Shndrrd Curv€ of lrcahctumric lcid
Hundred ms of D-solactulonic acid was dis$lv.d in apprcximarelt 70-30 ml di$lled
sarq ed raised rhc finrr vohne up ro 100 dl wirh distillcd w'et so thar each ml of the
solution contained I ng of salmtutunic acid (0.1%). Td appropdate dilutiois (10
100/d wcrc md. in 3.0 ml of distilled w$er ThB millilit€6 of DNS @g€nr q6addc{l in e.ch t€d tub. containing dilutiom, The mrxnnes w.r. boil.d for l0 minurcs ar
100"C on a warq b6th, @lcd ar tuh tgnFB@, wnh a bl&t tu tadllel h0' I nl of
dh(illed walr. The ablorhance was n€aulcd at 550 nm on sp€ct@photometer. A graph
*a ploncd b.s@ ab6orbd@ and D-Cal*nn@ic &id @n@tllio6. The slopc oftevaridble was calclld.d and ued fo. the n6urmdt of rcduci.g sug6 eleased by the
act'on of p*tinollric eEyn6. St nddd cwc of D-grbcn@nic a€id is Srren in
2.4.3.2 P..tlnly$e tus.y
Tbe adivity of pectinlrase ws indicated by the fomatio. ofa ied chromagon whrch had
nrxinm lbsorbee at 548turl (Peis Dd Asnselt 1963). 0.5m1 of cnzFc rv6
inclbared lor I hou at 30'C widr 0.5fr1 of 0.5% p@tin md lDl of 50nM Tis'HCl
buffcr of pH 3 md lnl oa 0.2nM C.CL. An€r I hou absorb..ce *as ndured ar
548nm One uit of lhe PL aclivily wd detined 6 rhe mounl of €bzyme that rnolased
thc .bsorbance by 0.05 in one hr at 548 nm. Controls conhn.d autclaved enzymes
ioltcadof aciive cnzlmes (Jdn e/d/., 1990).
2.43.3 Pectine3terase tursy
For P*tinesledc etiEaiion, loDl of 0.5olo (Wrr') p*dn io o.lM NaCl was added m
2nl of fiftdtc. Tl| pH w.s .djusEd to 4.5 vitb 0 lM NaOH sd miitur. was incubatd
or 60 nin ar 350C. PE activily wd mdutd by dct minin8 cdboxyl eDup, releascd bv
25
tiF.tid *ith 002 N NaOH' (Keftz' lg5t Oncu t ofrE v6 dcfin'd $ lhc omou
of dF. cl.sins o.c ni@cquivtlcnr of 6t'r hldrclvzcd lc&boxvl gbup) Ps
2.5 MINIMAL INHIBITORY CONCENTRATIONOF BENOMYL
corc4fttion (MIC), Pacnirl 8|'!s w€re
vith tdo6yl ( 0.00 _l.qr/nl rt tuDIn ordd b fr.d out the ninimal i.nibilory
goM on Cz.P.k Dox nedrM dtrichcd
l€@dr@ (Bok ed BoYDd' l9?4)'
2.6 CULTURE IMPROVEMENT
lor th. sdkc of clllw inpbv@ac
w@ rre.lcd lo vdiout nut gtu
2.6.1 Ultrrviolet irr.di.tion
dr. el6.d olt p*tiroltric at{M of ''l 4gs
FNn lh. p&nt t tliin (5 d.F old @lt)F)' lO 61 of co'idi'l tup'Gid @
!!nsfc.r.d to @.icat flsk @dainiry 25ml of ctdiliz'd Czeg't Dox broth Th*
onidia w@ rltowcd to grcw at 25d C on rtlfy shakd wilh 150 Dn for rbout 6 hF to
set Aeih gtowitrS tunsrl nv.eli{ Fiv' rniUilit'r of lh' brclr cont'inins m}telial
suspdsio! vs toEf.ncit to t staili2cd pcEipllt's 'nd
th€sc dv'clb w@ crpostd to
dFdvioL{ im.ti.lror fq 60 binut s wiu l0 Eidtd i1tu illdvd unda lh' bed
G-253!n dd 220 v .t 50 C.€e) of W l@9 ThG ndi'non do6' sivd to Oc r]@liat
su5?6.io! v6 l2rtdt/n':ls
Th€ disiancc bdw@n ldp dttt susPdtuion ws adjult'd al 8'Ocr for '!'h
tdd ro g't
moc than 95% dcdh dte (Azin ed Nobozi' 2001)'
2,6.2.1 N-nethvLN-.itro-N-nltrotogu'nldlne (MNNG) trertm€lt
Th. Da@tlveild strsB v@ eioM for 5 dtt! to gpt lwinu sfmhdm Cddir
from the cultuG w@ sugpald.d in tldilit'd Cz"o& Dot brert sild rh' t'llel or
A.shly sroM 6y..li! (6hF tlshlv soM) ws obt'in'd Thde m)$lir w6' dpoced to
26
1'o mrrnl. MNNG ed in rbet !' 2fC on wdt! brh for 50 niiut's wii! liE nn'rvd
of lo;dc! lo iiid. lDc dath owc with |trb_l'tld l'v'l' vlulc k'?iry t[' r'lciid
Eldr c.ri.nl .! 5.ool {ilb 0 lM cir't' brfr.. (Adclb"t 1955)'
And s!.ma{ wirh MNNG' l0nl of cFcin (l o % v/9 w 'tHGd
to 3toP th'
n.too. T d of MNNG *tit Hrovcd bv caarin4rtioo of r! 'rioo
ni$ft 'tl0,oo0 rPm for 6E miNt6 .nd tllc
'ltoFitrc *rClin8' wilh 0 l M pbo"holc citrd'
bufilr (pE ?.5). Tbc lat d m)Eclit w'c dllpadtd in 0E @c bllTcr'
2.6Jl Ethyl dethyl iulPhotrrt' tredo'nt (EMS)
Ethyl Mdhyi sdttdn t (BMs) wiot osM !!strl! ws 'ddcd
to tt' fdstd/eild
nr.li. (6 h[ n .l v gl!u) tur 3o oiluid *i6 6rc BtNrd tiot inlsnl lo alics 'cuft wirb ,lb'ldh.l coidilios (Sb'!trI'n
'td Nflfo' t9!9) AIia i.vi4!d tit!'
iniqv& LOol of E did nix!!! ert sitLL'sn ed dtld'd 2 bld in 0lM phoqDd'
citu bltfa 6rt ?.5) io @ lbc t*iion lhc E r'ct'd wdtilg. sio! 0 t M
tlFlph.t cittL bufLr tq! si6 to Sc EMS tF 6]! li' &d r!'' *@ rha
@t tdcd in ltc .&|. hofi..
2.63 S€lection of Mutrnb
Ant qoGur'ftdnot *irh mrt|gaiic tS'nE! rbou 50d of ach $!t(t'on
6ai.i!in8 r..lcd nlt.lit {it drtti'dlv tr&tfitfd to tlt' *'lb in ildividltl
@ipbld clil.iiint et?* Dor 'g!t
o'diltD wilb 2% citr' t'din i tcd oft|!'Dc
|! 1h. only c{ton sut!.' Th! Pld" w'(! itBuh'&d d 2fC I 2 '!d
{q! '*!ni!cd
Fsut..ly rfra q.d.d 2a h[ lirc i nvd io ttudv lb' glwl! Pd.rr Tt' colooi!'
; ltlcd.d qu.lit liv.lv' ltowioS lhc cL"t@ rmrior *hid wr viribk 'nE
n@diry {ith 0.05% nrh@i6 nd di' std w6! Eri 'ind
on Q'9'l Doa 'tr 'lai&SinSl. 3pd!. w.. Pick d uP nob lIS.r @lonict '!d
scft rlli,n d Cz?* Doi
mcdim {ilh O.S rgnnl co|Ftrtio of B'lbmyl ro 6ad di U' Bomvl ditlrr
.E iD. s.l.d.d d.i *@ t€lcd quniitdEly fd "zyrn
Dtod!'liod bv tLtL' Ilti(
2.7 OPTIMIZATION OF CONDITTONS
Slbncrgcd f.mcnltio p|!.* w optinizql bv 6udvbg th' ell@l' of vsrvils
f@notltion pqiods, pH 4d indulm sie, with vlring 6@ntr'lion of adilrtioml
dinoSptr eutq yest qF..t ,!d Twa-Eo (a surf-tdt) in Fplicd'd fltsks' lhc
sr6t 8y ws !o mainlain the pdiouslv oPdnizcd pannct'ls in th' $bscqucnl
ilv6tig.tioB opliniz.d in one qpdim@r ws Mintri!'d in lnc subsequ4l stu't6'
2.7.1 Ferm€ntation P€rlod
To d.tcdin. rh. oDtimun in€ P€nod for mdimm prduction ofp6tina6 bv ''l- 'i8!th subnaged mq{tim of ciE6 pel 2% of growd cilru5 pel wd used *ith o$d salt! ir
all sampl€ fldks. Th. indolalcd fl6ks wd i@bat'd al 250C ed duplicd' mpl?s
weq e^yz.A ^ftet
24, 4A, n,96 ed 120 bols of liquid stale fm@laiion Pati@ca
poduction vs optinm !ft.r 72 ho6 incubalion
2.7.2 Optimal PH
Optidd pH for nditul a.tiviiy of cnrdc pdtiruos wd delmin€d bv n€6uir8
@F. &rivity ar v&ious pli i.c 5, 6, 7, 8 ard 9
2.73 lDoculum Size
Th. fl6k coriEinirg medium werc inoculac'l with 3, 4, 5, 6' ond 7ml of
iroculm ro s.l@r $c mosr suittbL sit of h@ulrn fo' p*tinas's Ptldrchon
aftcr ?2 houB of fennentation bv ,4. tlier'
2.7,4 Yeast Extract
Yeisl cillct wrs us.d lo ctrlmc.lh! f.nn@t tion mtt To studv ihe cfi'ct of
y.st.xt act for oPtinum Produoion of pectinar6 five diffdnt concentation of
y.!sl €xtlct (0.1,0.2,0.3,0.4 $td 0-5%) *qe B'd in duPlicatc flask to dete'mtu'
irs oDlinunlev.l forD.ctinascaprcductionbv-'{'it"i
2a
2.75 Tween40
Effdr of .liffcrent conccntarion of Twcco_E0 on subm€rgcd f€rmcnlstio! w$
sturti.d for maximun Production of peclin$es aft.| t2 hou6 with 6ml ino'ulum
siz. at pH 5.5 under pt@Pdlnizd cultute @ndrtim \
2.S MOLECULAR STUDY
2.8.1 Fungrl samples
For th€ study of Sdetic div*itv, among th' Parent md their mut$ls' thcs'
w@ ana!'z€d by uing RAPD lccbniquc st Institut€ of Mvcologv sd PlDr
Pathology U.iveFity of thc Punjab Lahorc.
2.8.2 Isolation ofc€nomic DNA
The total Senomic DNA isolalioo wu pefomed using CTAB m€thod (Dovlc md
Doylc 1990).
One gm of youns nycelium wls cul, wdhed witb disliu'd wai'r, {tried and
grcud into ! very fin. powdct wilh liquid ritrog.n iD pr' @led ed e!o'hv'd
pdsrcl and monai This grolnd powdd wrs ttnsfercd into 15 ml bluc cap
nlb.s(Fal@n3 tubc). 2.5 ml of !rch..t d 2x CTAI (65"C with 02% p
[email protected]) wo! tdd.d lo tub. bcfoc Ao?,n powiLr sitrt thawins n ws_bixctl gdtly by inv.nitrg thc tub. fq s.vdl timca and itcubtted at 65 C for balf
d houi2.5ml of cholorforn: isouvlalcohol (24:l) w6 'ddcd
ed mixcd 84dv
by invcrti.S the tut! to fom an einulsion and ce rifugcd for l0 minut$ d
9000rpm.Su!.mata (abov. naldial) *!s t'.ntf'td io t ncw fal@D tubc 06
volume of lsopropdol Gr.chilled) was sdded ed mixcd gcnilv to pr'cipitdc the
DNA. Cdlritugation w!5 donc d 9000 rpn for 5 minutts sd suPqnat$t was
disolrded.Pollel wa! iaketr and wdhcd with 700lo cthanol twice Pcll't wls an dricd
dd Frsp.tdcd i! 20li o.lxlE butrcr. Thcn 2ul of RNA4C aa5 add€d '!d
29
incubated fd I hour at 3?C (inixed gcntlv b'fore incubadon) F4ual volllno of
chol@form: isoamvl atoohol (24:l) wa! .dded lnd mix'd s'ndv Spin'd for l0
minut s at 13000 rpm in micrccmtnit8p and sup€matant was lsk'n l/t&h of 3M
NaCl es aitded.trd mix€d g.ntlv The DNA was P@ipittt'd wilh lblolulc
cih.nol (cel.d) Slin d |t l30OO rpn for lO hinut6' $lpddl w!,9 <hst&d dd
warh thc D€llct wilh 7oolo ctt6ol. Th. Plllet was rI. dticd ud r6uP'Ddtd i!
0.1xTE buffer or drHp. Thc @n@nlittio! ofDNA wB ncatu&d !t 260trm
2.8.2.1 Erqn.tiotr of DNA Concentr.tlon
For lh€ stinltion of tolil gcnomic DNA con@'tntion of six sMPlcs'
$sortdce wls lne3surcd !t 26OM on sPeottoPholom'ier' A solutiotr wilh so.D.6 of I conlains 50Fg of DNA Fr nillilitlr. DNA quetity wds c{lculakn bv
DNA Con. in !g/nl = Absolbde at 260 nn x Dilution ftclo. x 50
2.82.2. Qu.lity ofDNA
Qlltity ofDNA w.s chdk d by Mning 5 ul DNA on 0.8 % !g!!sse 8cl Pt!p6!!d
in 0.5X TA! b!fid. Th! DNA s.nplcs Sivirs tnd io thc sel *@ nj@t d
Wnh ihc .dvent of rcR Echnology, it has becom€ po6sibl€ to siudy gcnclic
dificrence in tunsi 6nd othd o.ganisms. RAPD is ! new techtiquc b$.d on rcR
(Wiuid ., a/. 1990). It utilizs singl. shon mdom prid6 to @Pfiry !ud@
DNA in rh. PCR. Shod oligonocl.otid. Primd in rcR oclio6 wih ldcEd
melitrg tanFnnr mPlific! i sletrun fiom .ny tdnpbtc DNA. Oae or norc
of trd ar! oft€n polynoDhic and this Polytnorphis (RAPD) can b. scn.ticallv
2.8,3 Condition Optimization for RAPD analys€s
DNA @c€ntation in thc *oddrg solution of approxinately 80'l0or8/ ul in
distill.d tlro w6 @nfm.d by spe.boPhotom.ler. For RAPD Mlvscs (Williffi
JO
er dl.. 1990), @ncatnlion of gcnomic DNA, l0 X rcR butr€r with (N'HrlsO''
M8clr, dNTPs (dAT", dcT?, dCTP, dTTP) 10 mcr mdon Pritnd dd Tlq DNA
polym6$€ w.rc optimized. Th€ l0 hs. oligonucl'otide Filn6 obtain'd fom
Gcn€ link cripdy (USA) s/cre us.d for thc ar plificalion of lho gcnomic DNA
2.83.1 RAPD (PCR) reaction
Th€ following PCR r!!g.nl! with oPtimirld @nccntdidr/Mount wft us.d i!
RAPD (PCR) Prolile
Daatu izing ti 94qC
D.utuntion at 94 qC
AnncilirS at 29-36'C
El&nsion * 72'cTolal cyclcs 44
Final extension for t0 minutcs at
2.E.4 Agarose gel el€ctropboresis
for I ninutc lfor 2 lninuies
1Z'C.
Thc rcR producis w€.. clQtophoc.d al 80 V in 1.0% a8re gel for.pproximarcly 2 houE using lX (TAE) bufrd ltong with . DNA nd@ld siz.
2J.4.1 Agrrose gel elcctmphor€slr tnvolved the folowhg sreps
An .dcqu!. volumc of cl@trophoEsis bufi.. i.c. lX TAE buffcr ws !rcpar.d to
fiU clerropboBir rAnL ud to prlplE th. a8@se 8et, Dcailcd uout of .Astosc(l.09) wa trto in ! lst, @lsiriry €l@tnphodn bufft! tx TAE (l0ohl),loneh i. miorowav. ov€n for 1,5 lninurd dd ws lwided to cnlurc .v.n lnixing.
Gel ca.ting lray wa pEpsred by wlppin8 lEhing tllc on il! borh 6id.s and !suibblc comb was adjBlcd on it. Th. mclt .t ry.rc6€ wa! cool.d lo 55"C ro 45"C
bcforc pouring otrlo thc 8el crsting !!y md .thidiutn bronid€ (5|lj) eus added inio
3t
it. M.ltcd !€!'w pour€d on thc 8cl-ossting !!y; Epls tnicdlv pour bctlt€.n $c
thickns ofO.5-l.Ocm. Bubbl.s w@ ftmovd. undm.ath $c conb d otr thc
ru.fac. of gcl and g€l was solidificd al roorn lemr@turc. An€r solidifioatior ofgel al rooh lctnFlatuE fE cohb wB mov.d c{rEtully to avoid learing ofwclb.
Ttc rnrstiDg hF wB .l$ Emovcd from lhc aidc8 ofoasting F.y. Thc gel-.asting
tray @!t!inin8 lhe g.l wa pl&cd in electrophorcsis r4h hlein8 lx TA! buff.rido n. Bcforc l@ding thc rcR producr! i! lh€ Ell! ofgct, 3 ul of6x loading dye
(Brcmophdol blu€) wG !dd.d io rhc rcR producr!. Onty I Oul of RApD DrcductswcE lord.d on lhe gel. Mskd ofhrom molccltar sizc ws told.d on bofi sid6of thc 8.1to oEp@ ahc .ia of difrd.ni lEDlifi.d nlgErctrts of 8clomic DNA.'Irlc wi6 of .lErlphoGir urt wa! [email protected] ro !ow6 suppty lld wcr.sdjust d !o a c.rtlin volrag. (80 V). Movcm.Dt of dye indic.bd mignrion of DNAfton dode !o clthode ihrcug} gel.
The fitrgerpritrts w@ crmincd unde. ultra viot4 hnsiltuniDior edprrologrryhed using thc cll Documctrtario! sr!0.m.
2.8.5 Ditr ADatyris
'Irle badi wcre @uncd by sbrii.g fion top of rh. Id6 k! th.ir botrom. AIIvisible ud unmbiguolsly s.o6blc ftagnents .rnplitied by rh. primcs wcEs@r!d uder fi. hejditrS of lorrl scorabb Aagncds. Amplifi.{rior Frofile ofsixsamplcs of/4. ,r49ef w.rc ootnp.r.d wifi @ch olha.nd bdd! otDNA tiarmcrtsw.r. scor.d s prBdi ( l) or abs.nr (0).
In odo to ec6s $c ov@ disribltion of g.D.ric diiftity, Minirlb soft*lr!vcFior forRAPD daits.ts wcrc us.d.
J2
2.9 PROTEIN ISOLATION
2.9.1. Precipit.tiotr of Prot€in
Tt. culnrrc fiIrlle of mutar.d stnins wer. p@ipilat d ai diff'rcnt oolcmtmtio$
of aDmonium sutpbate (Nt!) ,SO. fiom 30-80%.Thc aqucous Pha? so fomcd
ws scprmtcd. Prctcin ptcipibtion vs canicd out oa ice bv nddition of ult a
gmde ammoniud sutphsl€ lo thc lqu@us phase. Tho aqucous Phas. s"s dtd kcPl
ovo Dight at 4cc.The potein fsctions werc prc.ipitded out by bindl.8 with
mmonium $lDlat at 70% satumtion. Thc pEipiilic6 werc collected by
@tiin€.tion at 8000 |pm fo. 30 mirures. Th€ slid pell€$ oblaincd al ff/osatuFtion w@ th.n dissolve in dislill.d Mi.r ed dirlyzed against distilled w.tcr
st rcom t mpqaluF for 24 hous to derllr th. !rctcin,
2.9.2 W.shlng of Dialysis Mernbrrne
The dialFis thdbmnc was washcd proP.ily bcfoi. dialyris. Abour 156 td8pic6 of dillysis tncftbdc wcrE cut and dip.d in solution of 2%NarHCOr edtmM EDTA ard wB boilcd for l0 minut s. Thc m.mbm€ eas.id.d wirh
distilled water 3-4 limes. The membEne was .llowed to cool a|ld dipp€d itr
distilled wakr to be sloFd h Efri8cl?ror to b€ usld again.
2.93 Molecular Weight Determitrltion
Molccule weiglts of isolated p.clinrs.s were n€sEd sing SDS-PAGE.
2.9.4 Polyacrylamide Cd Electrophor€sis
PolFcrylamide 8el cl..trophorBis (PAGE) itr rh. plts€n@ of l% sodium dodecyl
sulphrtc (SDS) w3 pdforn.d on veni@t rtab g.l. The prcrcin srnpl. wsprcp!rcd 4: lqrl ozync sntrplc w6 nix.d inro loopl- of T;s HCI,30 rL otthis mixturc wd nixcd inro 20 !L of loading dr ad heted at tOOF,c fd lonitutq i. dry bad. This nixtule we then loa.t d inro wels of ihe gcl d€pcnding
uDon th€ comb size. Thc .lcclrolhot€sis wls conducted d conttant volhg€ of 50V
for 3 touE at room l€mpcrature. The eel wa! slthed wilh coom.caie bluc fo.2-3
houls and d6ttined wifi destaincr for 30 minutes.5 !L of prcstlincd pmlcin
Iadder ws llto in €pFtrdorf aod lEatcd for l0 minutes al l00qc and os.d rs
2.10 Large Scale Poctinasos Productiotr
Thc p|l)Gs ircludes Drcpostion of itr@ulum dd f.menration mcdia or which ro
cultlre 0F orgoisn for terinases producrid. Thc fcrtnent rion @nditioru srch
aB pH, t€mpemtuE and sutgtEtc have alrerdy b@n optimized !o obtaio rtrc
naximum ield [email protected]. Tlc crude erzym. ws hrresred ard coacenrnted
by an4oniutu sulplslc pEciprtion.
2.10.1 PrepsratioD of Inoculuntspore Susp€trsion
In order io prcduce @pioll3 number ofspores fo. inocutm, fresh citrus pects wer€chopped into sDdl pic6. About 50 gm w€d [email protected] in a ro0 ml flask for 30min, A looptuU ofbyetium frcm CzC.k Dd n.duh prare w4 !dd.d ro rI.ll.sk dd w sh!&cn to €BuE equal distiblrion of inoculuh. ARer ircubation ar30 t2'C for 8 -10 days, the infccicd citrus pecls werc lgirltod with distitt€d waGr10 give a resulting sporc suspcnsjon of I06 sporoymt.
2,10.2 Fermentation Medium (Solid Substrtte)
One huDdftd gEnr n@ lra! ed I OO gn chopp.d cirus peel w4 moistcncd wirh200 ml 0.01 acctate buffcr, !H 4.5 (nrio= t:l:2)stE diQ.nsed p€r plljtic rty(30x15 cm) and stsilized in dioctavc. Tllcn 50 nl of spote susFnsion wsE@ulaled per rray of thc feddr|lio nedium. The @ys wcre @vered wiihsienl€ news papa ond ircubared fo. 4 days at 35.C.
2.103 H.rv€ldng of P.c6!rse5
ric mld. p.di c5 i,.m cf&t d bv &lding oo' littr drsli cd w!t" F trv of
fcrm.flt|tion n diutn. Thc moist ncd f.rmcntalion mcdiun w!5 slimd for t{o
lnur rt ubisl room tcmpcnnrrc. Tt mixtu! wtr filtc&d though nylcn [ct to
!€paraic thc ric. bEn .n{l ciitos D€cl. Th. oru& a|zyhe w!! colLcld od pbccd
ar 4ic.
3.1
Chopter 3
RESULTS
ISOLATION AI\D SCREENING OF
PECTINOLYTIC,4JPEX G I LL U S NI G E R
Finy $o i$lar* of,&p{8t&, ,,8.' wa i$lat d aDd id.nrili'd ton difc€ir eut'ls
(TabL l)- Isllca of,'{. ,i8dr *cre ereM on Cep€k Dox n'dim suppl'b6&d Mt}
2% C?P. Enz}r. lcrivity of tudgs A€ CF s inv6lis.t d bv wU pld' Lchliqle 3!d
.nzlre as}!. All iehlld si.iE of,4 rr8tt lw $d.d bv PtliDi!'ry $tgitg by
Dlate m.thod. IJnd€r lhe eiv.n .xp€nrcd.l conditiN' pc.lin d€gndation bv ''{ t'gct in
wll plat {4 .viddt by cl6 dc mud tlF nugal @lony whd 6 dats old cdtuG
pld6 9@ loodcd sit! 0.05% rulh@im t d toldiotr lhc ElstiE dd 20!6 (Potclcv
iod.x) {€re cslculated lrE rsuli. shoM io th. rlble I indieted lhat livc culiG had
Elative cled @n€ abovc 1 70, eirtt c! cultw bnd rcldivc cld zonc betwo l ?O
1,38 .rd twcny ninc cullu6 hld FlaliE clco 4* bclow l 38 Th* ielal6 wG
cot 8oridd d hi8b nod.E|. 6nd tow Fclines pddu6 Es!6tiv'ly Hielt pelil@3
prodeing sfAins €xhibn€d I 905, 1.161, | 743, 1 742 and | 74 rcldivc cld bft md lllw i$laLd from ci.s ed .pPl. liuic. Thc hqitEl aldivc cld 4G *s ob$ry'd
by strai! No H2? ic. l-905 T!. nidnm clatiE cld d6 sd I llE I ll8,l ll3and 1,090 by H43, H49, H40 .Dd H4l csFdivelv llt$ fou srrains wqc isolaled Iion
sta dd u. Thc hig! &d nodaale p@tintrd podl|cing st?is wF sl6tcd for
ilnrF sfr](lia od low D*titlls prcdEing snid wG djstd'd- ft' clzvn'
Dodstron bv 23 et.cled smiN of,4 ,js?t w snldi.d in !ub'n'rs'd f'motdion wilh
36
Trblel! Dirm€ter ofclear relative mrc by'4'tigdr ltolates
Edi6t-EEdG----Tiffi n' s"ttr't ndr v'.'.i drc : : , !'+Di€
H21 l-905 ! H09
lt 50 l.?61$ H08 1.338ep
H5t H18 L288 f{
H02 H14 r.283 kH12 Hlt L2?6 s-q
H05 1.625 b. H25 1.L15 elrll45 r.570M H0l r.88h{H29 t21 1,235 i-q
H06 H39 1.221i{
Hl0 H03 1.221i-c
H3l 1,461c.g H38 l,2l0i-q1.206 t{Hl7 1.446 eB H04
H72 r.426 d-i H3t l.206 L{H42 1.425d-i H44 l.l98l{H46 r.420 d-j H20 Ll95 n-q
H48 l.4 d-k H37H 22 l,4l0d-k HJ]H19 L406 d-l It 30 1.168!-q
H24 t,406d-l H47 Soil
H13 1.395d-n Hl4
H36 soil 1.393 d-m H2lIt52 1.386d-D H26 l l30Fq
l.ll8 qHll H4lB7 H49 Lll8cIt 16 1.351ao H40 l lll q
ll2a H4t 1.090q
vrlc *idr difid ke rhow .ig'io.d diff.t.* s d't'i!'n bv DMr Td
37
3,2 PECTINASES PRODUCTION IN SUBMERGED
FERMENTATION
Enzync poduction by 2l stnis of,l. t{94 vd sMicd bv subm'rged fmQ !tio! in
rh. n diM with p€lh s a el. cdton $l@, ln ordd to invetiSab thc q@titativ'
l.reh ol DolyCetM.!." pdtinlve sd !.ciin6ldse, dl lh' *l@t'il t!!iN w'E
gFM fo! 96 hoM in 12 ,l8k! for.!.h 6l4in !u8B &'c c![w nltrat6 w'rc
6ll4tcd .ftcr cv..y 24 hou6 &d $e etivitv of pdlioolvlic 'nzvn6
ws ddmircd
undcr oprimjz.d 4sY @nditionc
3.2.1 Polygal.cturonrsc Activityi
Thc qudtitativ.lael ofpolvg.l&hsNe ss inv6tigatcd dift@t sitgc' of fogd
erewth, rtl lh. pFliminaty *l4led ttliDs scr. Sroen for 96 houc dd on' lb* and
d€ry 24 hos ss withdnu for @r!c Mv for all individual slsils l w6 obw€d
rhat ndinu alivitv vdi.d sigrili@tly fbE on itoltle lo the other' R6ull! cl€dlv
indieled lhat stnis H06, H13, H45 ed H46 Avc ndinD &tivitv aft{ 'ncubdion
for
?2 hos i.e.0.492, 0.620, 0.392 dd 0450 U/mlmitr r€sp@tiv€lv (Fis lc) 4d $a a
d@line w$ obwed al 96 ho6 i.. 0.a82, 0.400, 0-350 dd 0 402 U/nl-min E3P{tiv'ly
(Fig. ltt). In thc cse of trtining ield6 rher€ ws s conlituous in Ede id aclivitv up
10 96 ho6. Thc cullu. Iittrr'rd of rais Ht3, Hl9, H22, dd H3l' H40 ed H45
d@ict€.t lh.l4l &tivily i,.,0.400,0,420.0.420,0425,0.350 dd 0,402 U/3 _nin
[email protected] (Fig.ld) aftet 96 hou6 of,ncub.lio. Stoin ltl2 glvc 07E6 U/nl'ni|\
srai6 Ho2 &d Hso showed 0.686 u/al-bin whil. sinin E5l s.vc 0 ?20 u/mlnin (F 8
ld), Tbc Iuimun activily of polyg.leturcnsc ws dhibited bv ltzin El2 which
show€d 0,202. 0.49. 0,620 sd 0.?86 U/nt-min ana 24. '{8. 72 ud 96 ho6
38
Artvily of PG.s. rn r2IHrs
=0,'.u5
adl
En 16 Ea H0 Xll m2 rrj tm H$ r@ lol r?r l!9 ril llr 16 xa l|5 ll llll L0 l5l llit
Sr.b
ns. l. & b: Pdygdet@ne ..livity rftd dift.at iMhoti@ Fiodt Dv 23
i!.iB or lsp.tsi B tttsd t 24 r|rs, D 4E HE Vcnic.l b@ $owsta lad aror of m.G of thEc..plioB. Bd with diff.Ml lG$cE !!oesigif.rlr dific|!@ (FO.05) G dddnit d ty DMR alt
bAc0ily ol PG{o .ltfl lN tln
08
:01
ro rG 16 Hro ilr fi2 ffi ftt H9 rc rlr rrt r!9 |3r lc r0l rr2 rl5 rl' r{a r!0 rl1 rlt
39
l.fvf oaFb..t 12 Htt c
Etro
:02ior
r& 16 r[ m0 rlil rl2 lltN Hlr ltll l{l |{r |l' Rt rat l& lla l{2 1116 lld rxr l{0 lBl rlz
,hlr
Lftt of F€t &,tl Hn d
;07E06
;-"
aor
r& lG r{a l{0 Hl H2 tll! rilt l{! l@ lol Rt ll9 tll $2 La u2 x" tr3 |l|l E0 l51 15?
""'*liJX?ffiH,f,,#lJ'ffr"'trt #ffi''#*1'3;;d *i "i*
;f o; 'loliclt
.. B{t with dil&@r Lda' ltbw.r-rce"r diftdqc! (Pl0o5) B d.itEird tt DMR G.r
3.2.2 Pecti yase rctlvityIlic qudtibdve l€v.b of Pctinlve w@ d.o dltdnincd by 23 straiu of''{ t'!9t 3l
rn ilrqv.l of 24 bow in tipli.d! thlts Tltc 6uls of lhit dPaitMt Fvalcd lhtt
only onc srrein i... Hl7 showcrl its Fqinm !'tiviv and lh' in@Mion of ?2 lbu$ i a
8.2 U/mldin which *4 Fd@ed who quetili'd 'fter
incubttion of 96 tou6 i c E 0
U/ml-nin whrle tlt olh.r 3lt!i$ shw.d sndud ilr@e up lo 96 hod of inculdion'
Th. cults filrrte of 3t$iN ttl3, H22' H2a,lt3l ed H42 8a!' lh' Ld a'rivitv < 5
U/mlnitr 'tlE slr.iB ltt3, tl19 slrl tD4 .howcd s@ lttiviti6 i ' 4 6' 6 6 dd l6
U/nl-min rc3p-rivcly .lLr ?2 4d 96 ho!6 (Fi& lc-3d)'
Th. slr.ins Ho2, H27, H46.nd H5o gw. .nzvmG activilv >12 U/nl'ni4 out of ih'F
fou.thiB H2? dhibit d dE !@imd &livhv rho etiviv of H21 vs 42'7 2' r4 a
ed 163 U/ordin .n r 24, ,18, 72 lnd 96 to6 t 3?..ri*lv (Fi8 3t'3d)'
A.fYb of PL.. rtu 2a Hn a
t3
:la5129O:s
|olcHa Ho |nl R Hr r, ffi la llr llt rI' r3l lta 16llo ls ll. lis ro rll L2
thb
fu{vt of PL..18 fltr
13
;$
=12310
!r
t0 16 rs Nto rfi H2 3 Nft h! r!2 llt r!? rI' |nl lla lo lll l$ fin ||! lo $ rd
lldB
Fi& t. & b : Pc-ildye srivity.na difaat iEt liod Fio& bv 2! ltliri or- Awr2ills nlt r: a)24l1B, 2b) 48 trB V.nictl bd 3hd tLtrdi(l mr ot
-;- "f 6* Hlic.r8. B.l| wit! dltrdEnl ldd slbs ligtifi@tdift@D. (P9.0t .t &tadii.d by DMR r.c.
42
,d,t of PL...l.n Hr c
lt6Er.;12
irL
llc rl! lo nto tit ln2 rfi! Ht lri &2 lq lo l?t l3l lc l$ l{2 rr3 rxr rlL lls t6l 162
L ttol?t Jbrl.Hn d
13
itt:rl910
ie
nt ln6l0 H0 lll Hi2 Kt fil illt E2 lla llt l!! $ Ka 16 lo rx5 l{l rxt lio 151 r52
ns 2c & d: P*lintv@ lctivfv sft.r dificrat iE$'tiod Fiod! by 2l slniB of"- -- iiiii,i"*' ct tz H* "nd
d) e5 H" v'nlc'l bo's show 'rrrdld 'mr.iil- "i-'ti'.ett".r-.
B; wirr di&rrdt l'd'r3 rhd' 'ieni6odift@c. O9.05) s d.t dii.d by DMR L<'
at
3,23 Pectinesteras€
R6ut for 23 strsi$ of,{ ,!:qa pm<tucing petin st€rss€ d' shown in Fi8 2a-d Thc
oulrlitadve )d.ls of petin6le@ *fr dccdin d and th€ int'rval of 24 hoB in
ripli€t llslc. It ws obaotd lh.t !r.iB Hll' t122,1t29 d H3l shryqd ihc
nini'rM a.tiviry l*lt of 6.9, 6.8' 3 9 .!d 5.8 Ulbldir GpcclEly 'ft@ w4 E
@tinmu in lle in tclivitv otpetin6tqu. qc.!t fot srais H19, Et9' H31 od
tI52 which sho{.d 10.5, 8 4, 9 2 and 1 l.s U/nl-min Gpetivelv (Frg 2c) tnd 72 hoN
ofinobation sd rcduction in &livitv is cl.u fiom Fig 2d i.' 9 8' 6 8, 3 9 &d 5 8 U/nl_
min Gledvely, Th. strAic Ht2, Hl3, Il5O sd E5t ildiclt'd cnzymc &tivity >12
U/ml-nin, AnonS lh@ four sl..ins' H5o s!v. th. ndin@ etivitv i ''14'5
U/bl-ni!
(Fig 2<t)-Th. adivnie offl5o *€G 8-2, 12 4, 12.8 .nd 145 U/nl-hio 'R'i
24 48 72 dd
96 ho6 t!.pdlivcly (Fi8 2!'d).
$
=l2.
A.tvty ol PEs.i.f 21 Htl
r{a rG 16 Hro lrr h2 ffi t ftl l@ lo| l@ llt l3i l! l! nu l{6 |{! ll{a l{0 151 162
Bt lr!
b^.fi&
of PEr..fu.a8 H|!
l3
iri
Io:.
t3I ts ts rdo 1lr1 xlI 1ll3 |ll7 K! l{21{l lct l{! tll & 16 Ho l{l tli 116lio Ll l0
r| 3.&br P..dcro$ &rivitt dd dificFat ircubcio p.'iod! bv X- 3tt B of Aqqsiths ds.r: .) 24 Hn, b) 4t tk3 Veihd t3show$D&td c.t!r of nas of lht!. rlPlial4 B&! wilh diff@t lctLreshow si8nificst difct@c. (P9 0t u d.Lmied bv DI!,IR L.i.
45
A.&0 dPE!.fr12 H|l c
Efl€12
:r=a:.
ra H! tic fi10 Hrr 2 H! |nt fi! ra |o |g l{' lll l@ li t€ lx5 fi l{l Lo l5l loSblr
dLtvt olPErar .fb.3a tl|t
t!Ztlzt2
it
rc roa rG Hro Hir $2 ffi 7 H$ ru r{r rlt Ee r$ le rs l{2 116 lrc Hl rl0 ll! 16l
r!.3c&d: P.di6l.'cetivity.ndditrqt .ilo$.tidPaiodsb23ttlin ofttpednu,nsq c)n$rsun d) 96ltB. V..!cd bltt slow st tdnd ffioft|lad ofihF Eplicd6, Ars {ith difttat lddt *oe !n8tif(d [email protected]) a dct@ild by DMR t rt
:-Sdid diludN b. eidioo ol,ldwin8 $g..!,
?id of midi.l rlraEid of .r. ,{F
l,sring Degadarion b! high p.{rines pntluces
3.3 CULTUREIMPROVEMENTAid q@$r.tiw *l@rg bv oPtimiz'd aEvnc
'!sv lbG stdic (Hl2' H27' dd
H5O) haviry tNims Pot r isl lor pdtd46 pFducliotr w@ npbvcd bv vtio6
nui.gds lo .nnd* p*li!66 prcdetio. nE gttds w@ dltar'd bv UV iflrdidioE
dd two chciiqts ic. N_nethvl, N-nilro' N_niloe8@idine (MNNG) & BthvlM'thdc
Sulpho.ale (EMS)
3.3.1 Uttr.viol€t lrradiations
Dilur.d @ni.tisl sus?@sion (115x ld @oidir^!l) or fnallv *latcd ltr'jF @ u5'd
ld mutagd'c lr.![ndl The dilln€d 3u5pdBioc b stoiliai P'tipltt6 s@ pl&'d
ud{W tdp.t 2?3m (edicins lhc 6er8v of l 6rld ftn'1rs6 for 5'30 ninur6) 'Iae
di*dce betw..n lahP wa adjurc{t al s 0 cn tu 4h tdal b ed not! lh$ 95% dcdh
rat€.Ai€r 60 min .xpGW noE thd 95% kill ntc *s obstd (Fig 4!) ed ndnun
liequdcy of two bmomvl 6s|ul mutDls wd Ehieed An{ Epestd aposw 4
bamyl Gi'lmr nut{ts w.@ sel4t€rt which w@ test'd fd pdti64 poductiot by
subnsg.d fcd@talio! This q@tirdiv' tdt fo' p6til,s Poducnon dqlcd ltd
nurarr; 12 srvc aisrili@dv nisi pmdu$on orPolveala'ffil@ i-'" 0 68' I 2l' l 3s
dd t,48, U/nvnin afid 24' 48 ?2 trd 96 ho6 r"p€livelt Thc &tivilv of taal stnit
ws o 216, 0.50, 0.64, 5d 0.786 U/nvnh aRd.24. 48 ?2 .rd e6 ho6 E pedvelv
(Fis5!).
33,2 N-methyl'N-nitro-N-nltrosoguamidin€ (MNNG)
T!€ genlal 36rin w@ 8om for t d!v! to g't dsituM s?oolation' Conidi! ton
$# culru6 w.E 3lsFnded in stdiliz'd Clc?tl dor bmth with 2 f'4 pdrin dd dlc
p€la of &shlv gowrlt ott'lir (6 h ndhlv gom) *R obr!i!'d Tt' n]@lid
;rioi or wild 3tain tt? ws E!r'd ro '!b-tcrb'l
do6 4 de/ol of MNNO '
0 07
;rNaNor solulion in o2M &etde bufrd ai Ptt 45' And the rcpdLd tr.ltnotts of
stnin H2? nf.lia wnh MNNG foi 40 tninuG' fou b@nyl abl4t tnutarts waE
eLcte<t and rcslcd quelit'tiv'lv in shdkc flst lor ft'h potcnid ol P4linlvrsc
biosynlh.sis Th. data showert th nut4l H2? po<luc€d 58' lO?' 24? &d 290
u/nvmin tnd 24. 48 ?2 ald 96 hod '€P@tiv'lv
in @'irst 1o p'r6t slr'in llat
prsluc.d 4.1, 7.9, | 42 sd 16 0 U/nvnin GtPlclildv e!3 5b)-
333 Ethyl M€thyl Sulphonate (EMS) Treatmed'
Bhyl nelhyl sulphond. (EMS) *irh 0 5 M llr'nsth wd {tded to p'Fnrd nvcelb of
fl50 (6 h frlil y sloM) for 30 Dinute tilh t niNt6 tihc intcflals Morc thm 95%
kil ed tdibm d6iEd dulrlts w@ !''hia'd and 25 niluld And E at'd
rarbot fou Drt ir! *F !.ldtcd. OB tt of ecd@ hirluE ws *ithdam Dd
dilutcd 2 frld in 02 M PIF€Phrrc citrrl! buffct (ItIl ?5) lo 3lop th' Exti* TbE
rcp.ltcd rashin$ with o l M phoqh.l. .tuic hlfq w@ Ev6 to sd EMS nE
mycelia dd ths. w.re lh.n @spddcd in th! s' bufid' Tl|. c mu|.nts w@
qullilativcly a.alyzcd tor P4dncs|ffi. mil Minuh enviri*wcE64 106 207 Md
21.6'lr llniatflq24,18,72tttd % hou Thc pelin6rda3e &livitid of Pmlll stan
w@ 2.q l2.l, 13.8 ald 14 4 Uldvoh t.'etiv'lv (!ie 5c)'
50
.8lr.r c@ .lbr lrv Tutn.nl s
:r:$
lc||cm.lttllioli!ln..t b
ldx.r c|d .nr Ea Y..!rnt c
Fia4.-e Swird/ril cs. of lh@ 3mi of ,1 '/ett
ft12 H2? ed H50) lnd- mlndid: .1uV irndit'tior! b) MNNO' eEMs
5l
3.4 SELECTION OT MUTANTS
ARd dpoclr!.od !!.lDaa vilh Dd.gdi!.ga&i rholt l00d ofad .||!p@ioo
co .inirS iralld nlElia w@ .!.plic.uy t nf6Ed b individurl Ptitipl'&'
co .it:int Czc!{t Dox m.di'd epPtcoa&d tilh OEPLtd badvl ft! Ple rwitulh.tcd d 25oC dd dlnitld rsddv.n r r4!.t d 12 how l!n' in&rd io
'h!lvt!. eo$lh P.t6!. rlo @loni.. ti{in8 t@ dtrClt 4ott w6 sletd a if' ba6nci5lln nu!$l.. Tbac .tni!, *@ dlow.d !o gw o C4P* Dd m'diun tLat| br
cono.tLbn not Polv9.r.sbFn... unrkr'^
II6
comp..i.lon of P.cltnlv... b
t-t'"tlo
com.d.lon 6, P.otlndt E.-
a
FlLs,-q Comodisn of wild &d nuitnr lBid @ th bo$r of peli'|!s prodEtid_ (r) Polic.L.n!6e, b) Petidve dd c) P.cirEt @)
3.5 OPTIMIZATION OF CONDITIONS
Liquid state f@dtatid pltJ.s *s oPtimidd bv studvirg lh' 'f'ct of tding
fmdr.tio. pqiods, P1{, in@utlm sizc. v!5sl 'xt&i ed 1wttn80 in Lipli@t' fl6k9
Th. slralcgy sE !o MiDrain ihe proioulv opdnied p'@'t'G 'n
th' subs'qucd
3.5.1 E{tect of Fermentltion Pcriod on Enryme Activity
A set ofliplic.t€ flsll conbininS liqoid stat. f.mdrldo' n€dia wm inoculated with
5dl in@ulm ofvild e.l nutat.d sinic (Hl2m, Hl2, ID?n, 927, II50n tld HJ0) ed
w.E hddtcd tn6 24, 48, ?2, 96 dd 120 hoN ofircubltiotr ar PII 5 t Fig. 6t 3h43
rhit slrun H50m disllsyed l@6t p.lvsalrctorclN elivitr6 amng fi€ mu|,antr al c&h
[email protected]@ pdiod i.. O-41, 0 63, 1.21, 1.36 4d 0 85 U/mlnin'^d 24' 4E 72 96 atd
120 nos rcsDdivelv T!. &tiviti€s of tt27d *.F O5l' l 12, l '25' l 19 ard 1 13 Uhl-
nin afi6 24. 48. ?2, 96 dd l2O hou6 of incub ion 6P@tiwlv- The ruinm&livi1i6 of pobltct@tre w@ @rde<t bv Hl2n i c O ?l, 1 2t, l4l, I 49 dd 0 98
U/mlmin an r 24,48, ?2,96, md l2O hous cpotiv.ly' l1 wd obwcd thallh'
prcductior of poly8Aldt!rcl@ imrc6€d wffi an inftGc i' fmdlltion timc ton 2+
96 tolB ed ir ws lwihal !ftq 96 tou6 in thc ce o{ !t lis gl2n, H27m ed E50m
whe@ ach of lllrn showcd a srgrn@t tlduction in th. &tivi9 ofptlyS'l&lNne
all€r 96 ho@. The 'wiDun
4tivirv of Polygrhansnse w4 @orded by Hl2i and
lhe incubation of96 nou13 i.c, | 49 U/ml-ni.. Th. polvg.la.tllt!|w a'liliti4 wte al$
foDd to vsry siSDdcdlly .nd difrMt fed€ntstion pdiod! bv lh' wild sFAins ed
mdinun lclivity wd obtain d by Hl2 |nd 96 houts of ircub.lion i.a 0 79 U/nldir(Fic.6a).
Fis. 6b shos lhe 6ult of p@linlts. etivitiB by Ure mltants Md th€i. pdts(Hl2n, Hr2, H2?n, Iu7m, HSOm $d H50) hdBted afla diffdqt fm@ttion
pdiods.'Ihc @lG ofnut tcd straiN indicatcd thar strain g50m shw.d 360' 9.30'
12.80,16,60 ed 13-50 U/mt-mi, afr6 24,4a,72, Nd 120 hos EsPativ.tv Thc
dEjn Er2o Fvc 3-80, 5.80, 6.,|0, 9.50 dd 7.20 U/nt'min anq 24{a'n.% ad 120
hou6- HN.rer dt nuinun &tivili6 of pednlt@ wde rddcd by g2?m i.. 5 90,
11.?0. 23.80. 28.80 dd 23,20 U/ml-min dts 24, 48, 72, 96 sd 120 houB apeiivdyTh. Gult in Fi8 6b cldrly indiqt€ $at sll oa lhe 3E ift qhibil.d deir ndinm&tivily lnd 96 ho6 liId simil{ b€hlviot w4 ob*dcd by wild slraiB wh@ U27
cxlibit d 16.2 U/u 'uft of peclinryse and 95 hours wtich wt' lNind .llorg t[ lh'
Frri 6c {ds lh. t@lr! of Pelitclrdt& |.rivfiG by lbe qild tdd thlir nul|di'
(ltl2m, Hl2, H2?n, ID?, HsOu !d BJO .na $t i4dlltd for ditrcEl ftn'olxion
pdiod{A$on8mui4edltrairuHl2nshrycd63,E9, I? 8,24 604d 140Uto -nhof
D.cri&dauc dci 24 1S, ?2. 96 &d l2O ho|l6 E5p€tiv'lv $rai! H27m g.vc 68'
lO5, 2l.t 25.0 |nd ll.4 U/nl{in dld 24, 4Et ?2, 96 'nd
| 20 tou E?edEly' Th'
&tieitid of P.cdnd@. bv BtOd n@ 6.8. 10.7, 2' 5, 28 5 &d 14 9 U/Dldin tna
24. 48, ?2, 96 od lm hos n4@livdY
At!|y br dd.tbir.lid ofoptind eddttliol Fiod 'lc'tly idic'tt tld dl lL' sttinr
.how.d |h.n nuidM lcrivitv aRd % hour' E si8!i66! Edudioo w|! ob€v'd
I '.I";gta
a.rtar ot Pod .t v.rllc F.Nni.don Pdb!'
&t ry ., t{5.r-rhr F.imda.rbn P-bd.
I
I
a.t|rtiy orPE-o.t!.rylhg F.md or F.rto.l.
I!I
Fb. 6r+ Activide! of Pdygdetuord., P@iiBLre ad PetidtB. d dif.Gntf@nbtior pqiodr by rte. wild !d dEir mur.nt3 ot tt ..eitla nlgra) POe, b) PLe, .) PEe. v.rtcd t6 ltw stdt hd au of n|cc oftltc rpliete Bd qirh dillcFlrt lcrd! sho{ rig||iliotrt dill.cM (Fs0.05)!r ri.t din d by DMR l6t
56
3,5.2 Effoct ofPH
Th. initial pE of blsrl ded'ud hs srcal infludc' on nicobisl nttaboli' trthwav Al
ft. iner.dimts of lh. ne.tim w@ dislv.d lnd pH was tdjust€d at 5 G? 0 individullv
rith 0.5 ptl difidoce i! ordd io liudv th. cffcct ofinitisl pH d llfte mutlted ar w'll
4 wild srBins (Ill2n, H12, H27n, H2?, H50n ud H50,
Th€ r*uls psentcd in Fig Ta show 0tc polvgaleiurcnsc @tiviti6 Ac@rdiDelv Hl2n
dnibiied l 06, 126, l ?0, 123 dd l.l3 U/Fl_min at 50' 55' 60' 65 dd 70 pH
6periv.ly. Thc sFain H2?6 !|v. 0 95, 1.00, l 30, l lo md o'95 U/nlmin 't
5 0' 5'5'
6.0, 6.5 od 7,0 cPdtivcly Th. strain H5om thow'd o 90' ll0' l l0' 0 90 dd 0 85
u/nl-min al 50,55,60,6,5 sd 7.0 plt r6P@tiv'lv 'Itc hish*t &livitis of
polyed&tuone @.dcd w@ l,?0, l 30 tnd l lO U/ml_nin bv muttlt stliG Hl2m'
H2?n dd It5On eperivclv at pB 6 0 (Fi& 7r)
Fig ft snows lhc .fi-r of Plt o! Pr.drction ol poti!ryst by tlllc nuttrts ard l[cir
wildr of-r. riA.t wh@ 3lr.i! H l2m .ho{Gd 20 d 21 6, 2' l' 22 3 ed 18 9 U/bl-mi! at
5.0, 5.5, 6.0, 6-5 and 7.0 pH 6A6livclt Slt'in H5On 8rv' 19 8' 2l-1 198'198!'d
18,9 U/nnnin at 50,55 6.0,6 5 !:d ?.0 F ptclivclv IE h$imM Elivirv or
tedrl}a$ w l@dcd i! th. c& of sn i! H27b ic 26 0' 26 5' 29d 25 5 4d 18 5
U/bl-nir !t 5,0, 5,5, 6.0, 6 5 &d ?.0 tH t!.Deliv'lv'
Fig ?c .t Focttrrd lhc ..tivitic. of P@tindrcr& d difiqldt PH wh6! slt'i! Itt26
gave 18.2,20.8, 25.6'.40 dd 13-6 U/ml_min 't 50' 5t' 60' 65 dd ?0 pE
rcsDetiv.ly. Tt. acrivirid bv Hnnvct l72'202,242'265 tnd 16 8 U/ml-nin 'r5.0, 5-5,6.0,6 5 .n.t 7.0 pH spdliv.lv Thc Fdidun
"rivitie ofPehn6lcn* w@
qt$itcd by stlin H50ni..21.6,25.2,31 8,2E 2 'd I? 8 U/ml_nin at 5 0' t 5' 6 0' 6 5
dd 7.0 pl| r.sP€ciw.ly,
Ase'€ fd d.tdni@rion of opdmrl PH for p4tinoltlic 'nzv!6
indi@tld ndibtl
etivili€s by wild d *€ll s nutml! at Pll 6 0 s thoM i' Fig T'_c'
51
rcEry .l to-. .idrh.-l tn
5
5!I!s
a.dvw ot tL.- .t d!l!.rit Pn
E6
t'
Allvryof ta-ddlt ndt||
f:
Fis ?.{ : Acriviti.t of Pol}tdlctuntrsc, Pcclhsrsr* ad ft<rialr* at ttrfr€Ht-" -.n w tr."'"rra ,iti-,Llt alt'fi oI Aqqgtlhs nte'r
" Pc'd" b) PI$c'
5 pii* vJ"a t-" 't"* n-t tettuof D'!E or <c Eplicdc Bs
ii a'rJ-i*i ,1"* 'ls'tl-t ditrttdcc (P3005) s d'tmilcd b'DMR16!
5E
35.3 Effect of lDoculum Size on Enzyme Activities
Crewth media wae inoculacd wiih wild Md tch lnulst slrains (Hl2m' Hl2' H2?n'
H2?, H5Om sd H50) wilh vdvnS iDoculun siz' ftum 2 ro 8 ml ttd inclbacd in
tiplicare fldk at pH 60 for 96 hou6. Th' cfi"t of id@ltun on lhe sctivilv of
polt€acturo@ by lhree mullnE as w'lt t! wtld slEins dcphled ii Fig 8a ddlv
iilicatc thal slrain Hl2n eav. th. twihM a'tivili's ic l32' 140'184edt60
U/nlnin et 2, 4, 6 od 8nl of ito.ulm !iz. Gpdtiv'ly Th' eliviti4 bv Hl2n v@
1.00, 1.20, l.40 ed 1.24 U/mlmin lod o 86, l l0, 1 36 ed 1 20 U/ml-nin st 2' 4' 5 and
8nl of i@uld tlspdtivclt Th. ndimm &livity bv wild dtaic w als' oh6av€d at
, 6nl,of [email protected]. l 06,0-?2ed0.84bvHl2,H27,H5OU/t -mitnsP6titelv
;s 8b Fpr@nts U. &tivi1i6 of Pclitrbsc bv th' cnxm 6ltd6 obt'ined Eon
subndacd f@cnt tion i.@ulat.<t *ith dirf.Mr iNnhm tiz6 Thc atEin Hl2n
shoeed 22.5, 26.2, 2t.6 srd 20.4 U/mt-nin d 2, 4, 5 tnd Enl of i@ulM rspehv'lv
Th. ndinun elivilid wft cxhibit d bv H2?n i' 24'4 2E 6' 33 6 ed 30 0 U/nl-min
d 2.4.6 drd 8ml of indulw siu Fsleriv.tv' Th' &liviri6 of Petinlt@ bv H50m
sft 20.2. 27 0. 2?.6 tnd 24 6 U/nl-min .r 2. 4, 6 !d 8ml of Mulm ti* cp€tivdv
Howwd lhc ldinM dtiviti6 dhibircd bv *ild 'tnjr ecG E 2' 18 tnd 10 2 bv rlr2
H2? rnd H5o nlD@livclv .r 6nl ofiMllw 3iz''
Fig 8c indid6 rhlt .mng lh. Dubrc{t 3tr'iB Hl2n garc 26 a' n'7'25a &d 244
U/nl-oh d 2,4,6 tnd 861of i@ulm 3i& mc &tivili6 bv 'j27n
v@234 240'
28.0 ad 27.4 U/mlnin whit. tuihm etivitid sF dhibn'd bv g50n w@ 29 2'
29.8, 32.6 and 302 U/nl'6ir ar 2, 4, 6 .nd 8ml of ino'ulm stc t!'!etiv'lv Thc
rFd@tion of petiebric cE 'r4 Sndudlv ircq!'d ion 4 to 6 ml blt rt w6
ddticdly r.dw. at ilsrlM or 8nl (Fi8: 8.'c)'
59
rttg6
A3!Jt ol F3-. .ttrvha 56ul!t 'E
A.l|'lt .a P|.s .t v.dlte hr'clrn tL'
t-IJ
t6
a.dvry or PEs .tv.tvhc h*lrn 'E c
r$E$*;
[:
-F: Aclivirig of Polvg.La!8o@' Peddv!,. '!d
Pedntdre tt difrc@t'T;fi;; il#;'J.'d rrritr Fu'nt! o{ 'r'P"g'tl6 "" d-:9::rr9
iiil'il,"i]ri,illv;J'u" 'r"" -'a'4 dror or "* or u*.-ri; ir" "it' or'- r.!.'r $6w ti8!i66d diffq6e (P{05) adetemin€d bt DMR l6t
3.5.4 Effect of Yeast Extract on Enzyme Activities
Y4l cxirr.l wd us!.t |o €nndcc thc fcmdr'lio! nle of polvgd&turcne' P@titltac
ed pccrinest@e Prcduclior' F e differfrl concmrJlDn (0 2% 04t" 06t0 8%dd
l,O%) of y€.< .rtr&t w@ add.d into ptcoPtimiz'd subndg'd fm'nttion n'diA in
r'Dlicdc ost' Tn .on|rol did nol @av' rv J@-rdrr&t ft ws notcd rnal 04%
t€st oxtrd cxnibilcd 2 3 I U/ml/hin etiviiv for pobls!'clmrse bv Hl2m ed 38 9
U/d -min &tivity for PdtitnFe bv r9?m Mdimud &tivitv of 316 U/bl-niD for
t*dndLN w6 qhibit d bv H50m with 06% Jqsl d[sL ll st! Fordcd 6t
:onhl (wilhod yest €xtr&o gsvc mininum aativitv Snnild behavior *6 obedcd bv
wild 3t iN wb@ ruimm stivity of Fllr'l&tuona!6 was @rd'd by ttl2 at 0 4%
of YE i... l 16 U/ml_nin whiL tltc !@inu!n eliviry of Pcclitb€s w !8 8 U/ml-6'n
by H27 at O-4oZ of YE. Among the wild sr'i$ mdimun cclivitv of p'ctincslqre w$
l;.2 u/ml-din bv Hto at o 4% of YE n wd 'lto
ml'd thrt &tivitv *s r'duced 't
hi8l
@rcolrdion a!ov. 0.6'l" of v.d "t!d (ng 9a{)-
!t dorv-rEr.lt rd tG-. A.d"p
E
tI
!r.dd n-r !r!td 6 trr. ^.&ry
:d
a'€'l:.
Hr..r., Yot Edtut on P€s a'wry
lo
t?t;;
d PEciitr6la!!' !t dif&t6lEtr. 9.{ : Adivili.t of Pobttb'1!lou'' P@Mlv"' e
@c.'t id of v.!3t .xmd bv "*' vT.* 9* llg: "rj%'g::##m*:x11ru#x*ffi.:ffi(P9,05) u d.t nir.d tt DMR Ld'
62
3.5.5 Effect of TweenS0 on Etrzyme Activities
Tvd8o t6 ut d to e.hloce lhe subnc'ged fdndlation nle for thc pmdedon of
!€tins. DitTcdl @di..tion of twdso i e O 05 0 lq o't5 0 20 rid 0.25 l*
add.d inb 1i. [email protected] f.ftolalio. n dium of cirrls p€l A conttol (wilhoul
t{c680) ws run DrEllcl Addnion ol lw@o80 into Itow$ nediun ohatued th'
D41ir$6 pFduti@- Amolg tlrc nuhlcd stnins EdiNd i'ld of poly8ll&tuMd'
ed pdriltlrre by gl2n and H27n wc 247 &rd 30'13 U/nlmin cp*tiElv rt
0,I Ov6 whiL mDddm &tivitv of P6tincd@c by lt5o Ps 3?'6 U/rnl'hin tl 0 05% of
lven8o. Anong tle wild stnins ndinun lctidtie w@ @rd'd tt 0 1% of tw€d80
Th. groqlh d.dit @rairlg noF lhd O tO% of r*680 'hN!d
bwd &tiv nt of
63
Efi.ct ol ll're.6o on PG... Acdvltv
tz5
I --5o o.oa* o.lotr o,l5x o 20ra
c.E.d{or.rnrDrrao
b
E30{,5tmgr3510
Fo o.o5t oro4a ol5* o5*conc.rtr{lon .t TK.6!o
En€d ot lv,e.6o o. F|.- &tvny
E nec-t ol fuecnao on PEa33.cdvlt cffin& llh+. Activili6 of Polygtb.tlo4rq Pcddv4 .ad Peddrar$ tr dif@n--- *q'rdo "rrdoso by tc wild s'id of ,1. /lsE .) POd.' b) Pb''
c) PEs. v..!c.l bo .!F; drd.d tu of r6t of tld cdida BcG,riaidi*m t"IG" .t . nsnifiot dm.E@ (PS05) s d'rdnird b,
@
DMR T6i
3.6 MOLECULAR STTJDY
The yi.lds of lhc DNA cxtnclcd bv using CTAB cxlacton nctbod vcrc
detemined by sp4troPhotom.hioallv !, 120' 260 ng/Fl md qudiry of DNA wd
evaluat d by agaro6c 8pl el@ttoPhoiisis.
The .epro<lucibilily of RAPD lMlvsis is tjo$ lo be highlv influ'ned bv
€xperimenlal conditids lt is, lhcrcforc, dccnlill io optimiz th' PCR condilions
Aner oDtiniznon of r€action cotditio$' polForyhism among the wild nnd
muianis w.re detcctcd Eins diffcrctrt tnndom prim'$ Tweltv Pnhe6 of oPcro!
siet w@ optimizcd for nAPD ml)€is for {Eir Abilitv io prcduce suftici'lt
adplific{tio! ptlducls ot of 20, dne tudm primd lMcly OPc3, Orc6'
OPC9, OPC10, O?C11, OPCI4, OPCI5 dd OPCIs 8sv' the $frtcidl
polymorphis Mong the wild.nd mutaits Th@fon tunh'r cfforb wd foc$cd
Fo. the assesment of genctic divesitv, among {ild md paEnt, PCR producl3
wcr. run in gcl, thc atnplilicd DNA bd& w.rc olwcd, thc abs'nc' ofband w"5
recordcd s "0'.nd PrBd@ as'l" Thc d.ta l@d'd in this man'r wa
analr4d lhrough Minitab 14.
All thc princts showcd dimorPlic bmdilg pa8an for motT rts wtrctlts
monomorPlic banding Pattcn6 w.re obs€rved among tic !€rents' DNA
firgcrpnnting .t t clucidatcd ll|e variations atnong thc !.@ts and mubnh The
6ults indica& lB getelic div.6itv .$otg lhe Patatt of ''{ tt?a and more
genctic divmily amdg the muLnts.
3.6.1 Dendrogr.ms wlth single linkage and corr€lrtioncoemcient
The d.ndrogrms were @nltruoied with lhe help of coinputer software MINITAB
14 for individul pdmeE o! thc basis ofp.imer Pattcm The r'sults ofthc PEsent
siudy gsve Prclihin!.y irfonttion about DNA ofsi( i$lales of 'lsPergi 6 nigq
whcrc tb@ w.rc wild @d thfte wcrc nutanls Thc ninc random pnm'6 glve
Eproducible dd very sltbl. r.sults Peolid to rhc ss' stai$ It ws concbd'd
th.t distinct RAPD fingerP.int! anong the dif€r.nl sPlcies wcr' obtain'd whcn
suiEblc primeB w.rc us.d and PCR conditions wd oplimizcd'
65
It is revciL-d ftoh $. dctrdtognm (Fi8 lh) corstruol'd bv th€ r.sults obb'rcd
from pdm.r OPCa thd sirdleitv levcl rangcs b€tween 100'37 5% mong aU ibe
strais. strain Hl2 and H27 werc l00p/ similar while st'ain H50 ed H50m 8!ve
84.5% sinilanry, $litr H2?m showed 40% ald sbin Hl2n showed 3? 5%
similuty wid! st of th. $rais.
Thc ftequdcy of gdctic drGity monS wild dd mutant3 !tr!i6 is shom iD
&ndrogram (Fi8. t2a) by primer OPC'. lt was obd€rv€d that shsin H12 dd H27
werc 100p/o simihr to c&h otltcr but th.ir simil{ity es 84.4,76 5, 53'0 'nn
29 5%
with t{50. H50m, Hl2n dd H27n re$.ctivclv.
Th. Deldrc8:m d.Pict d itr Fis. lJ. ws co6ttucled a@orditg lo thc b6ndi!8
pllrm oulinc.t wirh primd Orco. h rhis dddegnm 6ults cldlv indi€t'
lhsr fiv. sfains iocluding th@ wild and two mut!tris (812, IO7' H27m' H50 ard
H50m) werc 860/" simillr white Hl2m showed Et.6% snnihrilv witl rcst of lh'
High la€l of gdctic divc6itv m@s lhc wild dd lt.ir nublt! *4 cld Aon
th. d.!db8nm shom in Fig l& wha! two wild seain.s ID7 6d H50 showcd
l0f/6 simil.rity while H12, Hl2m .!d H27m werc 8? 5% simih bul l['irsimilaiiry wid' Hl2 and H50 was 8?s%. TIE strah H50m showed 866%
sinildity with o$er slnins
The data of gctrctic simildiry fiom dddrosEm presal€n fi8 l5t revals that
slsin Hl2 ud H27 s@ lQfl' simil& ond solher clust!' of H50 tnd H5Od
*@ also sdc. Whilc 927 aad Hl2m wcrc 44 5 dd 25.6% simillt with lhec two
The data from th. rcxt dendrogram as sho$n h Fig 16a show'd 100% simileity
b€tw€en Hl2 od H l2m ed .ls moDg othcr lhrtf, stai$ (H27, Hz?m ed H50)'
The stain H50tn ws 25% simild to rtsl offie stliffTtc rcsllts oflhc dodrceram in Fig ltu clclrlv ildioL lhat two Pails of sbains
(Hl2 and Ht2n) dd (H50 dd H50m) wcrc l00e/d simild whib F27 ad IDTn
slow.d 77.4% similarities.
Tt rugc ofg.n€iic limildiiy (Fig l&) q!! highlv divdsificd wlllrc Hl2' Hl2m
md H50n ftr! 77% similn b c..h otb.t wlilc H27 ed H2?n lti! 45'5 %
.iDihr b Hr2 Hl2m '!d
ttton rld 37.5 dEiLr to H5o'
Thc ft.olt' fton thc.l!trdmgrn 8.t t!.d!fi..lhclta t3ofOFC,iodictt'dtblt
dl rtjrr etrc disinibt, dd lhowing .inihitv b rho tugc ofdtv 23'648
67
20u01650
t0o0850
650
500
.100
300
200
fir.tl: RAPD tiodEts ty PcR dplifictti@ of DNA isbr.d Aon fte wild .tdO.n E n nt3 Nirs FiE orcl Mdta ol I r.b it tl|o@ lo lb. bn offge
8!,lrr; Dddrcsm wilt sirglc linlrgp ,nd clrebiion @!frci@t disttF ot lhcb6!k ofbading p.ncm prcduc.d !ftd Pdnq OPct
68
H50
M1650
1000850
650
t00
400
300
200
Figl2. RAID podEs by rcR mplifietior of DNA itoLr.d toD dre wild ed- d.ir h;ids Bins D.ibd OFCc Mrndof I lb it ttod io ttE Ln of6ge
Fis.ur.: Dddrcg@ wirh ri4l. fint|8c dd coGldion d$ciclt ditlrF on rh.b.!ir ol h@dils rliLE Fodu.Gd .ns Fiffi OPC'
69
Hr:. 'u7
20001650
1ofl)
850
650
Ir4l!. RAPD pr!du.t! by PCR dplificdid of DNA iel.t d tm lbG wild sd_ 6.n o'ir.d! uili FiG OPC'o MttaotI kb i! tbom !o th. t'n of fisw'
FEl3.: Dddosrd *it[ 3irgle lixloge ord c@l!rio! ceftcial disltE @ lbc-
h€lb ofb.!db! p!$m prcdu..d rnq prir Orcro
5(x)
3002N
r6ih
m
HZr- tl50
2txl0t650
t00o
850650tm400
300
200
Ifi
Fi&U. MPD plduE bt PCR mPlificdio! of -DxA
itolat'd tlm tltc sild tld''-- i"i, ^"i-" *i'g p'i.r oicrr' Md(d of I kb k elFwn to th' bn ot fisw
fi€.14r: Dctdsssn wit tinsL lidi,ge .!d corcldi@ @r6ci6r dili|@d |nc bosi. ofba ins Dand pmd*.d ancr pnffi urLr'
7l
H27 rO7. H50 H50d
2000.
10008J0.
650.
500,
Flcl5. RAID poducB bv PCR Mplifelion of DNA iel ql ion thE sild &d'-"'il;;;;;"'t"r.. oPtD M!r*a of I rb i3 dDM to rh' bn ol6sw
300
2@'
v*rbb'
Fl&15.: Dsdogld wilh si4lc Urrlge ud @Elttio-' dlficidl dtbe 6 th'-
hatis ol b.dins D.n@ FodE d !n rPri@OFCD
72
2000r650
t0o0350
500
3m
2@
Fi&16: RAPD prcducis by PCR uplifiqtio! of DNA islat.d non rhrcc vild lndth.i! Bul{|tr Bios Ddda OPCI. lvlrLd of I kb ii slFM lo tL l.fr ofrige.
ri9r5( DcddmgE n *i$ singl. link gc ,nd .tnlation @trci.m disrle on th.brik ot boldils pltd pt!du..d .ns Fdcr orcr+
tt
13
II2 HrL H27 m7! H5O H50i
2000
l6J0
t000850
6J0
JM,t00
300
200
Fk 17: RAPD prcdu.b by PCR dnplifiotion ofDNA ,lobLd ion 0tr€. *ild lndrh.n nul'aG uing priDd Orctr. M&td of I tb is sboh to t!. lcft of
Iaa
Fi&17.: Mnoge wilh sbgl. liot!8. .!d cofrl.rion cflicidi dirr.@ @ l!b&eis of brndins p.tcn pm<luc.d sn r prinq OPCB.
N@r650
350
650
500
3@
200
Fi&rEr RAID prodsa by PCR ampli8€rion of DNA isotrtcd tion rltr.. *itd &dlhcn nut ra Biog FiDa OPCD Mln.. of I kb is rhom lo rt! bn offru..
n&r8r: D.r<Log@ wim dnSl. lirlre. .d coGLlion e6ci.nr dj,re oo rheblstu of bedilg p!!.h F.du€d .na prilE OPCE
PCR mpli6€1ion ofDNApnM Orc! Mdk of
ilohi.d ioE thE. qild 3dI !(b ir shoM 10 if. lci of
2000
1650
t000850
5m
2@
Fi&19: RAPD podEts bylncn mutdl3 uirg
I
Fig.l9.: Dadegm w b sbglc liilogc &d cdErdi@ @mcidt distrrc. on rhcbair of b€dirs p.tben produ.ld .nq Din€r OPC!.
76
UNWEIGHED PAIR GROUP METHOD ARITHMETIC (T]FGMA)
Hl2
Hl2m
H27
H21on
H50
H50mn J tr ---- -1
Flt 20: Ov.6ll linilairis mong ll. rbra *ild slrdB ard rt it Eddrs otr I[.\tb of b. inS Fltn podkd by dl p.teE.
3.6.2 Unweighed Prir Group Method Arithmefic (UpgrD.)
UPCMA tle is v.ry simile to &ndsem obbi!.d by .iigr€ tiikrse lid @Ntdion
coellicieDl dislance .Dd eave ta ov.Btl dircl€nce in Frd&gc of rihitdity ln$trg lh.
strliN. Acording ro UIGlrtA .lt rh! $niB hlE beD divid.d in to re min gmuF
@h gFup @ntddlg thd s.i6, IE sr"i! Hl 2, H l2e ,!d tD7 seE pta..d in gDup
I whne H27, H50 sd H50m kE plMd in gDup Il, Th. mutts of UPCMA indicdls
rh.l sinildity tErlrq llt2 ed qt2n E 86.1% ald bod of 0,e st.iB s[o*b.l
76.4% similditie! vii[ lD?. SiEild behvior @ obcend h Ooup u *b@ It50 6d
H50m indiclt d 85.?yo sinileity &d lb.i. simitsity ril! H2?m lae 76.2%. rr. jis
depicts tht gDup I dd croup lI wF 70.8% simitd ro dch ortcr,
3.7 PROIEIN ISOLATION
tDS PAOE edyds of th€ prein i.ol&d fto mubtt of.ltPtSr&l' ,{ga *r.rbo crri.d od nnic& 6or,!d th. F!*a.. of r l1!t ir Tt .l6.tofhddic
F!.m! obldr.d fion rllaltz.d .n?nc adrca (Frg. 2l).xtiti.ld m difrGlmog oudrt rlriE Pml& p@!! !.odll..d bt n|b-n rged $'4lad-lhowd lh! nolcculd riz. ofFor.i! ?8 (Dr.
f lrybdy,_ggb"a*!' vlibL only i! r.ry|r' ilolrr.d ffom 70 % .dmdn'hlulp[!!c Fr!'lrrg. ldrdions of CfS. The tdd dct .t d oo SIX PAOB ofrflld 7t XIh ioDlL.th! F@ of,oCiE r loteiD. Tt &qtl.s oblliicdrtv.ah rhlr tho mued l!|itr! .dnibtsd ,@. b.h|vi6.
IIPG SlgPAGl gn a F.ar dbr br -rh* .f/, .tt .d rrr,
7A
3.8 LARGE SCALE PECTINASES PRODUCTIONi.irr. 0J"".* tt .r"t"a stsins of'1' 'i8e'
in elid stale fcnncntation wa!
studied h the medium witi citrus peel as the otlv carbon source The aotivities of
Polygal&turo.6., PcctiDtyasc al(l P@riflBt'resc werc signiiicandv high a!
corparc to subm.rg.d f.rmdrdion. Polvgnlacturcnse activity (Fi8 2dA) eas
ndimum afld 96 houb i.c 3.55 U/nl-min bvIIl2n s in thc 6e of submcrgFd
fmentatior and ninimum bv H50m i €.2.73 U/ml-nin.
Similar beh.vior was obw.d for Pectinllasc !rcductron whcle all thc mutrted
strains showed a S.adual increase in the lctivity with lim. Thc maximum lclavitv
of P€ctinlye was rccorded !ftcr 96 hou6 i.e 352 U/ml-nin bv H27n and
minitnun lctivity by H50n i.e.24.2 Ulnlmln
Thc ndihun &tivily of Pecnncsterse ws slso recorded !ft€. 96 hours which
w641.0 U/nl-min by H50 dd minimun was 32.4 U/ml-hin by Hl2m
79
IActlvlty or PG|.. In SSF
115t3{ !.5!.
bAcgvlty ofP!..o h saF
!to53s;30
Irs:rclo
Ac{vltydPt In sSF c
:.:.o135;30Em!15lr0l5
@Pc.tccaabY $lid tnt'flfutrtio! bv thtlc nrlmta ot 'lau lwnISq
80
Chapter 1
DISCUSSION
Pectinses @ a gtoup of @ymd rhat all,sck ed depolvn'rt' p@rin bv hvdolvlis dd
u"Nliminalion s vell 6 by d*sletifidion @ctions *hich hvdhlya lie 6ter bond
b.wecn th. @boxyl sroups ol pelin (ceci lnd t$ano, I98) vadoN g'nd of
micoorganisms can produe P*tincs, blt Uld.nto8 fungi ot spcci* 'lsPctsi|E,,se. aE monty wed b6.!* il is clsified s CFIAS G€ndllv ESrrd'd a sf€) by
U.ircd st tcs F@d ed Drus Admi.istation (USFDA), which h$ povcd ils use in f@d
indusiy (P.riz ond Fosk!,1983).
Pr€ently ahoig Ulty two sldins of AsP.rsilbt niE r, 23 wE $l€ltcd !n r PElimin ry
$cning and lindly th@ straint wilh tuinum nduhl polc i.l of pe 'rc
Fldu.tion aftd qumlilalirc ealysis, Atut nuli-srep $@n'ng, thc elect d srai.s
wec muat d by UV lnd cheoicols t.d bmomyl ci3b mulanls with inprov.d
psdnsd poducrion wE pick.d up. Th. nuttnb w subjet d io vacly of .3s1i.1
p€dnc6 lo oplimi4 Ihe fmc.torioo @.dilions. Th. huimun eliviti.s of nubl.d
$r.in lr@ r.ard€d .ft€r 96 hours ol feroqnlation, ,t pH 6.0 dd 6 ml ot v.gebtirc
inoculum riE, lt ws ale notcd lhar 0.6% of Y6t *lrd ed 0.1% of lw80.nnoc.d rh. dzymc etivity, And oprimiznio of f.mmt.rion @ndniont rlE nleads wll s wild sttliN weE o.ly4d BpnetiElly thtouCh RAPD ling.tprintin& The dalA
€lucid.le th. vdidion Mong ih. p@nb dd dulanls lxlibiling l.$ Cltidic divdily
mong ltE psEnlll,,l, rielf md nor. Scnetic dircRity lnong ilt mst nl!. Th. Pdcinpan ns pdduccd by nuuntt of,l. ',sc,
slE*€d ! holcul. ol 78 (Da a cnzync
Izgc slc pmlinM peduction on slid slolc lcmcototion moli! by the mutants
qhibit d not then +fold incee ofpolyenlad!rcne, 2-fold ofp€ctinlyae dd l-fold
Among rh.52 t61solic of ,1 sp.rstllu ,iae. hign ed nod@te pmdue6 *Eslet d for fiml $@ning by sing optimiz.d ebzyme asy meilods lh. i$lol.s fom
citrus and apple fruiB wE obed.d 10 t high peclines podueB Simil.r tsults h.v.
8I
dia bd Eponcd by M.ldontdo., al (1986) ln 0En stlrdi6 it s sb'm out
Asperyi\$ nigq ieLl.d fron ot'n l.md wa tU' lo Dr!d@ norc
polySd*lNns. dd pdtin6Lle Out of 52 eldted !aait5' 2l w@ s'l@rcd for
turrh€ !.udis in $bnergcd lemololion witn cihs peel pccrin'
Studi6 on th. t.ldtion of,4rp.4!/rc ,te,t ltroin! for poducrion of pedlle hw
pFviously bd nEd. bv 54dd s,o*ds Dinu ud De (1994) @Uated 109 3trtis or
,{. ,'!cr dd subjet.d itd lo qu.lira$vc od qundLliv' eE ing fot scl"lion of
th@ slr.ins whicl w@ $c b6t p|.duc4 fot p.ctioascs AU stttins w'6lsred fo'
0Ei. dopoltsrh.$rcN. p€dtD.s6. .td P@ rlvdc lctivnid sd for mins wcF
cho@r cotty ., al, (1995) dctdnin d th. petid6 Ptldlclion bv lsPcralllBIm
by nEuing lh. cld encs forEd euId tlF @toni6 tbin'd wirh dh@ih nd Out
of8? ielalB l61ed, l5 podu.d r.d an. 3nd 3tr.i!in8
PEndy, ilordqlo iov6tiglt lhc q6titalivc l*lt of polvSdlelmn@' p@trolvrse
ord p€lindlc@, 2l sFairs selectcd stdins of -r. ,rs€r w@ $bj6t i to tubdged
f.menhtion for 96 hous. Il ws obseded tnrt pFdetior of polvgetumne wi.dcosid€dbty mn8 lll th€ 3.1*t.d strli6 of ,l ,t!€i Th. nsimM acrivily of
polrse.t@E w$ Folded by Slr.in H l2 i... 0.?86 U/trt'nin Thc qulit tiv. Lvdls
of peirllye .nd pddnGt @ w.rc lls d.cmin.d by.ltadv s.lelcd si!.is. Tb€
6ulls cldly indicrr. $ar $.in H27 dd H50 Savc lwimm 4tivitv i . 16-4 md 14,5
Dud ., .r eOOO) haE rcpon.A 6.r atP.rsiltus jaPoni$ 586 9v. tnc IMibal
polys.letoom. .ctuny at 48 hou6 (0,500 U/nl) vs obLincd in ihe pBebc. ofo,5ol'
patin ard 0.5% glu@s..
AIt r rhc fml scrcenins of23 stnitu of,r, t,?.t in slb_ncrgd fcm.ntari@, d'@
sFliB (HI2, H27 md H50) witn mi!t@ n.tudl pordnl tu Pelilts 'lldEli@w@ el*r.d Sinils 3tr.c8y on @ing of fu4.l culuc w .dopid by Bocu er
ar, (1994) fo. !t!drtn8 pFdEtion of leti@s ftotu !olI4 pulP i! 3olid4Ltc
fcmenrltion, Thelc thEc sdr potntial shtin! weo duroted by tw id.diadons ed l$o
ch.micals (MNNC rid EMS)
li6 dlffurc of 60 marrt6 und.r W lonp 95% lill nlc ws obscd dd four
bdoDyl Gbt !t nuLrts w@ sl*t d ed r6rd for polyS&tN@c pFducdon bv
a2
subdrg.d f.|]Mt tion Onc out of tls fou mult |! gavc nainu Polvg&hftne
rctivily i,!.0.68, L21, I 15 dd I48 U/n 'mn.Iler 24,48, ?2 dd 96 ho6lt+6ltv'lv
wh'l. the etivity of plmt $nin ws 0216 O 50, 0 64 dd 0 ?36 u/dn-hin ttd 24' '18'
?2 rnd 96 hos Glcliv.ly Ara t[c Epdt d tqlndie of lEtin H27 ntlclit wilh
NO,n{C fo! 40 ninntes, 95% mntlity ntc wd obw.d, four hdomy' B'sttd nurdF
!@ st*t d ed 16l..l qudtitiliv.ty in sh.t [dk for pctirlyls bi6rvnrh6t rhe
d!l. rhopcd rtltonc mur. of H2? Pddlc€d 5,8, l0 ?, 24? dd 29 U/nn-oi! dlq 24
48, ?2 dd 96 hou6 6p41ivclv whit. lhc tcivnv of p€Mi stttin wB 4 1, ? 9' 14 2 8nd
16,0 U/ml-min, Th. sclcctit nuunr ol slBin H5o bv EMs dow'd 63, 10 6, 20J a'd
27.6 U/r .mh *hib iG Pedt g!v. 2-9' 12 l' 13.8 md 14 4 U/n!{in af|.. 24 48' 72
ed 96 hoe rsPetiv.ly. TtE Bboque ollow6 quick *ldlion of muDtcd s@i'&
vhLh h@N obl€ lo 8rd uda nw qonditioN, thh 9!t ofnuralts s'l*tion ha b@
rpon d by saol F&.4h6, Bo* dld BaFer, (19?") sler'd lh' n td !&ds or
/V@dp,ro sa$a on $? bsit of b@mvl Bistac' Salvrr a at (2001I lle *reded
rhe gdetically inpovcd pccfircl)4ic fugi bv t nonvl rdi6t!'t sh!i$ Coe'onng
g6.ric impovmol, k@[email protected] MlF (199) EFn'd $o ed thc fold
inct.e oa PCe sd Pl& PrcdFtion in nul! 5 of '4tt€r'rb nAer' FdEds"
Salom a ar., (1996) obhined n anls of P4ErIli!'' dPdltlu wiin ! two fold
incrcosc in Pt,!s aftt PGsc prcdwdon G compcred wnh o'i8jml 'tnin
Hoq ttdl'(2004) tr!.d sinil{ 3tnt gy io inqFvc ciEi. &id ptodetid bv $@ *l6t'd stuia of
t:p.rsillu ,ieq noh @c tul.s , Codlial suPdsion of 'l "igdt GCB-75' shich
eroduoed 3r.l gl citric a.id fsn 15% (*/v) tule6 slerr, w6 subj"{Gd ro w-induc€d mutagd6is. Atun8 thc 3 vdids' CCM-45 6 foDd ro bc t b'd6 prodw
of.itrc eid &d slve 5OO gn cimc eid' sd it wG f!ftbd nipov'd bv ch@ictl
mul.qrlruhuins(MNNC) o!!of rhE 2"doxv'D-slwo"rditn Ei&l! GCMC'
? I el€r.d s lh. bd mt .t, whicb PDducld 961 e/l ciLrc sid 168 b tnd
Itubrid, lt wG ob$.d rh.t $. nult!ts w@ flsrd gosi'8 org&is dd
prcde.dnot citic acid.
Mdy dp.dtuts htv..t|.'dpt d to i4@. t!. p<folrrc of dzvbe podwtio! bv
irpoving cullF ondihoB A0.r nulti-stcP s@ing ud tutalon l,te nullnt! w6'
8l
optihiad by !tudying subn rge<l fmtnlttion 4t vlrving fmcitltion paio<b' PH 'id
Thc l@illM sclivities ol PGe, Pb& od PEe e@ l'49' 28'8 lnd 27 5 U nl'oin
Ecod.d an r 96 houts oa fc@ntalion Friod' It *s obs@td thal dlc prcd@06 of
pdtids imE!.<t with d iftllE h lffin ion llm l6m 2+96 ho@ sd ir wa
didl ,n r 96 i.us wbdd lhe activitv thowed d signific!'t d@lin' !n'r 96 ho6'
Frictlrich er dl (1989) r.poned sinild rcsulG of peline ptodulid in l0 lilt
bioidclor sing ,rtP?4 &t ,ig€l dut nt Al38 Mdinral 'clivilv
*s &hi'vcd lnd 95
bou6 of f€metlitior ed lhdbv th. &tivitv E@in'd @tll'!r' lri'dricb tl d.| 0 994)
in rrcthd study ltttb6td peLitl,G dsirs crliv'tion of'4 'iaet
in '
nFdim with
gl@* @tMr.d@ of 140l8/mr. Mqibw eiivitia of p@lins! w@ obllin'd
wirtin l4Ol8O hoN. Thc diDud siivitv of polvSaladwns' *G 3 U/cm ' l3
U/cmr for D€dn*lers dd 0 5 UhD' for Fotinlvd'
fte high€sl aclivili* of ?€e, Pl-lse nd PEls w€o l ?O' 294 atd 318 U/nndit
rccordcd al 'niti.l
pH 6 0 ThB lhc initd PH 6 O ws ued tor cnztrn' svnth6it &om
6u1ant sirlim in nnlq 3tudi6. BlllJ@o et zr (193) foud $ar optinm lcrel of
adivity for p*tins dud€d st nd n uF,l DH l4ls tnd lt hi8! ldnFfi!6
Th. ffiimd &tivnv of dl $F nutai.d sE ils (Hl2t4 H27d '!d
11506) fot PG$t'
PLe ud Pr4 wa. 1.S4J3.6 ed 32.6 U/nn-oin rdoided in cul$F fntltc obioin'd
feo subn rsed f.lrMt tid nedi@ itrocullted wilh 6nt v€8d'livc inooullm The
FDducdon ofpetines g.dualtv incE$cd lbm 4 b 6 n hul il wB dralicaUv !'d@
Patl ad Drvan@d {2006) studr.d pt..6 vdirblB lEh 6 !i4 of iNdurD' PH'
tcdp..lturc, pani.lc 5i4 ..d mtt@ @ttcnts oflimiz'd with d 'in
lo achi€ve Oe
@inM prodEdon of Pdftjs, Thc iF@'d ld'l of petines ptoduc$on sG
@dcd rr pH 5.0 dd klDd.@ l4'C in slbnqg'd and $lid4t!G ls'nLlioi
@di1io6. fllc i@ulm st NB lo5 d _r 5po$ for slbhdscd dd lo7grsporcsfo'
did-sralc ituntation Fivc hundred mi@netq pdiclc dz' md 65% nd$e ontcnt
or slb3ditc wer. oprinun for IEi'M Prodlctiod of pcti|tjs in $li&trdc
fendration. Undd opdmm 6.didons, tdit|M ptldEtior of petilllg wa
34.2ulg in SSF od 12.6 Urnl in SnF.
Ir s rcred lhal 04% tdt 'xlFcl 'xhibircd 231 U/mUnh activitv for
*,rg.r"**" O, Hl2n $d 38'9 U/nl-nis &tivitv for peti'ltrs bv H27n'
Mrx;M !.tiviry or 3l.6 U/t _nin for Pari'o$'6e ws dhibitd *ith 0 6% ttan
dnlct. lt ws t*ord.d lhtl cofiol (vilhout vlsl qtBc0 e!v' nhi6u elivitv lt wd
sle .ol€d thd lcrivitv ws G<tuccd at hie! @rc'nhnon tbovc 0-6% of tqd qtr'ct
Additionoftween8oi o grqth icdium ennanccd th' pedn8a lr'duction Mdinm
yicl(t orpotyetbctlFrc oa l-dnlt€* bv Hl2m a l H27m Bpoliv'lv wcrc 247
u* ,0.,, U^-an ", O,ta"
"t ndimM rclivnv ot Pddn6c@ bv H50n w$
3?.6 U/nn-min .1 0.05% of tw€tn8o Thc sowlh medi! co lining lr@ $s 0 loqo or
tqen8o slDs.d lo€ &riri!6 or"2yn6
fte cxtol of goeic divdrrv mng dE 8!n'ti' trEtdiis hd b@!
doering v.rios @thott! ovd ! pdiod ol ti@ 6irg ' widd 69'
Rccd devctopndts in mlduld biotogy 'tubl'
c to find oln spei6*pdific DNA
nErke6. qhich d€ indtpsddt of 4vnoNd|rl vdiaii63 Dui'g ttQ ldr d6!de'
dcrl Ghliqc .ub s Pi "
Mmoillitt' RAID 'd AI!-P dc hw b@
.ndiv.ly .dop!.d lo id.ndfv tho lPtciflo g!tr'tic @l(6 Th' ultidtc fom of DNA
ch@ldi4lid is th. @nPletc s'qrcncing of thc g@na Amola $c rook id'nlifi'4
ug6of RAPD Eq.td hs ltt incd '
widd m8p mt only in''l tlitt but tl$ i! d'ry
li!;s o'sonisi. RAPD m*cs scnordsl !t DNA ldcl !rc kiown lot thcir lcchnical
u*i",o * tgo t*tto*- (willi@ d al' 190; wclsn and Mccl€ldd 1990)
RAPD s. @t or tcs @don v dislnbut'd thre!8hout 6c gcmtu (KdL t', 4i'
194). RAPD @nd lpm thc lsjorilv of thc chreno$nE Nd mlp borh gnximd and
dinrl b lh. c.olro|]tG CaMs " ar. 197) Also R PD dtds hrye bdn
'ue ro
mp nov.l chnmsonal rccios (Moda c' a'' 1994) dtd' dEetoF' mv b' ablc ro
t8g.t Sorc.pdr rcsioe of 6e chremetut It is @nvenidt' clticidr dd snsitive
r*iiquc rd aereting rhe pot'rmiphis of slnomic DNA (wcldr t' ar' 1990) rnd u
hor. @onomicd tnm RFL? (tE er al' 1993)
Wi|h thc .ttv61 of rcR l4helog/, n nc hcomc po$iblc to studv g@cric dilfcr.Ms
in ftlgd st itu RAPD is a od r@!niq@ bts'd o! PcR (w'dlio t' al l99o) r
ltilid sinsle snon ddm Fid6 to ahplifv tucl4r DNA in th' FCR Short
85
oligonudeoidc priner in PCR Eactions wnh low@d &n€aling lemp.EtuE anpliUs !specmn fmm my tcmplare DNA, One or noG of then @ ofte. polrhoQhic and this
polyDorphFm (RAPD) c& b€ gcn.rcalty napp.d.
Ceneic diveuity is cohmonly nasrEd by gcnctic dnffce or eeneic aimitanty, borh or{hrch Inplt that thee @ eitnei diffcmcs or sinitditie or $e Ecncrrc tevet tweirr990)
For lhe assessnnt of Beheric divcni9, mong wild and paffit stmins of,4 rrsrl nine
rineu gavc propa suhs, PCR produc$ w.rc M in gcl, lhe mptificd DNA bands
*w obs.Beq the absen€e of band eG @rdcd d .!, dd pBenccas..t. lhis innns
system eanier mployed bt Kandzi .r a/,, (2008). Thus rb. data vas recorded in lhis
mamer and maltzed through Minnlb 14.
AII tbe primd sho*.d dinorphic beding !6ncm berw*n p@nr and rheir nlranrs.
wher€a mooonorlhic bedjry pait ms werc obs.ry€d @ong rbe parcn$ by nosr of rhe
pnm€6. DNA fngerprinting d.r. .lucidlbd rhc wi.tions dong d€ [email protected] and
nurdu. L6s ge.eric dieeGiry adong rh€ p@nral -,1. ,,g.r dd nore genetic drlc6rryahong rls mulanrs wa foud.
It is Evqkn foe thc 6d.r shoen in dendrcgms rh.t rwo wild srr.ins i.e Hl2 rdH27 d 10070 sinild {hile othcr dc.drogrcns showed then simil&iti.s up lo 75 j%.
The ndimun s;oildity level bctwtu Hlz ed H2? ws arhined when DNA of borh
stFins seE mptncd witt primd OrcD, wnib d.ir @Jmc p'oducrion w6 drlf.Mrunder all condidons. It prcv.d rhal rh.se srrains hale sone dijleFhccs in on\€r pan ofther goomc. The sinilriry ldets dong all thc nutsnts i.e. H 12, H2? and H5O 6ns6iom 88.s ro 4l%. Mon of rhc dddrogms showed lbar de nurrn$ exhbtr.d hrsh
%age of similarity lelch sitn thci. pd€hr. Howevq thc !€snlr ct.lrty indiclre rhar
nnians wcrc rorally disiincr od divergor iom lhc Bl of rhe sr.ains as rr did .ot cllstcr
Ac@rd'ng ro UPGMA all tne strains have bed divid.d in ro iwo maio goups eeh grcup
containing th@ sn_ai.s. The srain Hl2, Hl2m sd H27 wF plcd in grcup I *hrteH27. H50 and E50m wee plac.d in Croup II. The Esut|s of UPGMA mdrcarc rhar
slmilariry b€nv@ Hl2 dd Ht2n ss 86.3% rnd both of rhse strlins showed 764%
smil&irid wirh H27. Sidilr b.i.vior m obsdet i. Crelp II whm Fjo md H50m
indicated 3s.770 sinilarity and then sinilrity wnh H27n w6 ?6.2%. The resLrtts depjct
lbal Sronp I Md Gsup I I were ?0.8% sinil& ro .rch orher.
N,n'd Aiat, (2006) ralyz.d gd€tic relationship mong ituee 7sle€tld rpl using lour
trbibiry pnm.6. DNA .xtlctcd fron my..li!m of,4pe.e?/16 spp was succsslutly
amplih€d &d preisc infomarion a glin.d abou! tn€n emer,c simihnrcs rnd
dissinn&nies, Thc rcsuhs Everled polynorpbism of 37. 59 aod 5t% of an.lyzed
Like aU oth$ techniqu.s. the RAPD-PCR hs limimtions, anongsr which ac the
compl€xity of sultant lingeryrint pan.Ds dd lh. facr fiat h€t€bzygores .aMor be
dningusbed 6om hom@ygot6. Anorh* shonconing of RAPD n ns lo\ band
cpearability and @!Gne of p6odo bo(h It 6 howcvd be impmrcd by apptyng
su'$ble conditions to Move lhe inpuriti6 in cxbacted DNA and keprng the
amplifi caliod condidoos sbble.
SDS PACE malysis of rhe ptuGin holaled from ftlrlnrs of ,lsle/gtls ,igl ws atso
caded out whicb lhowcd rhc pEsoce of a ptur.in. The el@tropboElic panems ohaioed
lom dnllzcd .nzFe cx!"cts €xnibned no diff.cnc. don8 murmt srBins, poGin
panem Edued by suLmased lmdtarion $owed 78 l(Da a nol*utar si4 of
lnleEst'ngly, tbn ba.d w6 visible only in sampl.s isl.r.d lron ?0 % annonium
sulphde pccdtage saturations ofCFS. Ilc band delechd oh SDS PAC! ofamund 78
KDa inplies tbe pre*nce of p@tind6 protcin. I! indicalB rhal the nurat€d strains
wnh the in€Bing appli€tion of pdi@6, its @sr efiedire pmducion has b.cone
on. of the nost imporr6r dgcrl. tllge s.l. .E mc podlction by nueEd shrns ot,,1 n,s€r in solid 6l!rc fmotation w sMicd in thc nedim witb citrus pel .s $e
only qlbon $urce. Thc .crivitiB of polygalacturonde, p4nnlyase and pecriicstedse
werc signincmtly high as compde to srbm.rgcd femotation, potygalacruronase
acrvity ws ddinuh aner 96 hom Le. 3.55 U/ml-nin by Hl2fr and minroum by
ll50n i.e 2 7l Uhldin.Sinilar beh.vior w6 o5@ed lc peiinl,r* produdion vhft all the nurar.d shi.sshow€d a gmdnrl incF6c in ihe activity wiih rimc, Thc ndinuh aclivity of pcciinlrse
was recoded aft€r 96 houu i,e.15.2 U/nl-ftirbyH27m md mininum activny by H50n
Thc mximum oclivily of patinesteN Nd rlso @orded after 96 holN whrch *as 41.0
Uhl niD by H50 &d nirinum w6 32-4 U/mhi! by Hl2n.
Childlal "r
al ( 198 I ) produccd pelblg on hrgc $rlc by solid shre f.menrarioh The
rcsults revql€d $ar rh. prodncrim of Nyrc by Asperyillus cofianc'ilr CFTRI 10.18
on wheat brr medruFw.s marmm at 2lhoua offemenlation al lo'C rrth an acii*yof 10.49 U/cmr Aco pdrison ofenzFeproductility anons two nost acnvc stla sof,r ,,ae. reponcd by Paul and Darddd (2006) a.d tne nuta s in $e presenr study was
cadred or The tcsul$ tcvel€d tbal ndihlm oMyne activiry by 'brcincDnonedsulins in opanM coidnions for €xopetin6e wos 14.2 U/s under SSf d lor
endop.ctine w6 12.6 U/nl uder SnI, whercs tnc muhr stnrns r ,2n. H27m.
Il50d) rn ihe cu@ study €xpcsed mdinum enzyne actiyities ofI55,3t.2 and.4l
U/dlnin for lcase, Plase dd PE$e, especively.
The nain objalive of lhis G*cb ws to inplove lh. strains of Aspqgillus niat b.\
IncEasihg lheir yield of peti'M. d import nt Equnenor for sevesl natronal
industi6. For a cou.rry lik ou6 vith norc $d 160 million p.ople dd w'lh yc lrpopuhnor growth rtc cxc..ding 3.2%, a significd anout of @ym6 will be Bqu,Ed
to tulnll the ned of th€ induttid in tute. This study will pmvrde thc gound for
developmenr of industrial .nzFes ro n*t $. loc.l d.ned. ft will slrengthen domestic
hdusry wiih consclu.nt s@o @nonic benefits.
88
REFERENCES
Acu$-Alguell6 M, Cuu@'Rojas M' vinicrgB{on4l'z G' Fwelt_Toc E'
199?. rroduclion md propenies of thr' Pedinobdc activitica poduccd bv
,ltPeteiltu ni|d it slbrdgd sd solt ! ' fqrMtfi[ Appli'd Mlqob
4d Abt d$ol a3 (5)t 8OA'8r4
Acu.-Arg@ll6 M, Guti.n4-Roits M, Vinicg!4oEzlc? G' Flvclt'ToG g
tqg+ ercct of war"r crivitv on exo-p@tinde Ptuduction bv '!spe'gl/F
,is.r CH. on sotid sraG fcmenta\i$ Biotecl[ Lett'rs 16 (l)t 23'za'
Adel; EA, 1965. oprinal condidoN for nutas'udis bv N-mcthvl-N-niiro'N-
nirroeoguanidinc in t aoliK-l2 Bioch'n & Biopl'vs R6 Conn l* 7aE'
795.
Aeuibr G, guihon C, (1987) stimulalion of lhc prcduction
plotinolric @tiviries of '{ sP'rsilhs sPP Enzvne Mi'tob'
Huitron c, (1991). lnflucnc' of PE on 'ndo
and
by Aspe4'ills tW CH-Y-|O43 Cm Jd|
Aiiloo KE, Smilh Jq l/ood BJB' 1994 lldslial sp@B of sov saucc
foncnbtion 6ing 'lvelg/fa From taonoot ed gen'tics to indariat
application (.ds Powcl I(A' P@{ick A sd Pcb'dv JF) Pl6m Pres'
Irndo!! 155-169'
Azin DA, Nooozi RF,2001 Eff@t of chonical! d $' improved gl@nrtc
predetivitv bv e,lrP'4i16 ni!'t fitii' Appl Biochzn & Blot@htol'
6l(3):393-397.
Chapter 5
696.
Asuilar C, Trcjo B, o&ci! J'
sop.crin$e Producron
Mtcrobiol3lt912-9t7
E9
Bai ZE, Zhng Hx, Qi t{Y, P€ng Xr|'/, Li BJ,2OO4 P@tiae producdon bv
/lsperyilld niSel \singv&sr. wal4 in solid statc f'rmdlstion fd 'liciting
pler.li*ts.rcsist D@ t,bterource Te"nologt 9s: 19-52
Bai zs, Zlds EX, Qt gv, P.n8 xw' Li Bt' 2004 Petin'st Plodrctid bv
,1sp.4ill6 niger' Vsing *^cc wrter in slid tttlc fdtrMbhon for clicitiDs
pl6t di*dc teis|!n4 r,bres o!rce T@hnolog 95t49-52-
B.il.y MJ. P.ss B, 1990. Strain md proce$ for Productim of polvgdacluton4
Eazyne Mi.rob Techhol l2t 622'624
Baka RA, \victd L, 1996 CUlMt and Pot nti'l tpPliotion of cnzym itrtulid
in thc food indultry, fretdt Fo o.I S'i Tah'ol 7: 279-244
Bm@t Pdi6 MC, Coclho rLC, Silv! DO, 199' ?mduction ofpcctinlvue bv
Percillib 8i5@4*M cultucrd otr tudosc ed y'st oxtract for
d.sumning ofnaturAl lib€t ,€,rer in AWIE1I Mitob ra: lZ7'129
Barudawitl. Dtvid M, lg4g Dehination of serum Protcin bv mcans of biuret
rea.rio .. J. biochen lTT:751'766.
Beios GJ. Rott i8u.2 G, Tondin A, 1990. EnviM!ftnlll dd Ntntion'l ftct6
cortolling aflstoxin production in c{ssavt $lis shte fdrcntadon Jolz
Feneal Biodq. 7 0: 329-713.
Bltbglino RA, HrcrSo M, Piloef AlvL Bartholonai C, 1991- Culture
rcouircmdt! for the produclion of Prolee bv '4rye€ilur orizae m @lid
state flnnentltion.,4pPt Mi.lobiol Biot"hnol 35: 292-296'
Bes QK, Bltushan B, KaPod M' Hoondal GS' 2000 Production dd
chaoctcrizalio. of 1hcm6iablq xvlanN dd P€tin'* frcm St4pto"trys
W.QG'3 Jot Inda Micrcb a"d Eiotat 16 (Z):211'2t3
Bcg QK K.poor M, Tcwri RP, goontl GS,2001 Ble@h boostbg of
Etcatwtd *'aft pulp sing @dliDltion of xvlease and pectiMse ftom
rrrepron*e, sP. QG-l _i. Xa r!4 Puj.b univ'sitv 57: 7l -78'
90
B.hde A, Satre.rays V, Prdwal'Dcsai S& 1993 SePar*ion tnd litlt'd
chdcteriz.tron of thrce Polyg&|uonas of AsPeryiuw nig'i Entrne
Microb. Technol 15. t58161.
Berovic M, LoealDer€ncin M, 1993. Sofid nale fendmon of p'ctinolytic
.My6esby A. niseL J Chen T.chnol. aiokchnol s6: 209'2ll
Blan@ P, Sieirc C, villa TG, 1999. Prcducnon of pslic 'nzvmcs
in v'asts'
FEMS Mi.robiol Lett t7s. l-9
Bt&dino A., Davills K, Ca m D' ?andi.lla SS, W'bb C, 20001 Utilialon or
wholc whd lou foi th. production of exta@llulu Pectiiasc'g by sone
fungAl st ains. Pecar,rb.re,n a1:497'5Ol
B@cd, s, Rouss!,.Gurierez M Sen no L, Vinie86 GG. l94 kodrction of
p.cdde froD cor€c pulp in elid-stlte f'm€ntation 3vsLm: el'ction of
wild fungal isollte of hislt potmcv bv a sinPL ttft''sr'p $Gtring
E lmi+..J Food Sci Technol3r(l)t22'26
Bolk K, Bdymer HD, l9?4, Th€ Senetic mslvsi! of rcsi3luc' to bcnomyl in
Netdpaft cwsa Jou Ga M|tubtola1t 5t'56
BruMo MHN, C@lho ILC, tuaujo EF, Silvr DO, t993 P!'ti'I,!3t tcrivitv of
Peni.i iM Ariteolosqn rchted b degumming of hmi' Ra Midobiot
U.175'118
Cln JG, 1985. Tc., coficc ed @c@ In: B.I.B Wood, Editot' Mioobiologt of
len^t dJootu El!.vier scier@ Lrd' London (2)r133-154'
Ca5tilho L\ Alvq TLM, Mmdrotho RA' 1999 Recovery of !€ctinoMc
@Fcs lro.tuc.d by slid 5Lte .n\turc of '4 tt84 Pro@s Aiochen y:
t8l-186,
C.cci L, Lodo J, 199E. D.t€ninatior of cEvmatic activilies of com'rcial
pebnes for the clarification ofaplle j\i" F@d Chem 6rt 237-24t
Chilci G, Magro P. 1997. Pdtinlvlse dnd Polygalacturo'ase koenzFe
ploduction bv ,rfrlir cinqea &ti^s ih' €dlv st'g's of inf@tion on
diffdenthNtPleb-1 PtL,t Pdthol Ta/J)' 6149
9l
Cotty ?I, Medronho RA, Leite SGF, Couri s, lgg5 Partial purificdion of a
porySalad@n& Foduc.d bv $lid $^. atln@ 6f 'lsPergill6 nieer
3'15R. Rdista de Mi.tobtoloqia. 26t 31E 322
Dani H, Srzldyk E, Pachleeski & Rozvcki g' 1999 Cclhrl$e, pectine and
lroleine ProductioD bv rhc .ctomvcotrhizal fungus Ca'rioreUa ciboits'
tu Pedobiotosia. 13(3): t93'205.
Dalsa.d A, Palil SR, 2003. Detcclion of po&ntial fungd isohtes for lhe
prcduchon of F.the fiom d*.d.d &i€d lunoow'r had' 'io6
lal'94 (6): l0l8-1026.
D.-Gagdio A., Mdd.lari G, Arctu N' Nucita F, Tripodt MM. Lccurlo RB'
2002. SCP tnd crudc pcctinase prcduction bv slurry-$Ele fcmenrltion of
- IeDon pulps. r!b/"r' tcch"t S!: 89'94.
/ Dhillon SS, Gill RK, Gill SS, Sing! M, 200a. Snl(ti6 on lh6 utiliation ofciE$
P..l for P4tinsc Production Bing I$srs AsPeryi B ger l em J
Entitu,. Stutlt* , 6r(2J: 199'210.
Dinu D, Dd V, 1994 Sll.cton of,tlrsralir$ ,iAet .ttiins Producing P'clic
.Ey6.s. AtuleL U"iE6itatii Biologl. 4a. 33'17 '
-Dmu D, N@hifor MT, sloiat C, Costachc M,Dini$hiotu A.200? Enzvnes wilh
/ ncw bioheoel prop.di6 in t!. Petiototltic comPlcx Prcduced bv
Atperyill6 4i8er MIUC 16 Jomol olEiot@htotog l3l(2r' 128'l3l
Doyle JJ, Doytc JL, 1990. Isobtion of Plant DNA ibm fr6h dsuc '4'r'
12:
l3-15-
En{i TM, Mdk nm P, 197? In: [email protected] A. (Fn) Production of @Uulytic
@zymd by tungi.,'{dvar.e ti Biochem Engtn slt)t l'23'
FetMdcesalnlo T. Adorin M, Chas-Alv6 AC& Coelho .ILC' I 996
lsolation of peciinae hyp.Aroducing n't^nts of Peniclium *pa^tn
Rqda4.' Mic rcbit os ia. 21 lt ) : | 5' l 8
Fo&ny WM, Kelly CI, l9E3 P..ticet'zvn6 Microbial Enzvne Eiote'hnotos'
(Fosrrtv wM, cd.), 131-l 82. Applicd s4i6cc Pnllish's London'
92
Fnedrich J. Cinennrn A, Stein.r W 1989 Subndg.d production of p€'tinolvlic
.Mym.sby tsPqai B "ieet Eff.ct ofdiffccnl .@don/sgibtioo rcgim'
lppL Mi.rob. Technol. 3lt 490-494
Fn€drich J. Cimcmo A, Slcinq w, 194. Con@mitat biosvnlh6is of
jltp.tli B niler Wnnolyn .MFcs .nd citric acid on su@s 'trtd'
Mictob. Teclrol. 16: 703-747.
Gailing MF, Guibcn A, Combcs D,2000. FBcbon l faclorial d.sigts apPli'd to
.IzytBlic su8!r beet PulPs tressing imProveMl rioProe4! t'& 22: 6+
Cgfun, GC, Hous RA, 1992. Cirds w4stc an altcnatiE strbstilt for p€crine
producrion i. solid stat cultuie Bior6 Te.hnol.3gt 91'95'
chanem, NB, Y@f HH, Mahreu$ HK, 2000. Productiotr oI 'lsPerauttw Etu
xylan$. in solid slatc cultuet: aPPlication of thc Plachett Bumn
erpcrift al deign to .vdloale nuFitio@l requiM€nls 'ioB-
Tech1ot'
761: l13-121.
chil.bl NP, Prnakrishn. Sv, Nimat! Devi P. Lon$nc BK' Asth.na HN' 1981
L!r8c $alc productid of peclolytic Gtrzvnes by solid statc f.lmtrtion' JFood Sci. Te.hnol.rst 248-251
tlao YY, Brlck n RE, 1994. Pcctinse .ctivitv of v.getlble sPoilage bactdia in
nodificd ttrlosPhcrc. J. food- &i 59 (l): l?s'178
Haq I, Ali s. Qade.r MA, Iqbal .I, 2004. Citic lcid Productid bv lcl.cttd nutdls
of Aspergillus niSer lron ea. MlsdsEE Diorcsot e TeehnoloSor' 93(2\:
125-130.
r.l@ndal CS, Tiwari RP, Tiwdi R, Dahiya N, B€g QK, 2000 Microbial .lkaline
pcctinass sd fieir .PPli€ti@st ^
ftview- lIPPI Mi.robiol Aiotechnot-
59:409-41E.
Hous RA, Voget CE, Enol! IU, 1998. Som. f.ctoB .ffccting p@hra3€
prcdudiod from spPle Pom@ i! elid slat6 dlt!€ ,atl t/6,6 21: 147-
't57.
91
Idrid, M. Ricard,2000 PcctinN in pap€r maldng Solving ret€trld Problcc
in mch4i€l PulF bl.0ched with hvdrcad Paotid' Eazvne odt! Mictub
Tec h nol. 7(r\: | | 5- | 23.
Jai! S, Duran.t H, Tinbv C, 1990. Productio' of €xtlcellule pcctina* ozvrncs
by a mutait (Por6) of Petrtilliun o..itanis '1lppt
Mt'tobiol Biotech'ol
34r 308-112.
Jd-Fdcoi., Thiobault and Ctuistrne M' 20ol Pbvsioch'mi€l ProD'ne of
!4trns in ih€ ccl! walt and anct .xr.cti6 vo J' Lncrcb 'lntlEiotechnol. 16 (r4)t 112"114
K'poor M, Bcg QK Bhusho B, Sitrgh K' Dadich KS' H@nd'l GS' 2001'
Applidlion of alktlinc dd lh€lmntble polvgaletuMe from Sati[6
sp. MG-cp-2 i. .t€gumming of rmi. (t@"'en4 'iwa)
ed sunn henp
(Ctototano juncia, b"r fib.B, Pftces' Bio'hen 361 803-807
Kdgi F, Cumc AJ, 19E5. Solid sr.E fmcntation ofswc'l s!8hum lo €thmol in
rcrator drum fcrmdror ,Dr4L ol 8io48.27 | llz2'1125'
Kartanzi SK, St warl JMCD, 200E. A$@ittioo !'alv'i3 of fibd llail in
Gostlpiun aftoteud ^.@$iM,
Plant Breeding l2T (2): I ?3- 179
Kshyap D& ChddE S, Klul A' Tcwui & 20oO' Prodetion' Pwindion and
ch.ncr..iatid of P.cdw nom Dac ls sP DTl wodd Jotnal "Mt tub. and Biotech^ 1613)t 271-282
Kdhylp DR, Volsa !K, ChopE S. Tcwsn & 2OOl APPlictlion of p'cdnG4 ir
comdcial $ctor, a rcvicw. Aio 4!on Te'h'ol 71t 215'227 '
Kau G, Kumr S, Sltyanlnlaa T' 2OO4 Prcdetion' chtEcl4iafon ed
appli@tion of a th.moslable polvgstrclwole of ! ihennoPhilic mould
Sp o tot / ic htn I h e n@ P hi I e Apinis. A io rc Yu Ta' n o l 9 1 : 239-241'
K.rrdz ZI, 1955. Pccric EEytre Metlo.b En4tuol lt l5a-166'
Kolnd IA, SPtul.ling EM, RobiNn ltw, 1959 Apprcvld ltboototv &clniqu*
5ri.d, Appl6to4 lnc, N.* York !P. 54-60'
9l
Knshna C, &d Chtndhs€k@ M' 1996 Ba!'na wlsre s sub$are fo' a-ltnvls'
production by ta./16 r/titln (CBTK 100) under solid si'tc fem'ntation'
Microbiol Eiotechnol 46t 106-ll|
Ku|a N, Nagamu. Y, Ya@oto K, Hdshim Y, slc N' wu J' Anronio BA'
Slomu!! A. Shimi4 T, Lin SY, hou' T, lukud! A' Shimano T' Klboki Y'
Toysni T. Mivanoro Y, KirihM T. Italsata K' Mivto A' Mooa L'
Zlong HS, Tamm Y, Warg ZX, Mom'na T, Urchd Y Yeo M' S!s*i
T, Mi.ob6 Y, 1994 A 300 kilob.s intdal Scnctic n$p of n@ including
883 expGsed s€qum6. xdt!re Cenet AtX65-312-
Latif I, Rtjoka MI, Malik (A, lgg5 Prorluction of c'llulose !v lhcdophilic
fsEi gto\A 6 L.Ptochlod Isca fiw VJ Mimb Biotahrol' rlt 341'
34E.
ls SB, whii. TJ, T.vlor JW, 1993 Dcl@tion of Pit'topthcr' speci€s bv
oligonucleotid. hvbridizltion to anPlifi€d iibosomal DNA spacc$
PhrroPdthotog a! l77'|8r'
Lcon. C, Ve DtU, 1986 &68!latid bv dbohldltre of the s'qMtial t' vtr'o
prcductiotr ofP@tic @vD* bv Botvtit 'inerel
cm Joat Bot 65:2133'
2t41.
Lcuchtcnbqgd A, M.ver G, 1990. Svnth6is of difLrdl p'con&s bv
lilamcnto6 8rcwing ,&P€.4,,6 nigq lttatt Folio Miqobiologica 36 (4)l
362-166.
Lruchtcnberg€t A, M.yer G' 1992. ChanSed p@tinasca svnlhesis by lggregrted
mFclium of snc ,{lp"tail6 '49et
muttnts C'nfiil iFtilutc of Nublno4
B.rgholz-R.hbdck , G..td v. E'4de Mlcrcb Technol llt lA-22'
LO-Crrto R, Tripodo MM, Lcszzt U, CiufAG U, V&cono C' 1992 Flavonodi!
recovery dd SCP ptoatucrion fton ffig'P€tr;l Bios Tdhnot' 42: A3'87 '
[email protected], Las@ L, 2004 OrgtDc lcid Foduclid bv filmdtou fungi'
Advancs itr fungd biot@bnolog! tot ind\nry' 'lstidlur' and M'dictue
307-340.
95
M€1us, J, dd bru!. L, 2004. OrSonic &id ?rodetion by Iilanedous fungi in
thc 8cr6 AsrErgillus Froft Tqonomy lnd ccnctica ro indusrriat
applaicatioN (€d.. pw.rr, KA, p@wicL A, .nd l.b.rdy JF) pt.num pr€3!,
Londor 147-154.
Mald@ado Mg Navdo A& Cjlticri DAS, 19E9. production ofp@lina..! bylqaSdtur l? sirg ditr.m{y prct ar.d lcmn pcll s 0E cdbot Bourc..Biot chnot Left_ A, 5Ot-504.
Mlldodado Mq Strald.r.te Slad AM, 199E. !rcducnotr of !.crincsrem!. ddpolysalacturcnls. by,.tv€€ /6 ,/:9.. in subn rgcd srd sotid sllt€ syst€ms.Joumal of hdusnat Micrcbiotory) M.t Bio,ec hnotogl, zo. 3+38.
Mlldorrdo Mg Srzrld dc S!3d AM, Clllieri DAS, 1989. Csrlboti& r€Dressionof rh. synthsis of inducjbtc potygal&turcne dd pc.rintye bykpe,EiIB htgq spp. Cuo. Mr.mrrbt t 8: 303,306
Maldon.do MC, Stnss€r de Sa.d AM, Clllicri DAS, 19s9.thc slalh.sis of polygalactuom5. md FclircstcrllEspp. Sci dq ,ltinans. 9:tot-\0.
Mrrd.ls M, Wcba J, l%9_ Illc predu.rion ofelulos.,[email protected]:391413.
ManiB ES, Sitv! D, Da-Sirva &r.cdrrs6 frsm rhermophilict iocha^, 37 (t)t 949 -t 54.
2002. Sotid srrr. producrion of rhmrDsraltcQhen@r@ .ua,tiaaa). Gode, p,oc.s
Mayu O, Scon M, Conn.n@ I. cFecsm T, Bmch J, vilrer J, pickeEgin &Jdlim.r. 1997, Two cryshl srrucruEs of p@r,nty.sc Aon,llP.49r16racrlcd tB d.iv@ conjo.rutionat chra8. tud srikng div..g6c. in th.subsFar. bbding ctefo of Fcrin and p.cur.lye.s. wo d J. Mlcrcb.D iot..ttr ot. li(slt 61 j -8g
Milla cL, 19j9. Usc ofdinit osti.ylic !.id r.!g.rt fd d.rtrmin.rion oftrducingilg,E. Anat. Chen. 3t t 426430.
96
Milncr JA, Madni DJ, Smitt A, 1997. Two ltagc inocub fd Production ofo-
atnyla.. by Bei s aDtvlblrluel@ie8. E"qn, Micrcb Tqhrol 2l (5\:
382-386.
Mirjrna cA, Pcricin DM, zool, tuuctid of P6ints bv PolrPoM squtu@
in squ.oue tw6Pha* systed ,tt , e and Mi'rob Te'hn" ta: 467472-
Mncb.ll DA, Gr4ficld PD, Detl. EV, l9E8 D'veloPhdl of ! $lid sr'ie
EtlMb'rionnodt], Rioteclnol Tah. 2t 1'6.
Mohuib SA. Elsycd ST, Iedrv Ew, 2000. BEtu.tid of dzyrles Prcduccd
Ed y@r lvatru'& 44 (l): 47'51.
Mhid Aiat, 2006. Gmotic vmlbility anong lhr6 lPcci4 of 'r'ge4ttu Rddm
Ad|Plifi.d PolymoPhic DNA (MPD) M{k€rs fd Gm'lic Aslwis
Jounal oJ Appli.d Scienca R*dtch 2(10)t 109'711'
Nair SR. Rr*shil SK, Podr T, 1995 Efr@t ofc.tlon sou6 on th' s}ndEsis of
pectinlse by ,{sPl8irr. 113 (l )r 37-40
Nisio M, T6i K, Nagai S, 1979. rt}drcle Prcduction bv-'lqtailr6 t'?t' in elid
xa? cllnvtt\on Eut Jout.,lppl. Mioobiol Biotec hnol8: 263'270'
Orioc AHS, Allsop D, Esgitu HOW, 1981. Smilt's intr'duttion to ind!'lri'l
mycoloSy. ?'.d., Ed*ltd Asol4 London.
Oimb€ll K, r 994. Applications of ozvmcs io Aui jui6 t"lnolog! E"rme d'n
/@dpD.drii& (APPlicd Sci6c! Prblishds, Lndon)' 129147
?anza MW, Fo6l€r BM, lg83 Dct rmining th' 3af€ry of c.zlbcs wd in food
?r@*sing. Jout. [email protected] Ptot l5t 4$45&
Pdlcy AF, Plge OT, l9?l Diffeent indetion of p'clinot,lic dzym's of
Fsanun @n, Can JM Mkrobiol lT | 415420
Palsons BJ. Newbulv JII, Jacksn MT, Ford'Llovd BV' 1997 Codr4ting 8d'trc
divcrsity rchtidship6 @ r.vql€d in ri@ (ODa r.tu L ) using diffalnl
tukq W... Mot. Beed t: |ts-r25.
91
!t't S& Daymd A, 2006 Optimiatim of P8!ss for $c Prcductid of tun8d
Delirases f'om da*cd.-d sd0owd hcld in submdg'd dd solidslttc
.6din6s. Biu.tource Technolosl 91 (18)t 234V2344
../i.dl sR, DyaMd A,2006. opnhiation of Pr6€$ fof th' production of tutsal
' pcctires fron dc*cd€d sunflowq h.!d in de submdgld od solid-stlt'
c./ndni6t Bio6otrce Te.hmtost vl : 2t40'2144
?.ric,a D. Antov M, vujivic N, 199? R.Pd m.thod for d'lccting low basal
activiry of expo€ctinasc of Po Itpow sqtunosB. Biote'hnol Tah llt a3T
836,
Pcricin D, I.Fk M, tutov M, f,rozcl B, 199l P.ctjne fto'r PlanoPttt
lrliarrr8 rhc.scnr of stlrow '.6n.Nk6'
Eelia lT QOtt 2r'30'
Pr.iss I. Ashwell G, lg63 Potlgalacturonic r.id t!6tlbolism in b&t'rit I'
Enzyinatic fomtion of lHeoxv-Lthrco-5'tex@'uulo& uro l' ^cid
Jot/'
Biol. Chen. 283. ts? l-157 6
RGid J, Richatd M,2004. Purific<l p€.tint!. lowas cldonic d'ftand in percxidc-
bl6ch.d n@h$ical tulp, tntlt e Mtc.ofial Technol 34t 199-5M
Rcviua BD. [email protected] jos. ML,2003 Additio of P'clollic 'izvnct; an
enologist P6ctic. *hic} itprcvcs thc chrcDaticity sd stalility of red
ti]trt(*" Itt Jtur. Food Sri Te.hnol }at 29-36
Rob.rt , Hils.trof, P. Valt., Md4 B Dai$n elzanv Sitvt and F Jo* vicia
P6$os. 2001 PectiDlvts€ P'ldetionbv Pene'illitn sk@to'dd grcM in
sugar csne juic. in rep@tcd blth eliltwes wotld J Mictobiol Nd
Biotechnol lT,4134r7-
Rodriou@ JA, Ecldei! J, Rodnguc Z, Sicfl! N, Dani'l A' Murbne O' 1985'
So'id Statc Fcnn ntatio! of dricd citrs pe'l bv AsPeryill$ nieq
BiotAh"ot. Lett. 1(t): 571's8o
Rousos S, OldG A, Rlimbrult M' Stuc.do CG, Innsc BK, l99t St{'giB
fd ldg. sl. indulMs fo. SSF .Ft'n! cdidiotpG of ftc'od'ma
h.zian6. Riotechnol. Technol 5,4l542O
98
S.tilrna CCH, Pula EM, P.rcin PC, Borge AC, Silvt DO' 2001
ChDcl*iali@ of p.clinlye prcduction bv an mdoPbfic sttritr islal'd
iom cor€c chdi.s- 33: 2, I l712l
Salen L, JoFsinghe U, 1999- Fund.hotah of puili@non of Pllnt eires In:
Tcclniqud in Plant, virelogv, CD, TEining Manu'l' JO' virus
Puifi@iion. IttdBti@d PotaroC€n!!,Pcru':l-I0.
sco|l D, Gnyson M, Erlnh D, othmd r! $Ta EnavcloPedia of 'henical
te.hnologt, E^ztmslnd\etial Wil.v' N€w Yort: 173-224
Sem.mva MV. Crishuti! SG, Gusrkov AV, Okuncv ON, Sinitsvn AP' 2002'
Islatiod md propenie of petine3 from Fungus ,lrePagiflB .&?ori6wortd J.
'iotal'nol ar.l Mi.mt 12 (4): tl48_450.
Snivd<uN PD, Ned K 1995. At&bbic d.gadltio! of r.din bt mix'd
c@sriia 6d oDtimiati@ of f.mbtron plnftt F for high'r !'ctine
^.tNiry. L.tteB in ,1Ppl. Micrcb. Z0 (2), 117'119.
Shonukd OO, Nwlfor OE, 19E9. Ilolation dd panial ch@ccrtitid of
tcrrrate sitivc mutaDrs of a@i 6 euilit lhcrcbna k 42t 4J46
Si.sr v, Said S, 1989. P€ctic @ym.s Plodudio in solid{tll€ fcrmmiation
using citds pulP pellets bv ?tuladrts Jlarvt' Tabercalaio eutga'it Nd
Peni.illiun chade:si Bn ahnol Leu llt343'244
Soes MM, D!-Silv! CNR, Camom EC, Cones E.200l P'ctinlsc 'nzvm'
Droduction by ra.illd ry and thcn pobnlial .ppliotion ofjuicc dF&rton'
r7 (l\,79-42.
Solis PS, Fsvel. TE, Vinicgn GG, Cutiec RM, 1993- Bff€ct ofdilftrcnt carbon
soue on lhe synihdis of Pectinse! bv ,4rpe4iil6 rk" in $bner8€d and
$lid{rdte fmcntgtion ,rpl n-li'abiol Biote.hnol S9t 36'41'
Solt PS. Favela TS, Gulic@, RM. Rous$ S' Sauc.do CO' Gu@kt're P'
Vini.gn GG, 1996 Prcduction of Poctin&s by ,ftpttattld 'tget
in elid
srlrc f.ncntato! .t inititl gle@ @ndttrtion Wo d Jo* Mlcmbltl
Aio@chnol. 12: 257-2(fr
99
Solis-P€rcira SE, Fav€la-ToE* C, Vinid$ C, Guti@'Rojls M, 193 Eff'ct of
.liff.rcnt cu!o! surc.s oD the svDthcsi! of pcctinale bv '4tPe'!i'6 tiga in
$bncrg.d dd elid $t tc fl!1||l!Httd6. ^Wli'cd
Mi'robiol and Biotechn
J9 ( I ): 16-41 .
St€€l RGD. Tode JH, Duok€y D, 1996. Principks md Proc€durcs of Sillislics'
Mccraw HillB@k Co.,3d €d New York, USA.
Trieb NH. Al,rdi M, Tngit S, Aouabddt.! F, Gdgoui A' 2002 HFdprcduc$on
of p.ltinasc activitics by a tulv @Dstitutirc nulant (CTl) of Pett'tt'iiun
occitdnts. Enzytue ,nd Mltub Tah^ol lot 662-666
Tao S, B.ithui L, zuohu L, 199? Elnec.d @llrl@ Fod&ti@ in fed htch solid
i^. fq:.* rati6of Triclodna vilid. S!.l. J' Chen Tehtul BioEaol
69 lt)t 429-432.
Tcix€iB M!S, lilho JLL, Duln N. 2OOO. Cdbon sourc" 'ffcct
on Pcctinose
lroductid fiom AsPctci s j.Podicu 586. Bt@iliot Jouml of
Mtctubtolog al. 2EG290.
Thoms, B, D. 1989. A tqt book of Induttn.l Miqoliologv 2d 'd
Plblkh€d bv
Sinau.r Associat lnc SuitarledPP 207.
Tohlu, Kobryishi, N. Higstj, A S@ttstsu, K Slwada ed H gagihtt! 2001
Puificsdon ed Prcpcni.s of s hie)l nolccullr wciSht slkdine
exopolygElacturdas frcn a stain of Baciuus Enzrne and Mictob
Techol. t9: ?G75.
va(@ino C, cxno RB. Tnpodo MM' Pslsc Rrvq In84s G' Schachtd S' l9E9r
ScP fiom oruge P..l bv fel]Mtalion wth tun8i sumery'd and Surftce
t nttfitt l@,Biol. V6t4 29t279-281
viil@i L, T6ald.n M, Sumakki A, 2OOl. Biocchology in lhc pulP ed paPd
iftlBtry. Rchm EI, ,iorec,ro los. vCH' mler' 571'546'
viikei L, Tenataren M, Suunakki A, 2001. Biot€choloSv ir lhe pulp od PaFr
indurEy. R€hm HJ, Editor, ,io technolost'vc'l'Wi1.v: 523-546'
vilarino C., Del-Gidiro JF, Hours RA, Ca@n' O, 1993 Sp'ct'opholorEtic
nEthod fd fibgtl P@ti[c!t rs9. lctivity dddninalior Food fti T'ohnol'
25 (2)r 107-110.
wang, Z. G., z. Tons S Y ChdgedL M Cd8 1993 Studv m pcctila* lnd
S.l.miim producing abilili€s of two kind! of ''lqt€i1i6 &u isollt€s
fiomZh.jiangMycopathologia l2l: 3' 163'168
w.in{h.n C., H J. Hsi.h md T C Tscng' 1998 hrific'lion 3!d
chddcriAlion of t P€ctin bt!. fiom Pr"'im rPl€tdea iffect€d cucumbd
frils, Dep&tm.rt of Plut P.thology md EntoDology' Nati@d Ttiw4
Univ.rsrty, Tsip.i, Taiean' RcPblic ofQila'
W.i. BS. 1990. G6.tic Datt Anll)sis: Molhod! for Dboretc Populltim Cmctic
D.ta. Sinrud Ascirl.3,Inc Publishd- Sutddlsnd, Mlsctus'n!' 377
W.tih t, Mcclctl4d M, 1990. FinscrPrint g€nots lttng PCR wilh dbit.ry
r'irrffi N@lelc A.td: R4. lEt 1213'7214.
willi.D!, J. G. (., A R. Ku!.lik, K t Liv.lc, J A !"rftlsti ed S V Tineev'
1990. DNA polymoabism &Dlificd bv [tilnlv pdnm atc B'tul ag.i.ti. nt *.8 llucleic A.id X4 , rE; 6531'6535'
l0l
APPENDD(
CZEfAK DOX MEDIIJM
F6O. 0Olst!
KCIt 0.509D
l,lgsOr 0.509!
IQIIrc. |.00€D
N.NO! 3.008E
20,009n
20.00gd
308
o.8 tI r (op6onr!)
100 Dl
45.5 g
lg8
250ot
t4 8Lltgn'
IL
Mjun 66 ps b 83 wilh N.oI ICL b.f@ arlitl lh. wluno uPlo l!
rx rrus-c@nrn rurre* n" :,*Sodim dodayl sulphd. (SDS)
SOLUTION I
SOLUTION tr
sDs
tE
soLUnoN llrax14,125 8
t02
SOLUSIONS FOR PREPARING GELS FOR SDS.PAGE
sDs
DII
STAINTNC SOLUfiON
DESTAINING SOLUNON
?x gd lo.dlrS bofi.r (PE 6J)
T!i!
sDs
DibioltEirol (DTI)
l0% Relvins Ool
10% sDs
l0V. drnonium pcrulphr'to
TBMED
t% Sl..Li!8 C.l
soMid II
lg6.8
250 bl
0.5g
225fI2,5nl
25 ml
225m|
225nl
O-128
04a2bl0.3g
O.U2S
13,3n|
1?ml
25 dl8.33 ot625 olt02,{ bl
t03
STANDARD CURVE OF D-CALACTURONIC ACID
10?6 {nndiM FndPhr&TEMED
)30CoMnL.lim orD8.l&tusnic a.id fus)
0.9
!
?03
0.1
LIST OF PRIMERS
oPc-o3 GGGGGTCIII
orc-12 TGTC T@CC
IGCGTGCTTG
TGAGTGGGTG
r0a