WASEEM AHMAD BUTT - Higher Education Commission

OPTIMIZATION OF CULTURAL 

CONDITIONS ON THE BIOSYNTHESIS OF 

XI'I,ANASE BY LOCALLY ISOLATED 

ASPERGILLAS NIGER 

WASEEM AHMAD BUTT 

DEPARTMENT OF BOTANY 

UNIVERSITY OF THE PUNJAB 

LAIIOR.E, PAKISTAN

OPTIMIZATION OF CULTTJRAL CONDITIONS ON 

THE BIOSYNTIIESIS OF XYLANASE BY LOCALLY 

ISOLATED ,4SPENGZ LAS NIGER 

A THESIS SUIMITTED TO 

TIIE UNTVERSITT OF 1fiE PUNJAB 

rN FULFILMENI OF TTIE REQUTRf,MENTS 

FOR'IIIE DECREE OF 

TXTToR OI P{II'€OTIIY IN E{'TANY 

BY 

WASEE]II AI{]IIAD BUTI 

Df,PARTMENT OT BOTAI{Y 

UNI!'ERSITY OF TIIE PI]NJAB 

LAIIOR.E, PAXISTAN 

2006

Tlis is 10 ce.hry thar thc rcscarch wo* descnb€d in this thcsis entided 

.OPTIMIZATION OF CULTURAL CONDITIONS ON THE BIOSYNTHESIS 

OF XYLANASE BY LOCALLY ISOLATED ,{.tPERCltZU,t ,V1GE{ by M!. 

wasm Ahmad Buft is rhe ongi.al *ork .nd h6 b@n cmi€d out undd our 

sup.wisio.. We have gone thrcugh all rhe dab/rcsultvmaterials rcponed in the 

manusfiipt and ce.tify thei. co..€c1D€ss/aurhcnriciry. we also.e.tify ihai lhe tbesis 

has b€€n prepded under our sup.Nision accordins io the prescribed 1b.nat and 

wc cndoBe ils elalualion fo. the aw6rd of ?h.D. degte through the oflicial 

procedrres of the UniveBily, 

CERTIFICATE 

Prof. Dr. J.ved tqbal...............,..........,,,,,,.r. 

School of Biological Sciences, 

Univc6ity of the Punjab, 

Pref. Dr. Ik.lm-ul-H!q, SI 

Institute of IndusEial Biote.hnology, 

covemnent colleee UnivoEity, 

^ ,14 4.1-l

DEDICATION

DEDICATED TIO MYDEAN PANEI{TS 

,

ACKNOWLEDGEMENTg

ACKNOWLEDCEIEIIT3 

I m thankful ro Almishty cod for rhe durlss btesinss dd idinite 

ocrcf, rnich @lblcd ne ro @mplele rhh diss.nation. 

I endd Dy dccFr md sinffi$ AEritud. to my aoduic sup.ris6 

Prci Dr. Javed lqbtl, DiKto. scbool of Biotoeical Sci6c.s, Univ6iry of rlE 

Punjab, Latlo.e &d Dr. Ik@-ul-Haq. Di@tor, Itrtirute of rnd8r.isl 

Biot€chnoloey, C.C, UniveBiiy, Lrto.. for th€n enrhBidtic gddece and 

inexl8ulible iGpinrion wbich €nrblcd me ro anlin Oe obj6lives wirhour ey 

dific'ntis. I gMlly .pp@id. tbcn @Btlnt m witr lsilive suggdiios &d 

.ndcisD. whw ed wha I neded duins r[e otirc Fiod of my 6erch wd( 

I m gdetul ro Prol D.. Res Mdood Khd, Chrimm, Dopa.tm.nr of 

Bot6ny, Univdsily of rhe Pujob, La,horc md prot Di Amin,ul_Hao Khe. 

Chdm4 Depanmenr of Boiany ed Envirommrrt Sci.nces, C.C. Univdsity, 

LaloE who p.mi cd me to uden*e tnis iDporidt Bc.rch work. 

I eould a,ls tilc b pay ny sp.cial grdlilud€ ro Dr. M-A. e.d6 (Rrd.) 

CSq PcslR Labs, Lahore for his sinc.r. suida@ ad $iriQt sugg*1ions during 

thc rcse@h work. Th. m(hy hculq mmbe6 of Depannenr of Botoy, C.C. 

Untr euiD . tilorc &. scloDwledeed tor rh€ir Bflsos .dvis. 

I m deQly indebled lo Dr. Sit€rder Ali, Dr. M. Hm.d Asn at Mr. 

Mohsir Jaled ed Mr. Huid Muthrn ftr sitrc@ c@p.ndm md hetp. AI rhe 

M.Sc. M.Pbil sd Ph.D. es@h schot.6 in Insritute of Indurial Biot€.ltnoto$/, 

CC Univ6ity Lshorc e lleo app@iabd for th€ flcoungcm€ni 

I u abl. io peht this di$.rodon only due ro 1he utiring .ffons, 

p.lienc. @d ohd* pby6 ofny d.{t p.rdq betov.d *if., wing sistd nd 

innocol Wdit Tb.ir lov., pori@e ed sctrercE h.b6 e a foBer roEh of 

(,*( 

WASEET A}IIAD BUTI

CONTENTS

Ch.pter L 

Chrpter 2, 

Ch.pter3. 

Chrpter4. 

ChrpterS. 

Ch.pter 6, 

GOtftllfTS 

rNTRODUCTION....,........,..............,...,......,.,.,., 4-10 

LITERATURE ROVIEW............,.,.................... ll-54 

MAITRTAIJ AND METHODS........................... 55-72 

RESIILTS...................-.---..--..,.......,....,.... 73-152 

DtscussroN.......,..................,....,................ l5]l6a 

coNclusroN............................................... 169-l7l 

REFDRDNCf,S........,.,.......-.......................,... l?2-200 

LlsT oF RESEARCH PUBLICATIONS ,......................,,,,.........,,,,,. 20!

Table3: 

LIST OF TABLES 

Cornp6ition of potalo dextrose d8armedium,,,,..................,, 55 

Composnion ofphcphate buffer ......................................... 57 

Composirio. of xytan a9.rmcdium....................--.....-----.... 58 

Compositio. of vogel's m.dium.............-................---..-.-. 60 

Compositionofsalinewaier..............................-..............6l 

Compo6ition ofacel.te bofIcr............................-.-----.---.-.69 

scrcning of diff@t $ni.s oI Asperaillus niSer .. -. - -... -.. - -. -... 74 

Slb-grcuping of differcni sinins of,.{spelsil/6 ,,get according to 

xylanascproduclivily.....................................................E0 

Scrc.ning of mutant sirains d€veloped through UV inadiation of 

Asperyillw niser C(BT-35 for xylde bi6yDthesis in shate flask 

Sub giouping of mutant sinins of ,4spe/gi 6 ,,4?r GCBT_35 

developed throtrgh Uv imdiation fo. xylanase biosynthesis in shak€ 

flsk...................................... ............................ .93 

Scrcening of nut nt slnins develop€d tlrough chemi€l trE tncnl 

(N- m.thyl N-nitrc N-nitrcsogldidine) of,.{tp?rgtl/6 ,ig?r BRCU! 

45 for xylandse biosynlhesis in shake fldsk ..... 94 

Sub grcupine of mul.rt str.its, developed ihrcugh cbemic.l 

trcamc (N-methyl N-inrc N nit osoeu.nidine) of .tspersi us 

nrgel BRClvrjfdxylanas€biosynthesis itr6h.kc fl4k-.----..... 99 

Sc.ccning of mutaot shains developcd thiough diffcrcnl tinc 

iniervals of N-nelhyl N-nito N-nit osoguanidine healneni of 

88

T.ble I l: 

T.ble l2l 

Table llr 

Table l4l 

Table l5l 

Table l?: 

Asperyillus niger CCBTwrc.m for xylanas biosyn0Esis in shat. 

flask ...........--..---...................................__.._...__........ t00 

Sub grouping of nutant stBiN developed through difforcnr lime 

intewak of chemi.al tlatneni (N-methyl N-nirro N- 

nnrosoSuanidi.e) of AspztgiUus rieet CCBTMNNGT@ for xylanase 

biosynthcsn in shate nask -.............................------------... t02 

Comparisor of kineiic p|ralneteB for xylanasc production by rhe 

paicntal strain of,4qergilhr nrael GCB-35 and Dutanl GCBTMNNo- 

30inshakeflak.--..............................................--..... 106 

Effst of differcnt agricultuml by-products on the poducrion of 

xyl.na* by Asperyillu ,ige. GCBTmNG-1o in shake ossks 

Etfect of different inorganic nitrogd sourc.s on the p.oduction of 

xllaneby Asperyi us niget CCBTnNG-30 in shake flasls ....- I 14 

Effe.t of diflemt phosphate sources on the producdon of xylanase 

b! Asperyi us niset ACBT MNNC-3o in shake flasks ................ I l6 

Eflst of differenl conccntratio.s of magncsium sulph.r€ on thc 

produclion ofxylane by,.{rpe'a!16 ,tsl GCBTm.-30 in shake 

flasks ...................................................................... 120 

Effect of diflerenr concenintions of calcium chlo.id€ on the 

prodlction of xylan € by ,4s?arsiltus n,ad GCBTNG 30 in shake 

fl6ks .............................................-.......................- l2l 

Effecl ol differcnl conccntratids of c.lcium chlonde or the 

prodlciion of xylanase by ,4rpe4:iflrMisel GCBTMNNC-30 in shtke 

flasks......................................................-.--......-...t22 

I l2

Figure ll: 

LIST OF FICURES 

Sran.lard cnne o{xylos€................................................. 72 

Scffiing of culturc nedi! for xylanae lroducnon by ,{rpe€illb 

z,serGCBT-35 b shlkc n6k.................--........................ 6l 

Time cou6e srudy foi xyldd. producnon by AspeeillB nig.t 

GCBT-35insh.kellasks..... .....................-.-E4 

Erect of dirdent ini al pH on produciion of xyla.6€ by 

Asperyi us nigerCCB'r-3' in shake flasks ........................... 85 

Time cou6e study for thc xylanas€ producrion by,4speryll/rt riaef 

GCBTWT-30 inshake flasks..................-..................... 105 

Effect of differnl inilial pH of the culture nedium on the produciioi 

of xylan4e by Atperyilld rigel GCBTnN6I0 io shatc flasks 

Eflecr of diffffir nitrog.n soudes on the prcduction ofxylansc by 

Aspergi 6 niget CCBTi*rc-3o in shakeflsks.................... l0E 

Effecl of differeni concentrations of meat oxt act on the p.odrction 

of xylanas€ by Asperyillus niger GCBTMNNG'30 ii shakc flasks 

Companson of diffcrcni lericult ral by-products on the produclion 

of xyldde by ,{ry?rArl6 .t?e/ GCBTmNG-1o in shake fl6ks 

I Il 

Cohp6risn of dift c.t inorganic n'trogq so!l6 on rhe 

prcdetion ofxylanas€ by,.{rpera,//!i 'lsef GCBTNG-30 in shake 

fldks...................................................................... ll5 

conpari3on of diff.lcnt phosphate so!rces on the production of 

\yl^n seby Asperyillus niget GCBTMNNa-30 in shake flasks..... I l7 

t07 

r09

Fi8ure l2: Prcduclion of xylMe for five .epe*ed barch.s (in shate flask) by 

Figurc l.l: 

Figurc l6: 

Figure l8: 

Figure 20: 

Fig!rc 2l: 

Figve22. 

imnobilird strain or,{rp".Atl6 ,isa MNNG'7 nycelia in sodium 

alginateand pollaretbane foam ...---............................... . 124 

Time couBe of xylanase produclio. by immobilizd nyceli. of,'{ 

,i8erMNNG-7 insodilmalgi..re....-................-.............. 125 

Reusc of mixed nould mr!.lia for xylane producti@ by 

immobilized,.{.nigerMNNG-7..........--...........................127 

Screcning of subshre for the prcduclion of xyldnase by ,{Velgillrc 

,,s€lCCBTMNG'30 i. solidslab fementaiion..................... ll2 

Effect of differcor deptlB of wh@t bmn on the production of 

xylan se by Aspergilt6 nigal GCBIMNNG-30 in solid siatc 

''''''''''''''.,..''''''''''''''','''',''l]] 

Effect of diff@1 dilumts on 1be production of xylanasc by 

,{rpe.gi[,r ntae. GCBTMNrc-3o in solid stat fer@lation.....- I34 

Time couse study for th. p.odDction of xylana* by ,4sP4ailtut 

n,aerCCBTNc-30 insolid state fem€ntation...................... 135 

Eflet of diffftnl incub.tion tmpeBturcs on the prcduclion of 

xylanse by ,qasl/6 ,ig?r GCBTMN^c,rd in slid slale 

femerL1io...............,,,,................. ..... -- ...136 

Effec1 of different carbon sourccs on the produclion ot xylansse by 

Asp.rgi rs niAe. GCBTMN1-3o in elid s|ac fementation. 137 

Eff@t of difr?rcnt concentration of $aah on thc prcduction of 

\yl^nase by Asperyi us ,isef GCBTMNTC_3o in solid state 

femeniatioh......,,,...,,....,.,,,,..............,,.... .. 138 

Eftecr of diflemt nitrogcr sources on the prcduction ofxylanas€ bv 

Aiperyillus nic.r GCRT{N1 ! itr $lid sutc fementation --. . . 119

Figure 2l: Effect of diff€rmt conc€ntmlion of amnonium sulphate on th€ 

FiCxK 251 

Figue 26 

Fi8ue28: 

Fisxrc l0l 

produclion of xylM. by,{sp€agr76 ,tAel @BTMNrc-3o in slid- 

sbtefementation........................,,,.,...........,,,,,,,,......... I40 

Efiet of tne of iroculun on $€ biosynrhdis of xylana* by 

Aspergillus nigq GCBTMNir0instired femonto...,,.......... l.l4 

ElTecl of differcnt conccntratiotr of vegetative inoculum on the 

biG'nthesis of xylod. by Asperyillus z,gel CCBTeNc-i in 

sriredfemenrd.--..,,....-...........-...........---...................145 

Effeci of agilatio. intensity d ihe biosynlhesis of xylbde by 

,4spe.sr'llrr rrgerGCBTMNN6.I insiircd f.mentor............... 146 

Effect ofacdtio. Fte on thc biosydthesis ofxylanse by rspag,?6 

,ta€lGCBTMNNc-rinstin€d femenlor.............................. 147 

Rale of xylane biGynthcsis by ,.{i/e/g/tur ,taef GCBTNG ! in 

stirrcd fmcntor.-.-..-.--.,.............--.............--..,............. 148 

Eflect of different inilial pH of fementation medium on thc 

biG'dnesis of xyleasc by ,lverAriilt ,Ig€l GCBTmc! if, 

stirrcd fmcnto..........,,..............................................149 

Effect ol differenl tmperatuFs on the biosynthesis of xyldntse by 

/veryrrs uia./ GCBTMNNC-I in stifted f€menlor............... 150 

Prcdlction of xylare fo! five rcp.ltcd fod batchcs (in stirrcd 

femenlor) by idnobilized,.{s,ry/atll!s ,tg?r GCBTMNNa l0 - 152

ABBREVIATIONS

LIST OF ABBR.EVIATIONS 

% Pe|@t g. 

NH4)?SO. A,moniuE sulphde 

'S' Significant 

Prob.bility 

+SD Stand.ld deviation 

A. 

"isd 

Cr 

Asperyill8 hiee, 

calcium 

Ca(OH, Calciuniydrciidc 

DNS Dinilro salicylic acld 

g cnrn 

GCB Govemne College, Bot ny D.Patnot 

h Hour 

H- Hydrogcn ids 

HrSO4 Sulphunc acid 

J. J@mal 

J/mrS Jolle ps squd mct ! P€r seoond 

K Kalium (Pobssium) 

&HPOI 

lb/inchr Poundt per square inch (psi) 

mg/ml Millitraln Frmillilnte 

min. Minut 

dl MillilitG 

Di polssiu hydrcgcn Phqphate 

Mdoxd O.T- Di&.tyl qter ofsodium $lPho succinic acid 

N NitrcScn

{ Dcsn c.nlig.& 

FCSIR t!&iirr Cquoil br Sci.atific |rd l4n'di.l 

Rtl6|rd 

PDA P.ae dd,o.c |g|r 

pH Pown oflf .dc.ftaid 

P.rt !.( nillion 

nc!,tl||tior F rnidr. 

try t ltwiolct 

wa{G A rtrin dev.lo!€d through thc dtcn[i. 

b.turof tIv !d MNNG 

Vohn !.tr vohmc psr niDut

ABgTRACT

ABSTRACT 

T]'e preseir study k concem€d wnh isolarion, selection and opiinizalion of 

clhral condnions for rhe prcdudiof, of enzrme xylanas by Asperyillrc niger 

One hundEd and four stmins of,r. ,isef were isolared from differcnt sit smplcs 

and iesled for th€ production ofenzymo Ofall lhc strains rcstcd.,4 r,gel CCBT- 

15 eave naxiftw production of xylanase. This srrain was selecied for 

oprimi2.rion ofcuhurdl condilions in shake flask. Among rhc fou.ditfere culrurc 

mcd,. e\dluatcd. lhc ma:imum enzymc prcduction (225 U/rnl) was oblained wilh 

M-4. Cuhural conditions such as rate of enzyme synthcsh (48 h) and pH (4.5) 

Aft€r optimizalion of the crltuhl condiiions, ihe parcnral st6in CCBT-15 

as subjeded to UV inadiation ior tlo minDt€s. Ninety-lbuf nutanls wcre 

isolared by obseai.g the zones ofxylan hydrolysis duc ro xyldnase adivny ii rhe 

prripl.tes Ofall lhc muran6 resEd tor cnTymc prcduclion. mutrnt BRC(r{ 

gdvc maxinum producrion (319 U/ml) of xylanasc. The selected Uv murarcd 

stoin sas ftIth{ improv€d after kedtmcnl with MNNC (50J00 gglml) for 10.40 

min Sevcnry-four chemically murarcd shirs of ,4 ,iscr wcre picked up and 

claluated for xylanosc production in 250 ml shake flask. The hulanr ,r. ,,s.f 

smn, CCBTMNNa.$ being a be q producer ofxylanase (493 U/nl) was rlcclcd

2 

In shake nast, cultural condnions a.d nuhitional rcquircmcms such 6 mte 

of cv)me synthosis (4E h), iniiial pH (4.5) and levcl ofmear ext.act (l 0 %, w/!) 

!s ! nittosen supplcmenr $!re oprimizcd. I. anolho $udy, ditfcdt asric!l$61 

by{oduch Ncrc lesred for lhe producrion ofcnzyno by solid-slote f€menlation. 

Of all rhc substalcs examincd, whear bran noisrcncd silh dislilled waler gavc 

optinldl produciion of xyl.i.s (1850 U/g). Thc prcduclivily of €nzymc was 

tunho improled (2480 U/8) by thc .ddition of narch (2.0 %) and ammo.iun 

snlphltle (0.2 %) io rhe femenrarion dcdium. The productior of €nzlme ii solid' 

srare fcmenralion wds found to bemaxinumT2 h aft$ inenlation. 

Xylaiase fcmentation was caflicd oul by fivc{epeared b.tch cullurc afier 

ifrnbbilizarion of,.1. ,!iel cooidia in sodium alSinate or polltrcrhane f@m. The 

production oi xyl.nase was increas€d in lhe s€cond balch by ,', ,,9"/ slrain 

immobilized in lhe polyurethane f@m. The .onidia of ,'1. ,!gel mutant 

C( BTMNNG! were inmobilizcd.nd cullilated for xylanase pro

l 

Scal6 up studiG wlE coEied out itr a 7-5 L srimd fcnftntor. ThecultuEl 

conditiohs such as incubalion i.mpenruG (30oc), inilialpH (4.0) and sire of24 h 

old vegelafie in@ulum (4.0 o/", v/v) wcre optinized. Tbe opdtul production of 

xylanase (?81.4 U/ml) wd achiev€d 48 b alts lhe in@ulation vhcn agilalio. 

inicnsity $s kept ar 200 rym and ai. supply at 2 wm (dissolved oxygcn 0.? %). 

The prcduclion of xyla.e w!5 ale @Fi€d oui by fed'batch syslem in the slircd 

femenror. Ar the cnd of femtutation, 80 90 % of thc f€me.ted broth was 

rcplaccd by f6h slerilird n€dium. The batchd wcE repealed four rc fiv€ imcs. 

The prcdudion ofxylanase was decreas.d sha.ply afler the 4Li batch.

INTRODUCTION

ITTRODUGTIOl{ 

xylffi (EC 3.2.1.E) is an extracellular enzvde, which hvdrclvzes the 0 

1.4 D' 

xylosidic linkages of hiSlty polyme.ized hemicellulosc (Siha 20011 Juge d 4l ' 

2004). This cDzyne h extcnsively ued in mmv indtistries including biobleaching 

of paper dd pulp, inprcving animal f.!d, production of €rhmol and nelhde' 10 

decres€ he viscosity of mash md to Prevmt fouling prcblems in distillinB 

equipmenl (Nuez er al.. 200 t; Sudha er a/ , 2003 i Moen ", dl, 2001I Chst er a/ 

2oo4). 11 is aho welt known that xylanascs hav€ t posidve ered on doush qu'lirv 

(Iialho and Cmona, 2004). These uses have placed grea1d stress on rncreasmg 

xylande ptoduclion and scarch for morc efficidl Procesrcs fq its biosvnth6is 

Tlc xylaase biosynlhesis depends on thc st.ain. rype, 

age and size of 

inNlum, comPcition of medim, nelhods of cultivalioq nubie requiremenl' 

pH. incubation tenp€ralue md fennentatiotr period (Monti 'r 41. l99l) 

Micrcbial prcducdon of xyldase was prefened to pldl ad aimal sodces 

bccaue ot c6iq availability, sEucrual siabilitv and sst to getretic ndipulation 

(Bilg.amiand Pandey, 1992) Filamentous fudgi have been sidelv used thm veast 

od bactsia ro produce hydrollric enzvmcs (Baldi ?t 41 2OO3) /tspetsi 6 nieet 

is fou.d to be the mosl poleDl fungus for xvl@se production (Chen e' al, 1999i 

Wu er al, 2000i Recod er al,2001)

Some improvements have been nade in ozvme vields 

5 

either bv modirying 

culturc @diions c by improvinS lhe slrain by uling the diff.rcnl Phvsical ad 

chcmi@l mrllgonic tr€atrdts. Both UV ddialion 4d chemicals like N-me$vl 

N-nitro N-nilroso cuaidine (MNNG) have bd us€d to imProve A 'iget stîn 

vieiL-vis rflanase Ptrdudion (Gokhal€ ?, d/.. l99l; Milagres €r ol, 1994: Steiner 

et ol , l9gai Chadh^.t al., lggg.K.logens et ol 2OAr' Extensive screenrng 6 

reqrired fd isolation offte best mulanl witb incM.d Prcductivilv ofxvlaase 

Pro.luction of xylanase is Sreatlv affect€d bv &e femeDution lechnique 

Diffetenl tehniques have bcen used for fte ryluate bi6Fthesis' qhich includes 

three majo. caicgo.ies, i.e.. suface culture, subm€rg€d and solid sate ttmentot'on 

(Cai et al, l99Er cawmde and Kmat. 2000; Kansoh ud Gamtnal' 2001i P'rk e' 

4t, 2002a)- Solid{late iermetlalion holds uenendous potential fo' lhe production 

of x/ffi. h cd b€ of sP@ial inrercst in those Process {hw crude ferm'nled 

product mat bc used djrecily as enzvme soDrce (cai er al 1997;Dos"zl 2003) 

Tbe submerg€d femenlsion coNidercd morc benelicial having morc nltrienl 

availabiliry than dy other iype Tle tine requir€d for subnsged i'ne at'on 

*as les and rhcr wd sofiicient supplv of ory8en a3 comparcd 10 the $lid-slale 

f€rme anon (Gones ?r al, l9g4t v.ltz et al.l999i Gouda 2000) SDbmerged 

Ldndtation for xyldase production bv shake flsk hd been reported bv m@v 

{o.kes (Chen ?t al., 1999; Gaende dd Kmat. 2000; Mo'ica t/ ol,20o2i Sevis 

and Aksoz. 2003 i Coelho and Cmona, 2003) Therefo€, for initial scre€ning and

subneqed lementdtion ,s prcIened ov$ solid{Lat€ 

Thc optimiation of feme.tation mediDm pl.ys a very impon.nl rolc in 

en7yn]. produclion, lt is c.ftied out aiming.t a low cosl medium fo! xylanase 

biosynrhcsi (Siendcnbcrg et al., t991: Monica et al-,2002). The oreanirms need 

eseitial elene.ls such as carbon, nitogen, phosphoos .nd sulphur lor grcwlh 

and subsequent xylanase p.oduction. The concenrtution ofrh€se elefrents hds also 

a profound eret on fie yield ofxylanse (Mishn et o/.,2004). 

Agricuhrral bl?roducts sucb as wheat bdn. whedr stnw, .ic€ husk, com 

cobs. om sm\! or rcc stmw havc been used fo! biosynlhcsis of xyhnNe 

(sn{cnbcre pr u/. lr)98i Christo} !r dl. 1999; Abdel Satcr and El-Said. 2001; 

st'h! l00l) Cho er d/ (2002) srudied rhe prcducrion of D-xylose by .nzlmaric 

hydroly$s of agricultural wastes. Xylahas€ biosyndesis was found maximum 

shcn sheal bran s,as ls€d 6 snbstrate conparcd ro puB xylan polymq, slgar 

canc bagase and rice st@w (Fenietu et ol., 1999: Patk et al..2002bi H^q.t ul., 

2002). 

( a.bon sou.cc affeds nor only thc mode ol\ylane producrion, bur als 

$. !clocity wnh which c.don soude is detaboli2ed in rhe funsdl cell. Baki el 

4/. (2003) snrdied ihc erecr of nulrieir soures on xylandse femenration. The 

lddnion ofedily ncbbolized susa6 such as glucosc and xylos ro wher bmn 

enhunced lhe xylanase producio!, AmonS the sevtrul carbon sonrces tcsrcd.

1 

highcr xylanase production was v€dfied in xylan. xylose. suear c.ne bagassc. 

Rhedt bdn.nd comcobs cullurcs (Coelho ard Camona,2001). Cho {1997) 

obseNed thar th€ starch is rhc bcsr additional ca$oi rource fof xylanase 

Dillerenr organic and inorgaiic nitrogen sources and thcir conccnlElions 

havc a malo npaci on ihe biosyirhcsis of xylanase (Kulkami er a/.. 1999). 

T$een'8o (0.5 %. vr!) i.&ersed the yield of cn/yDc !p to 20 % {K!mi e/ dt. 

l99lr Balalsishnan e/ a/., 20001 Sun d {t, 2000). Thc (NHr),SOa was preved ro 

b€ lhe besr nnrcgen sourcc for mxidun xylan4c produclion (Mo.lcnccourt and 

Elclcieh, | 979; Bi e/ a/ . 1999: Kansoh and G.mncl. 2001 ). ,,t. ,tge. produced 

higher le\els of exracellular bera 8lucoside and xylanasc adiynies in 

submersed femenrdion when ammoniun sulphale. lmmonium di hydrcsen 

onhophosphate and com'stccp liquor wcE used as nilrceen sourcG (Gokhale "t 

d/ . 199 I ). Thc usc of y.a$ cxrract as a supplemcit ro rhc nitosen source rcsulted 

in considenbl€ inp.ovement in thcprcdudioi ofrylanasc (Couda2000). 

Time couse study is o.€ ol lhc critical faclo6, which d€t€fri.s the 

efficacy of rhc proccs along with prcdud fomatioh Th€ p.tl€m of accumulatcd 

reducing sugar at'ler specific incubution tirc is char&lerislic to each species 

(Mairai ./ a/.,2000) Cawande and Kamat (2000) demonstnled lhc maxinun 

xyla.ase dcrivilr 48 h oter incubarion. Somc work{s. howcver, achieved optimal 

xylanasc produclion 96 h after incuborioh. (Kohli ./ al.200li Oclavio and

E 

cordov!.2003). Kalsoh and Cammd (2001) rcPon€d thc mdimum vicld of 

xylnase afrcr 2 days ofincub.tion on a mtrry shakq (180 Dm) at 3trC. 

Th. lempc.d@ ed &e iditill pH offte incdium llso pl.v u inPodanl rol. 

in ihc bisynttesis of xyldts.. Telnperltw bctwcdt 25_30qC was usuallv 

empfoyed for cuhuring of /1. ni4q Xyld,s. produclid was fouod to b€ 

mximum at 30t by ,l- rige. (Palm! €/ 4/, 1996; Cano ?r dt, 1997i Ylun ed 

Ru$/u. 1999). The oplimum pH of lh. oft. m.jd xylanase SrouPs i5 in lhc 

range of 4.0-5.5 (Maria ?, al, 199). the crudc xyhn&tet hrve PH 6.0'7.0 with s 

emp..atue optimwn 30qc (Dhiuon and Khanna 2000) Colina .r dl (2003) 

exmined muimun xylmls. lctivitie at pH 4.E in fie sh.l. llask lnd fcmcntor. 

Optimal produdion of thc xyleolytic enzymes was foud in cullurc @nl.sininS 

oll-spclt xyle st 30oC and initi.l pH 7.0 (Pdhmsn., al, 2003) 

Stitrcd fertnentols of diffmt wo*ing clpacitid hav. rlso be. us€d for 

$alc ry productio of the xyl.n.s. (llias and Hoq, 1998i TehaPm ., al' 2003) 

EDlanced production of xylana!€ in bioreacto. w8s achievcd bv oflimizltion ot 

differcnl pamnelen e.8., typ. &d age of inculum, 9H, incuba0or LmP€Brure' 

*hrion .nd agitdion nt6 (Ped! l98q Sied€nberg e/ 41, 1998i Colid dr al. 

2003). TIe agilation sd acation iil€s w@ sdditiootl fsclo6 that influ.nce ceU 

grc{tb and xylme poduclion. Aetltim and agit riolr in submcrg.d cuhur. 

@ndnions nol only prdidc lhe oec€ssrty oxygcn for 8t@lh of$e or8$ism but 

also oromolc thc €mci€nr excrcdon of the enzym. into lhc fmdtation mcdium

9 

(Techalln ?l4r,2003). Reddy et ar (2002) have r€poded lbe eff.ct ofaedlion on 

th. production of xylee uDde. submerged @ndiliols Jhc mtximl ozvme 

aclivily was achiwed a10.75 wm. Merchant er al (1988), howev.r, achieved the 

oaxi@l xylande aclivity at 1.0'wm and 100 Am !€ldion .nd .gitatiotr rales. 

rcspectively. Ilias and Hoq (1998) rcponed lljal cultu€ time in bio.elcto. ws 

rcduced siSnificdtly 6 mmpar€d b sh.k. na3k st'l(ti€s. 

Tle typc md size of lbe inoculum ued in the fementltior ptucess dso 

aflst the enzyme biGynthesis. Sicdenb€rg ?/ 4l (1998) rcpon d that the 

vegetative inocuhm 8!vc hid€r xylrnase biosynlhcsh a @mPa€d to rhe sporc 

in@ulm. Xylaias€ *as pu.ified appmxinat€ly 25 fold by smmonim sulPhale 

preipitltim, get filtratiod lhrouSh S€phtdex C_50 and im qchange 

chromaiography on DEAE-c€lldose wi$ a yield of approximately 23 % (Ghd€ib 

ed Nos, 1992). chiverc e, ar (2001) ds (sied out lhe Panial pudfi@lim of 

xylaiase using (NEISO{ pre.ipilation dd sel filsation chrcnato8raphv 

OBJDCTIVES 

Bcing d agricultml country, Pakishn has vd1 r€somes of agricultual bv- 

prcducls for rhei. qploiiation as subshle in thc synthesis of xyled€ bv 

f@e.talior. The developme.r ofilis technolosy would b€ hiShly beneficial Tlt. 

pttMr work is @rcmed *ith:

l0 

i) Oprimiulion ofculluBl condidons for rhe productioi ofrylanase by 

l@ally i$lal.d stoins of..{. ,!i.r by boih subherS.d and solid stale 

femcntation tcchniques. 

ii) Scale op productron ofxylanase in slin€d lirmentor' 

iii) Optimization of mcthods for the isolllion and panial purification or 

\rtsnase fom fi€ femenred brcLh.

LITERATURE 

REVIE:w

LITERATURE REVIEW 

(omtat 4' a1 (1e74) poinred out tha! xllan is a consritucnl ofmany plair cclls. 

'lhe backbone of xyl.n is composed of D-xylose unit ihar is subsrituted by L, 

ffdbhosc.nd 4-mcthyl D ghmmic acid. Corbachcvc and Rodionova (1977) 

becamc succ*ful to isolale xylandc frcm tungi. Xylanae is insoh6tc in water 

bur solublc in alkaline solution. Johi ./ !/. (1979) srudied hehicctlulose as major 

group ol lrgnoccllulosc, which hrgely consists of\ylan. Microbrat xytln6cs hrd 

bccn pu fied frcm r.!p?,srrr s niger ^rd Strcpton)ds r'topras€r. Cornd ( I98 r ) 

selcclcd -,1 ,iser. nains I I 0 42 (CBs) .s a pro

t2 

with somc othe6 showcd rylanol)tic acrivny- Tan e, a/. (1985) sepanted hG 

different xylanase frcm Irichodenlo honia,t't. These thre cnz)mes were 

lcPoned ro be lhe najor compo.cnl ofxylanolytic systcn of T. hdEiaMn. 

Motu er d/. (1986) and Nco er 4/. (1986) studied enzymdtic dcsndarion or 

liSnocellurosic marenah. Lier@elltrlosc is rh€ nosr abundant o.ga.ic.ompound 

pruduced oD our planc! which pn'narily consists of rhree dajor potymes 

hcmicellulosc, ccllulose. and tisn'n. Ahong rhese hcmice utose is the second 

lars€n componcnt. cokhale cr a/. (1986) found rhal ,1. ,,a€f NCIM l2o7 

prcduced significantly high levels of &glucosid6o, secreted by hemicettutollic 

cnzrncs(ryl.nas€and&xyl6ide) inrheculrurcmcdium. 

Hieh yiclds of P-xytosidas wcre obbined shen n was groM on cnher 

xylai (3.0%) or wheal blan(4.0 %). Cetlulose was a poorinduce.ofr-xylosidase. 

Thc ptl rid rempemturc optima forp-xytosid.se selc 4.5 and ()5t, Especilcty. 

Dubeau ?r al (t986) prcduccd xyta.ase ot Cha.tonitu, .ethLt r.!,, and srdie.l 

ns hydrobtic poienrial. Maximum xylanase F.An rion by Chaerootin" 

.c//,/,l,tnn,r w6s obtaihed in culruE rupemaranl afld loh ofgrowrh ar 3?"C in 

basal ncdrum cont.ining lol, xylan ar pH frainrained between 6.5 and 7.5 

Xylanase producrion vas fotrnd ro be gloi1h a$@iarcd 

Crajek (1987) had prodnced D-xylanase by rhemophilic fungi usins 

dir€renr melhods of culrues. They cxamincd se\cn srnins of tunsi fo, rheir 

ubiliry ro produc. D-xylanase i'l tiquid snd sotid state femenialion. Colfirlee/al

l3 

(1988) isolated mulanls of,.4. ,€er NCIM 1207 by subjecting the conidia 10 ultra 

viol€t imdiadon @d some weE tsed for the produclion of x7la|N, CMC& 

and Fgluc6id6e. In Seneml, adorophic muldts s@recd lo* le'eh of all 

eMymes into cultue bfoth. Schiner and vonmem (1989) developed a method for 

thc quantificatior of xyl66€ ud CMC$e fro'n soil. Th€ det€nination of 

activity qas caEied out lia subsrale and reaction was peifomed al pH 5.5 md 

io'C rempenturc. Bhsd 4/ al. (1990) prodDced puified dd charlccded 

xyrmase ftom , qerg 6 ochtuce^ employing in both liquid and solid state 

Chen ?/r/. (1990) sc.eened a high yictd xylanase p.oducingstatn A. ni4er 

c-2 liom the soil dd tEared wilh combinarion ofutravioter ddialions Gry) dd 

.tlyl mcthme sulphonare (EMS). The submsSed fcmdiarion conditions wde 

studied. Tle inilial pH wd 6.0, remperature 28.C dd cDllilation tim€ N6 96 h. 

The optimal pH md empmrnre fo. xyloase reactjon werc 4.8 md 50-55"c, 

rsp@riv.ly. Xylame ws stable in lhe pH mrge t.2-l1.4 but had weat rhemal 

slabiliry when eMyme in@bacd al 55oc fd I h at which 40 % ofenzyme &iivity 

mnained. Cokhale ?r al (1991) sludicd that,4. ,Aa NCIM 1207, pfoduced high 

levols of exlracellular p,glucosidase and xylanase acriviris in submersed 

femotalion. Among the nirogen souces mmonium srtfar., almonim, 

dihydrogen onhophosphsre .!d @mit@! liquor were b61 fo.1he prodrction of 

cellDlylic dzymes by,,{. ,!ger

Uchida ?r a/. {1992) srudied prcducrion of xylose fron xylan wilh 

inlfacellulr enzyme system of ,4 "tgcr 5-16. The stain produced a xylanolltic 

en/ymc system! including B-xylosidase, o-slucuronidasc and c 

anrbinolnnnosidase, i. p.ttei-shaped mycetia when rhc fung$ srcw in a mcdiun 

including rylan of its panial hydrclysis prducrs d a carbon souice. Bilgnmia.d 

Pddcy ( ll)')2) dcscribed ihat microbial production of rylandse was prefeftd to 

phnr and drnnal sourccs becauseofeasie.avlilabiliry, strucrur.l sr.bitity and easc 

ot 8€ne.c naniprlations von a a/. (1992) sudicd rhe xylanase prodncing 

thcmophilic fungns, which poducs xytanase but docs nor sccrere.ny cettutases. 

Thc edz)'nB src slir.blc as blcachirg ai

l5 

rado corr€sponding ro 3i2 respeclilcly with 

fredium yi€lded highest e.zyne level aner 

30'C wnh inn'al pH of medium adjusr€d 1.0 

inoculum size of l0% (v/v). Straw 

five days of aerobic fementalion ar 

Haq e7 dl. (1993) studied the sy nesis of xylare ed ellule b|nold 

cullurc,.{. ,,ger. It €xhibiled m.ximun @llulylic eMyme prodnciion using wheat 

brm as subslrote, Diffe.eni .ulrual conditions such es rdre of szyme synrhesis, 

extEction by diflercnt solulions, efeclr of diluenG and nirrogen sources wee 

studied and oprimi4d fo. best prcducrion of x)lda.e and c.ttula*. Baitey ?, at 

(1993) investigared on the produclioD ofxylanse from rrijhod.rna rceyi.The 

orgmism was cultivaled on cettulose-xylan media a1 different inilial pH. 

Prcduciion ofxyldas€ ws favoued by a high pH_ Minimum conaot betwEn 6.0 

.nd 7.0 $a obseryed by bolh eltulo* ed xyte based medi.. The produclion or 

xyldase at pH 7 was shown 10 be delendanr on the natur€ of xyld in the 

cultivation mediuq howevq it wd independ€nr of rhe other organio conlonents. 

Mdimun produclion ofxytmse rvas achiwcd ar pH ?.0. 

Bailey and Vilkei (1991) prodn ed xyt.|@e ftn lsperyi B hnigts 

ed Asyeryilb,ryz4€ on nedm conbining rylm a fte cdbon source.,4. 

lunisatu! ptod\..d xylanrse on unsubstiluted insoluble xtld but groMh and 

enzymc p.oduction o. soluble oligoecchdide fron the srcdinS of hard wood 

*'qe poor due lo prcscn€ of inhibitoB. ,r. rrlsaru prcduced good Kyto6e ai 

lhe !H below 3.0. Ai higher pH valus $e produciion of proteolytic eDzynes

t6 

.aused degadation of already p.oduc.d xyl as. aclivity. A. olyzae s^!e lcss 

xtll!]e activitr lhan,.{ lmiAara on $e birch xylo mediuN. After a.laPlalion 

'1sa 

capable of emciot enzymc producdon on lhe stesmed snbstrale. Nakamua 

?t al (1993) prcduced exlrelluld xyleffi ftom alkalophilic 3aeil6 sp. slraitr 

4lM-I, isolaled from soil for cnde xylede prep@lion. Optiduln pH was.bour 

9.0 dd xylMes we.e induc€d by rrabn but not with rylos, mbinose o. 

glucose. ll wd noted that xyl.r6e productilily ws influenced by cultm H" and 

prcduction at pH 10.5 wd higher 1han at pH 8.0. 

Patel dd Ray (1994) produccd dd chmctcri8d xylme fim 

St€pldz)r€r sp. grown on an alkali treted com stalk. This xylanase was srable 

ror 24 h ar pH mnge of 5.0 to 7.0, had bed opriml belwc.n 50-60.C ad had a 

half life of 5 h a1 60"C. T1E xyldas€ activity s6 inhibired by rhe addiiion of 

xylce imo th€ femdted broth. Milagres "r ai. (199,r) Foduced xylM€ ftom 

PenicilliM jMthihe iM fiom plant maErid foud in temitc colony. Tte 

cnzyme revealed high xtlanolytic activiry, which was inducibl€ by xylan. 

su8rrt8n€ bagdsc or xylose. Oplimal remp€arllre ed pH for xlluase activiry 

(98 U/ml) werc 40'C and 5.5, iespectilely. 

sumakki e/ 4l (1994) nudied eMyme aided, totally chlo.ine-free bl&ching 

of dirermt indusfrial enrvood k6n pubs usins purified tichodm re.sei 

xylande ald menase. Both wde found n increse bleach abiliry of die pulps 

dep€nding on @king Delbod ued in pulp p.oduction, The eff6r of !yta!:e

l1 

rrcahenr oD bnghlnes \'as hishest in the convctrlional loaft pulps and in pulps 

prcduced by extcnded cooking m€thods. High lmounc of lignin wirh hiSher 

nveraee molecnlar mass also could b€ cimclcd frcm ihe pulps afier xylan.sc 

trcatmenr. lhe e.zynes serc especially ereclivc in implovinc the bleach abiliry 

of oxygen-delignifi .d pulp6. 

Shubtkor .r al (1994) pnduced xyldn.sc by lhc yasr Ctrplo.o..ts 

wdzolictt on n din with different carbon sources Thc enzyne qas ld.tcd 

cxhcellularly ard was dn induciblc dzymc by &nethyl xylcidc, rylosr and 

xylan can be replaccd by extrach frcfr planr i.w naren6ls. It sas also found rhat 

rheF ras r coftlnlion bct*en €nz)mc ploduction and grc$1h ofthc ye6t on the 

medium qirh xylan as ihc solc carbon source. Hoq er al (1994) prcdu.ed 

ccllulasc-tree xylanase by Thetuo,n!.es lanugi"os$ and studied the ef€cl of 

aeirarior &ddon and m.diufr ompon€nts on iis producdo.. In shakc flast 

culture al 50oC xylan or xylan,coiraiiing substance induced maxinum and 

companblc lereh of xylanase while glucose. xylsc and sucrN app€arcd to 

rcprcss \thnase synthesis. A1 pH 6.5 both grcwlh and xylanse producdon was 

maxihun. Aspects of usine the crudc beta xyhnase prepahrion fo! applicarions 

in rhe pulp and p.p€. indusry aere dis.u$cn. 

Pinrsa c/ a/. (1994) produced extracellular xylanase iron ,rqdlsil/ls 

,'i?,1a,s rndstudicd the induclion ofthe synrhBis ofxyl.ie byusirganumber 

or con,pounds, including xylan of diffcrent o.igin. Dorosaccharide.

l8 

xylosnccharide and xylose dcrivalives. Cenain xylan like whcat arabinoxylan, oal 

spch xyl.n. bnchwood xylan and 4-o'nerhyl D'Blucurcnoxylan werc found lo b€ 

the nut poserful induccB while aylosccharides such as xylosc, xylot iosc and 

xylorclb* also scryed as induce6. C6ra-F.rcni ?r a/. (1994) investigared the 

ptududion of xylanoliic cnzldes by an,4. ,,A?/ cCMl 850 isolale in barch 

culturcs. wnh 4.0 % xyla. as the carbon source, about 65 U/mlof&xylanase was 

oblained which rep.esent lhe hiehesr rcported activity for wild l}?e srniis of,.1. 

Comes./ "/. (1994) prodDced highly themosrable xylanlse by a wild strain 

ofrhcfrophilic fun8us lr.,?,aascrs aurartiac$ and nedi,um cohposilior for lhe 

ptoduction of cxhcellulaf xylanasc was optimized in sh.ke-fldh culture. 

Oprinri/ed conposition 3.24 % conprised whear sran Gream prcteared, pariicte 

szc lpprox'mately 0.25 nn), 1.32 % phama nedi. and 0.49 % KHrPOa. other 

conponnds snch as inorSanic nitro8.n, Mgsor, cacl:, r6cc elenctrrs, and 

vnafrins showed no marlrcd positive cffecl oi ihc cnz)me yicld while Tvecn-8o 

exhibned dish cnhancinr ellecr and rhc oprinized cuttu.e gavc 5t47.4 nka|]hlof 

xylanase. Ihe fungus prcdlced maxinum enzyhcs when the pH was nol 

conrrollcd and Gpesed nycelium inoculum was used. Thc oprimum pH for 

xyluuse sas 5 and tenperature wasabout 80oC. 

Ilieva c? 41. (r99t srldied the bioslnrhcsis of xytanase by ,4.?e/8il6 

4trar',,i K I durine submerged and solid-state cultivarion hy irvcsrigaring some

l9 

pa€mcle6 of cnzyne s the femdtation mcdinm treG optimircd for naximun 

xylandsc producrion. wh€.t st.aw and mixtu'e of ball mill€d fraize stems and 

shear bran (2:l) prodlccd tbe highesl ahoDntofxylanasc ai oplimun tenpclarure 

oflo'C and oprimuh pH wG 3.5 a0. Sinsh et /r (1995) prcduced xvlanasc bv 

Fuvdtnn a\rtpotuh. which srcted hiSh leveh ol xylanolvtic trzvmes on 

conncrcial xylon as {dl as on sereral 5gncultur.l residucs, ofwhich sheat bmn 

supponcd maximum enzyme yiclds. n was also obse^.d rhar addnion ol02% 

olile oil, an incxpensivc source of f.tty acids, resuhed in u fuflher 25% incrc'se 'n 

Yang c, dl (1995) pdduced xylanase bv an alloliphilic Ad.r/1!r sP' 

isollrcd from hdr.l$ood knn PulP and it lvas cdpablc ofgrc*ins in dilutcd tsfi 

black liquor at pH I I 5 The evyne producdon wts Preccded in dlkaline mcdrun 

rr pH 9. Varimtrm cnz)me acrivity r!!s obbinc't bt cultilalion in a defined 

alkalinc medium with 2% birchwood rylan and l7o com (ep liquor at pH 9 0 

Higho enzync p.odlction was aho obtained on whcdl bran Thc optlmum 

lempcmturc a( pH 7 sas 55"C and in the abscice of subsnae. al pH 9 0, fie 

enztm€ las stableat 50oc for dl l€asl30 hinules 

Jlin 4 a/. (1995) produced xylanar bv a th€nnophilic funeus on a8rc 

iidusriial esidues in solid-slarc fmcnniion Untrcarcd whcal smw and sugar 

co. b.ga$e supponed enzFc production whilc ricc nraw and rice husk did not' 

Alkali and.cid chlolide treatnent oi rice stras and rice husk cauFd

20 

delis.ification and enha.ced en2yne producrion. The productioi qas higher in 

solid-state femcntation than in subme.ged fcmenbrion 

Palma er al (1996) studied the infl!.nce of da on and asitation o. 

xylanase activity. 0.25 % H,SO4 tlcat d sugar cano baea$e was fillerd and 

adjustcd ar pH 5.5 wnh NaOH and supplemenred wiih 0.1 % yeasr erhacl. 

Penr,//n,, was clhurcd in rsuhiog mcdium for 96 h ai l0'C bolh in shlke flaks 

ar agnaion nrcs frcm 240 rp.m. and acBlion 0.2,0.4 and 0.6 wm.In shake tlask 

mediun xylanase activily {as 98.5 v/ml at 60..p.n. and thc maximum 8rcwlh 

occured at 120 r.p,m, The hichesr produclivity (351 U/L) was obtained by using 

the lower oxlgen supply KLA b.ing 1.24 h-l- lsmail (1996) st'rdied the 

producrion of mlkienzyhes such as pcctines. polygalacturcnase, cellulM and 

xyla.M, usinS six fungal isolates gown on dange pftt a sole carbon source.,{. 

"igef A'20 was the most polenl and poduced highly active multienzyde qstems 

aier5 days in shsk€n cultlre. 

Dua c ., a/. (1997) invesiigltcd more rhan 500 srni.s of sPBrqil wirh 

xylanoliic actiliti6 ielaled from seveEl sources of eil and wood 

deconposirioi mate.ial. Out oflhe srlains, 23 micrmrganisms w€r€ able to s.ow 

in bnch wood xylan. The obseNed bchaliof ofxylanase activity at pH 10.0 and 

5.0 indiclted lhat thK ben prcduce6 of alkllino xylde yicldcd enzyme le'eh 

in rhe ran8. of2.61o 4.0 U/ml bul oftcr folr micmren.isms achieed enzFe 

levch lion 1.0 to 1.25 U/ml. Two fricroorganisns p.oduced xylanse which wet

2l 

almosr acrive dt pH 5-0 od otre microorS0ism w6 at'l. to prcduc. enzJm€ actrve 

al pH I0.0 having diffdot chemical struclures. 

Mukhopadhyay et "1 (t997) hota@d a potenl xvlmolvlic fungal st.ain of 

,ltperyitts ltavipes producing both endoxylm.se dd be6-xvlosid6e in 

consid.nble atnomls ftom soil of West Bengal, India Anong the five differenl 

b4tic media resr€d fo. rylmolltic enzyme produdion ,4ryets ill6 l@ipes stawed 

maximum enzyme production in Mand€l and Wabeis (MW) medium The isolaEs 

produccd maimum xylmarc Poduction 

at pH 6.0 dd 2.0% xvlan as caibon 

sourc€. Sludies by changing in enzyme m€dim sho{ed no seParaie growh and 

e@ynepioducing Phe. 

Gdptu et 4/. (199?) selecled Peni.ittiun unesceB l0'l0c for dct.iled 

srudy because of its abilily lo prcduce an interesring ozymalic conplex in 

quelily. The xylmase conplex wa cellulase-ft@ and had o optimal actililv a1 

pH 4.6-5.0 and 55-60'C on bi.ch wood xylan. The b€st production wa on sovbean 

meal ed wheat straw. Exprcsion of lhe xyl46e sd rcPr€ssed bv glucose. 

xylos. and lactose. The oprimialion of cult@ medim ed mold imprcved the 

ploducrioi 3-4 tim6. cai .r 4l (1997) isolated,4 "tg?r A3 snd studi€d its solid 

slale fmerlation conditions. lDilial PH 46, lemPemtue 2E'C' lml spor€ 

suspension inoculnn, @lio ol wheat bre to b.gdse I I 5 ed fedentation for 3 

days *€re optimal. The xytanase actilily was 5147 IU/g Compansot of xvlansse 

prodoccd on both liquid @d solid cultu€s rc!€led rht oPtimun t€mP.rature wd

22 

55'CioprinalpH wis 4.6 ad 4.2, respccdvely. Halfacrivity los in I h w6s nord 

al 54 and 5lo(, €specilcly. 

Sianro ?/ ,1 (1997) noted rhat Aspereilla ta'nurii ptodrced exlracellula! 

xtlrnase and intaccllul P-xylosid.e indldivny in glucose. The prc-src$n 

mrcclia NcE iicubat€d $ilh iyla. .nd nerhyl p-D-xylosid.. clucose and 

cyclohexanidc were found to inhibii xyh.asc producrion by herhyt beta D- 

xylosidc Subraruniyan ?r dt (1997) isolared and scrccned xytanase prcducing 

nrcroorsanrsms frcm soil samples and hemiccllolose mare.iah in 3 s6gcs by 

using whe.l bran exrrad agar hedium, xylan dslr nedium and liquid medium 

Nlrlr xylan. Arch.na and Satlanarysna (r997) studied rhe producrion of 

.\rlrcellubr rhrmo 'rabk cclluto,e.frcc \ytanase by ta./ tu\ h.t,taLUrrhn \oa 

by solid-surc fefrenlation .nd fou.d wheal bran lo bc the besr substrate. Thc 

p.odu€tion of xylanase reached a pesk in 72 h. A 

prcduced in wheat brsn moisieDed with tap woter at 

ascnt rar'o oi l:2.5 (w/v). Thc oprinum renperature 

50"(- 

hish levcl of €nzyme q,6 

3 subslr.tc lo a most€ning 

for xylanasc production was 

Siedcnbc.g cr dl (1,),)?) invcsrigacd rhe p.oduclion of xytande by 

AYugillu! (trdhloti by xylan and xytose. No xylansc was formed l,ilh xytosc 

Thc hishest enz)rn€ acriviry vas obtained wnh filancnrous frycctia and smatl 

(0.1-0.2 mnr. dia.) pellels at rhe lowesr phosphare conccntnion. Fushinobu a dl 

(1998) detccted the srrucrurc of xytanase "C.' tiom AJperyiuu, kawachii. -the

23 

olemll srrucruE was sinild to I I othtr fatnilies ofxytanas€s- Aspergill6 37 and 

m acid-bse @talysl, Clu I ?0, ale loc.ted at a hydrogen bonding distance (28'A), 

as in o&er xylescs with low pH oplim. Analysis of wild tyPe ed nuet 

showed th.1 ,,{ryefai[6 J7 ws inpondt fo. ils eM)me aclivity al los pH. 

Samain er al (199?) siudied rhar ta.i//6 Sp. strain xE and its mutanr 

ddivativ€ strair D3 produc.d rhmoslable xylurse whicl was suitabl€ for 

enzyme bleching of kFfl pulp. Xylaae synthesis w33 shosn to be induced by 

the soluble p.oducts of xylan hydrolyses (xylooligos&chdides) ed equally 

@tabolic.lly ftprcss€d when thse oligM@haridg accumulat€d in th. n€dium. 

Xyldase productior ce6ed when $e aclivity re&hed approximately 380 U/nl 

due to e mino acid slbdag.. 

Kit moro et al (1998) cloned ald characleriz€d a xylflse 8oe xyr Fl 

from a shoF koji mold ,4rpe rgillu otyzoe KBN 616. Tl,e xytr F/ gene was found 

lo be comprised of l4E4 bp wiltr l0 inr.m- The deduccd minoacid sequen@ 

encoded t prc1€in consisling of 327 ai no acids (l5, 402 Da) which wd simild 1o 

rhe tun8al fanify F xyfdas.s such as ,,lrpersil/6 nidulM xln ., Aspergillu 

kawrchii ,n A A^d Petui itn chrysoten@ xtl p. The intd@lqon orgariarion 

of:7, F.l was very similar to thal of finsal family F xyldde gmes. Plasmid 

P)(PR64, which contain 64 copi6 ofrz f'/ p.omoEr rcgion (Pxyn Fl) in lhe 

sM€ direclion. sA coBtructed ^nd lntlod&edin Asperylll$ oryae,

24 

I-oper t "1. (1998) endied thar lhe rylanoltlic syslem of an alkali-tohranl 

ta./1t6 sp. consi.iing ofscvchl xyhnascs raneing from 22lo 120 KDa and pll 

value tiom 7.01o 9.0. Crude xyla.ase rebined 72 o/. ofinitial activity afier 5 h at 

pll 9.0 and 45"C. Xylan& prcducrion was folnd maximnm when n was induced 

by xylose and rylan ar 42'C and pH 7.8. Crude xylanase rele&d xylotriG. and 

xylorerrcse as m.in products of xyl0i hydrolysis, xllose w6 noldetected. Ph.m e, 

o1 (1998) oplimiad the conccntrsiions ofoal spclt xylan. casein hydrclPale and 

NHrCI In the culture nediun ofproduction ofxylanase fron Ba.illlr sp. I-l0lE 

by mcans of response surf.ce he$ods. The oplimun composnion of nulrienr 

medium $d found to he I I 6 8/l ol rylan, 1 .94 g/l of casein hydrclyaic dnd 0.8 

arlolNHrCl 

sineh (t998) found that ThemMtces /a,rst',56 SSBP ptc'duced rh€ 

highe$ xylanase acrivily ol59,600 n kat ml-I, when cullivated on a medium 

containi.g co.se comcobs and yeast exkact as a nilrogen source. The xylanas€ 

{N very srable ar pH ralues of 5 5 - 9.0 wilh superior themosnble prcPedies. 

Halflives rere 341 and45 min at ?0 lnd 90'C, r€spectively. Macabe ar /l (1998) 

slb.lecled thc,lqelgtld ,rd,/a,r xyhnase gcies xln A and rln B to regulalron 

b, anbiem pH. In the pesence ot D-xylose. xln A Nas expresed 6t acidic 

Cai .t u1. (1998) invcsrigarcd the prodrcrion of xyldas fr6m,r. ,isq A3 

tsinC liqu'd fem€nt lion wilh hcmicellulos. and solid srate Imenrarion on

25 

agricultural by products. wang (1998) sudied that muh,plc-cnzrme systefrs 

containing xylanas€, cellulase, pectinr$, a-glucanasc ahd prorease were produced 

by hypq xrlanolylic strain ,.{ ,rgel NFU'AO2 in solid state tcrmentation. Sreiner 

.. d/. (rr98) srudied rhal Penkilliu\ putpurogchu,h mutated wilh Uv lisht to 

inrclse xylanasc prcduclioni thc bcsi muianl Uv-64 was t.eaied sith N-tr€thyl 

N-.itro N'niircso guanidinc (MNNG) and a second sche.ation, of mulanls {'-as 

obraincd. The mnr.nts aho sho*ed a 2.2-fold iictedse in &xylosid6e as 

compared tr ilh lhe wild t}?e. 

Gonaneli ?r a/. (1c98) oh.incd genetically stable mutants of Pe,i.illiun 

.a"cs(trs 2ot7 $ith modified cultunl tnd morpholoeic.l prcP€nies by Uv_ 

inducrion h.d incrcused c+dciry for biosynlhesis of eit€cellular xylande 

SiedcnbeB e1 a/ ( 1998) i.vestgatcd the prcdudion of xylana* by,r,is€i 

inducrion oi enzymc prcduclion by xylan and xylosc at ditferenl timcs. No 

iylanasc sas fomed wnh xylose bul highesl activny obrained wirh oar slucce 

consumplio.. The highest enzymc activilics were oblained wirh filamstotrs 

dycelia and small (o.l - 0.2 mn in diamet€r) pcllels and wnh lowest phosphate 

concent.rrioi lhomas er a/. (1998) rcpo.t€d thal xylanases can comnonly be 

asayed by rhe dinitrosalicylic acid (DNS) or the dseiomolybdate (ARS) nethod. 

Howcver, specific.ctivjtics were manytines highe! with DNS than with ARS 

Archo,D c/ d/. (lqqq) prcscnicd s rcview reslrdhs ldrious aspccrs ol 

xyldnolyric cnzynes prcduced by themophilic nolds. Seleral lhcmophilic molds

26 

produced xylanolllic enzymes. Xyldidscs fiom thenophilic fungi did not appea. 

ro be dille@it from their meephilic councQan in lhek pH stability, mol€ctrlar 

weighr, amino acid compcition and s.qucncc, iwtecEic point. XylaM of 

themophiles arc genc@lly induciblc 6nd havc almost simila. Egulatory 

ncchannns 6 in ncsophiles. Rashid (1999) isolatcd Ad.illzsPz'i&s PIl9 frcD 

pinus lelres. ll showed optimum xylanasc p.oduction when grown in yeasl 

tryplonc broth at 37"C. pH ?.2 and was sh0ken al200 An 

aftct 48 h ofincubarioi 

Xylanase produclion by Ad.illrr pa,r!/iJ PJl9 was not sroslh-associared 

maximum enzlbe production was found after 36 h ofincubaiion. 

and th€ 

Zl\\ et at (1999) i$laled /tversil/lr ,sr/3 rrDm ll,r4 stnins of flngi 

prcduccd high xylarac actn,ity (2176 Uml) wilh ir 6 days ofcullivation on lhc 

nedilm (4.0 % *h@t spell, 0.5 % glucose, 0.4 % N.NOj and 0.1 % NaCl) at 

30'C. The enzrm€ showed o imrm tcmpe.atu.e of 55"C and rerained 35% of 

acriviry after incubation ar 45"C for lh. Thc enzyde.ho showcd a pH optimum of 

5.5 and sas nable ar the ranse ofpH 5 0 to 8.0. The enzyme was considcred ro be 

.n cndoslycosidase because hydrolysis ptoducrs of birch wood xylan were 

xylodose .nd xyloletrose. Ce$e$c and Mamo (1999) .eponed thal xylanse- 

p.oduci.s allaliphilic Micru.occls sp. isolated from an alkaline soda lake Xylo* 

atrd \ylan induc€d enzyme prcdlclion bd no acrivny *a detecred whcn n was 

gro\rn using other carbohydnrc sourc6. The enzyme was very stable in the pH

71 

rrnge ol 6.i - 10.0 ar 40"C. xyl.nase icriviry Nas inhibircd by Cu sups (2-) and 

veluz c/ al. (1999) screcned dilfcrcni strains of nrtolrA sp. on thctr 

capabilily to produce xylan.se undcr solid nale lnd liquid cullurcs. The hrghest 

xyl.nlse ncrivily of 516 U/nl for liquid culiure was exhibit€d by st.ain lr?:,Pxs 

p"la frum lhe Philippines. For solid-$al. cultu.€, the highesl aclivity ol 7802 

unns,nl $us lchieved h,! Rhirot)us sP. MKUI2 oiiginaled lrom Thaaland 

Rinspicil (19.)t)) prepaed xylandsc cont.i.ing ciTymc cofrplcx by culturins 

Trichad.rhtu in nodium having trcdlcd com-processing wasts. The liquid 

componenl olth€ com possessing*astc wd r€noved and lhe rcmaini.g solidsas 

auloclalcd $hich rcmoved inhibnory activiry ad resulkd in incB6ed ivlanase 

prodrction as well 6 increase in lhc 6tio of xylande lclivily lo olher cn7-vmc 

I.tu !/ 4l (1999) stud,cd rhar xylanas€ could be produccd sclcctivclv bv 

Tti.hodo"tu rcesei stnin lnder definit condirions. Th€ dcgrcc of s€ledive 

ptuducrion s.s dlaled 10 rt?e ofca$on sourcc, @rbon iou 

rhe rario ol curbon to nnrogen (C/N) Lo*enng ca.bDn source corcetrlnrron and 

rncreasiiS llrc CAI mtio, by takirg xylrn nixcd with verv liltle anounl of 

celluloF ds c..bon source, could Propotlionally 

nise il. Hish conccnration or 

xylaiase (15.5 Uhl) and low conc.itralion oicellulasc (0 2 U/ml) with high aho 

b.Ncctr borh cnzymes (177.5) could be obhinot in fed'barch fcme.hion bv

2E 

conlrclling carbon source under 5.0 g/l and CN 6tio o!e. I1.2. EudBgn ./ a/ 

(1999) cadcd our the separalion of xylanase ffom the crudc cullure filk.les of 

Asperqilt6 sp,5 ^nd Asperqi us sp.44 using alfinity precipitations. 

Varquez., al (1999) @ni€d o in\esrigarions ro oprifrize fie cuhorc 

condiriors for the produclion ofxyhnasc andB-xylosidas€ by,4tperyillts lldrus. t 

filanenrous fungus, isolat€d fton soil. M.ximun xylanase (190 U/ml) and p- 

xylosidase (35 U/ml) prcducdon w6 obtained whcn thc strnin $a grsn on 

minenl medium supplemenled "irh 3.0 % ({l!) @mcob powder a the ca6on 

sourcc. Yaun and Rungra (1999) nudicd the xyl.nase femcnlation prccess and 

fie ethd of lcmpcraure o. €n7ymc production by ,.{ ,ig"r An. 1-15. A 

rempedruF oscillatory op€€rion wiih a 33'C for the cany 24 and of27r for lhe 

larer conld shortcn $e cultilation timc by about 16 h in comparison silh thar of 

28"C for all the tifre withour adveNe eff€ct on xylanase aclivity. Labeille er 01 

(1r99) claid€d muliicnzyme product qith glucoamylase, p.oteolfic and xylanase 

d.tiviti6 lion whcat bmn by solid+btc fcmeDiation wnh,-{. ,ti?r 

ou d. a/. ( I 999) screened a fr uttnt stain of PseudontaMs louresce$ 0-9( 

by repe.tcdly tredting it sith U.V. and HNOZ. The xyldasc aclivity of0-96 was 

as high as 125.45 lu/nl and was almost cellulose free. Maxinum €.4nc 

podnctioD could bc obuined hy using agBnan esidue as thc culture medium. 

Chadh! ./ d/ (19t9) coDpa.€d sttuins ol Therhohlr.es lanq'itr$ fion dilTerenl 

cultuies collections fo! xyl ase produdion. T. ldtrginosut BSI, . soil isohte

29 

prcduced thc largest amounl ofxylanasc silh lwo foms, xylanase I .nd II *iih 

mol€cular m6s of 25.0 and 54.0 KDa. The U.VTMNNG nulas€n6is of ir: 

krlgr,,rrr BSt aleurospo.es /p.otopl.sls rcsuftcd in xylanasc'htper P.odlcing 

mut.nh. Icmira c/ al. (lt)99) evlluated thrce ag.icultudl wasics for xvlanolytic 

enrynes producliotr by /rP"/gil!r rar@rii in $lid i6le fmentatio. Oplimal 

innial noislnre contmls for xylanolylic respeclirely to wheal bnn coocob aid 

sugar caic bdgassc cultures. Frcsenius er dl (1999) pudfred xvlandc_de$ading 

cnryme 1o apPsrent honogeieity frcm solid{lalc culturcs of Asfrgillu! 

/x,,tgatdl 

h was mosl active on birch wood xylan. 

Claudio e, al (1999) ptified xyla.ase II to apParent homoseneitv rrcn 

solid'siat culturcs of,4rAT silldrdiSdr,r' Thc punfied enzvme was most acr've 

dr 55'C oid pH 5.5 lt ws specific to xvlm Johnson er d/ (1999) Purified 

cxrocellular xylanase o homogcneity frcn thc culturc filtEte ofa lhcmophilic 

fnnl\s.l h.tDtorices loMgin rls_SSBP and its bish'mical cha€cteistiG vcrc 

studi€d. A yield of ?0-80 % was achicved thtough specifi€d procedures The 

spccific dclivny .alculaled using the dinitrcsalicvlic acid (DNS) method *as 1500 

Il/ng. Mlria er a/. (1t99) nscd sugarcane brgdse as substBle for xvlanasc 

prodrcrion by mea.s of a strain 6f Tnchodetuo hatitrMn Rilai isolated ftom 

dccnyine .lspi.lavenna sp. (peroba) vood The srowlh 

p'o{ile or sinin was 

followed over 20 days on 14 % (w/v) bagase dnd hiShcsl xvldnase aclilitv (288 

LJrml) lppe.red on lhe sevcnth d,Y

l0 

sileira et a/. (1999) isolaled xtlmN aclivitv ltom crude exl.acrs ol 

Trichoderna hozianum $mins C md 4 erown at 28t in a solid medium 

containing rheat bmn as the carbon source- Chnslov dr 41 (1999) isolated ed 

screened seven tungi slmins fof their ability to p.odnce cellulase-frs xvlanases 

that could b€ used i. Prct e|lnoni of sulphlte Pulp pdo. lo blechinS. The 

potcndal xylan produccs wer€ subject.d to shak. flask fementalioB us'd tbr 

different carbon sources, wheat brm, comcobs. o.l spelts xvld and bleach Plant 

emuctrt. chen ?r dl. (t999) scFned 150 frmgal stEitr' 8 slsins Produc€d Bainlv 

xylane &tivily over 100 U/ml. TrB srain No 49 s6 high€st xvlane producer. 

shich lenlalively was id€ntified ts,4 rig'l. Ito (2000) m'de u rcview with 26 

ref*.6. on enzymB c-amytdq glucoanvl6.. tsgluc6idlte 6tsas' dd 

xtlanase produced by shohu koji mold such as ,1q./ai,6 tavachii attd A 

@ahofi otc. Biochemical chamctqistics of xvlande of AsPeryill^ tawa'hi 

meh@Gd of &id r€sist4l ed mehuism of qprcsion of \aluase sw 

oksanen rt dl. (2000) sludi€d that lensile st ength and libc! flexibilitv of 

thc pulp verc.ecovsed by r€Iining belwq the cvcles howevr. this aulted itr 

dete orated draiDage propenies. The !.cycl€d pulPs werc 1rea1€d with purified 

Trichoderna reesei cellDlds ud hcmiellulses and lhe cheges in fibq 

prop€ni6 duc ro enzymalic 

hemicellDlases wirh the enitoglucanase treatments increased tho positive effec$ of

ll 

the €n

12 

srowrh on wheal bran ardbinoxyl.n. Ca; et al. \2000J studied lbal higher 

cnlircnnent.l tcmpemtue is benencial for the grcwth of Tncholodarac.oe Ll 

ond its protcin synthcsk. The optimum tempenturc lor the fomation ofxylanase 

Nas ll"C under this tenpcrulure ihe spccillc aclivily ofxylanase was 92 I U.mg-l 

proreins, ancr cnllivared for 67 h. it was 126 I U.g.- I dry medium. 

Ca$ande and Kanat (2000) studied strain ol,4rrry,1/!! terreus nnd A. 

,t8er $at produced xyllnase with undetectablc amounls ot cellulosc. Xylanflsc 

was produccd from various lignocellulosic substrates by solid sblc feme.triion. 

Thc besl nedium for,.r. rczs6 wd wheat ban produc€d 68.9 luhl ofiylanase 

and,4 r€er.74.5IU/mlofter,1h ofincubalion. A mde culturc filtr.ie oflhc two 

,|rrEry,rzr slrains s€s uscd for lhe hydrclysis ofvldous lignoccllulosic mat ials. 

shich renolcd the hemic.llulosc fnction frcn all lignoccllulosic materials t.sted. 

couda (2000) i.lcnigated thc prcduciion of xylanasc by ,.ryer8ift6 

tuflalii in both solid statc nnd subne.gcd culluies. The use ofcomcobs ii solid- 

state culturcs gale lhe highest activitics (124.83 UB dry n.rtels) fo. xylsnrsc. 

Th€ addilion of fcmentation medilm ro the comcobs in solid-slale culture 

incresed rhc uclivity by abour 2-folds ofrylanase. The medium with yeasl cxtlacl 

,s nnrcseD source showcd marimum actiriry in soliddute whilc rhe best iirogfl 

source in submcrged wds mixtlie of sodium nitrate and casein hydrclyzale, 

Oplimum icmpemturc was l5"C in elid{tatc and 30'C in submcrg€d

l3 

Abdcl-Sate. and El-Said (2001) studied rylan-dccompositrg fungi and 

xylanolytic acriviry ii agnclltural and indostdal wastes. ,.{rpelsll/rs &v!r, ,{. 

niger. Peni. ill iun, .hrysoEenuD', P @rtlophilun, P. fuii.ulosunl. 

P- otalicuD and 

Iri.hoder,tut hdzlanun Nerc lhe most prevalent species on ihese subsirates Beg 

?/{t (2001) e$ewcd rhe nicobial xylanase .nd r heir indu srial appl icadons. The 

cnzync syslem uscd by microbs for the netabolisfr of x)'lan is the nosr 

ihpofianl lool for investigdting thc use of rhe second mosl abnndant 

polysacchdide (xylan) in.6tu.e. Rscni srudics on microbial xylanolytic sysicns 

have gencrally focused on ihdudion of en2ync prdduction und* diflcreir 

conititions. punficarion. characrdizrion, moleular cloning and expBsion abd 

usc ofenzyhc predominanily for pulp bleacbing. 

Aidre., al (:001) srudi€d extlac€llutar xytan dcgruding enzynes, namety 

p'illrnrsc, p-{yrosidNe and o-aabinotumnosidase frod r/,t odenta haaiornn 

sttaln 4. when grown in liquid nedium culruics containinc oar spelr xylri as 

indncer Kansoh and Cammal (2001) isolard direEnt s:tr.ans of Strapton)..s 

from Estprian soil for rhcir abihy ro producc cxtracctlutar xytansses. Ofatl 

isolates J /irdans was selected s potdt prodlcer of xylanase. A noticcable 

iicrcN in enzymc acriviry w6 ob$d.d in fic presence of(NHa)rSOr. Chcn "? 

r/ (2001) srudied the effoci of stitrins raie, pH conrrol and feed limc oi 

(\flr)?SOJ on xyfanase prcdvtion by Ba.illus pu,nilus A-30. Wnen (NHr),SOr

34 

wrs fed ar 0 conslant rare in earliei age and regnlated l5te. by detmining the 

conrent of(NHt,SOj. xylaas€ activity could €ach 616lU/m. 

Nuc/ e/ a/- (2001) a$e$ed sp.cifi. a.abinoxyln fncrions soentcd b)' 

xylonolysis in wh.at bread dough\ lo6f volume dd crumb tcxlure of bread 

pruduccd silh xylanase heatmenr w€re smngly comlated wift fte foamins 

prcpenies of lhese fraciions in whcai bread dough\. Addition of xylansse 

modified sheat flour anbi.oxylans and result.d in a I@f wnh nor thd. t0 % 

srcrrcr rolume- Kohli e/ a/. (2001) studied rhe producrion of.ellulosc fne 

crthcerruldr xylanase by Thennadinonyet rrdtopl,i/rr ar 50"C and pH E.5. 

Maximum xylanas. prcduction Ms achiev€d in femenrarion nedirm using birch 

wood xylln as substrate afler 96 h of grcwth ai 50"C_ Maximnn xyla@activily 

or42 U/Dlwds foundbbc at65"C and pH 8.t9.0 

Lenos et zl (2001) srudied ftc use of pu.ifi€d xytd s a subsrmre for 

br@onlesroD i.to xylanascs incrds€d thc cd of €uyme producrion. 

Consequenrly, rherc have been aftemph to develop a biopr@ess lo produce such 

cMymes lsing dircBnr ligi@ellulGic 6adues. Considqing rhe indlsrri.l 

inportancc of xyl.nases, thc use of nillcd sugarcanc bagsse, wirhour ary pre 

t.e{tmenl, as a ca.bon source was adopt.d. Endoxylanase a.d B-xytosid6e 

aciiviiies wcre higher wh6 sodium nirale w6 used as rhc nirrogetr $ur@, qhen 

corpared snh peptone, Dr€at and antronium sutpbale at the optimized C: N Ftio 

of l0:1. I h! use of yeasr exrracr 6 a supplemcnr ro rhe nnrcse. sources rcsuhed

l5 

in considcable improvemenl i. rhe production of xylanases, showing rhe 

ofthis organic nitrogen source on,,l. ar.fu,i neiabolisfr. 

'nlponancc 

Konig "121. (2002) wo cd oDl a simple. rcbust and highly reproducible 

mcrhod lor thc detemination of xylanasc, Fglucanase and cellulasc in 

conmcrcial feed cnzymc prepahrions. rhe ncrhod wls b.sed on measuremenl of 

rcducinS hoierics rcleled by rhe cnzymes frch arab'noxytrn, bcta-glucan, or 

cdrborymethylccllnlose (CMC) 6nd independcnr ofenryme slaDdards. Elia.. dnd 

'Idmboullr (2002) worked on xylaiase recovc.y tto"|. peri.iltiun janthinlthjh 

snh a rcyereed nicellar sysrcn consisring ot a cntionic surfacranl usine a 

conrinuous proccss. A siaisrical apprcach applied lo thc Esulls showcd lhe 

hlshesr xtlanasc recolcly (41.5 %) w.s anaircd ai anionic slrength of t0 os/cm 

lnd a rohrnrefic flow at0.5 nVhrn. 

\4onica drdl. (2002) prodtced and characrenzed rhc ce ulase ftee xyhnN€ 

rrcm Tl.rnon)ces lanusinosB IOc:4t45 in shaken cultu.c usin8 comcobs as 

substrarc 1500 U/ml). An oprimiziion oflhc mediuD in submerged femcnkrio. 

was c.fllcd oul aining at a los cost cohposition for cnzyne production. 

Slltistic0 cxpcrihent dcsign poinring our comcobs areds cnz)me produdion. 

Judiih and Junior (2002) so*cd on rh€ influencc of some susaB on xytanasc 

prcduct;on b\ ,lsperyillus atua,,o/i in solid siarc fencnkrion ,nd rcponed rhar ,.t 

(r\tn.ri ha\e high exraccllular edoxylanase (t00 U/ml) and ,-xylosidase 

lctivities (r 5 U/nl) *hen $own on nitted sugar cane bdg$e as rh€ pnicipal

36 

carbon sourcc wirhour rearmcnl. Julio e/ d!. (2002) srudied thc bioconveBion of 

soybean industrial residue by Baci B etbtilis BL62 sttuin isolalcd frcm *atc. and 

soilcollecrod in lhe Amazon region. Thc isolate BL53 show€d the highestsp€ciilc 

acririry for rylanase, 5.19 lu/mg prolcin wirhin 72 h ofcuhivatioi and specifrc 

cellulase .clivily, I 08 luhg prolein withi. 24 h of grcwlh. Thc prcduction of 

prcte.ses rhrt refe .sociated wilh thc Io$ of collulasc and xylanase actilitics 

$6 also obsencd Thcsc rcsults indicated thal thc *lecled micr@rganisns and 

the cuhivarion prcce$ huve great biotcchnological potetrtrat. 

Pa* a/ ur (2002a) oprinized rhc inidal Doisrur€ conGn! cuhivario. rinc, 

rnoculum s/c and concenlration ofbasal nediun for rh€ prcduction ofxylanasc 

by an .l r4r"r mulanl using slalislical crperimenhl dcsigns. Thc cultivation timc 

ind concdntration of basal m€dium were thc nost inport nt facton affecring 

xylanasc lctlvity. An inoculum size of txlOi spoes/g. iritial moisrure conteni of 

65 %, culli\alion rime ol5 days and l0 limcs concentarion ofbasal nedium 

containirrg 50 times conccnraion of com slecp liquor were oplinun tbr xyl.n.sc 

prcducrion in SSF. Under the optimiz€d endnions, thc acrivny {nd prcductvny 

or xylandsc obtained aftcr 5 days of fementation wcrc 5,071 IU/g ofrice nraw 

and 14.790 lLi I L h . respcctively. Tle xyla.ase .divny predicted by a pol'rcmial 

nodel w.s 5.4E4 IU/g of rice stdw. 

Dudlc "/ al (2003) studied rhc crrncellular pbductions olB-xylanas.. p- 

rylosidasc. ,-glucosidasc. &mannanase. cl-a.abinosidase, eglucEonidase, c

31 

sulNtosidlsc and FPase fiom Aa.i//,' r!,rtld CBMAl0008 wirh rhee differ€nl 

\ylan $urc.s as substrale. lhc cnzymatic prcfiles on birchsood, Erzlrpr6 

grur./rr ard oa1 wc.e studied at alkaline and acidic pH conditions. 4/ril/f 

/r,ild (BMAI 0008 crown on lhe rhree carbon sources prcdrced nainly d' 

xylinase. Prcf'minary as)5 canied ott on E en\.lis krafi pulp f.om an 

indus$al pnpff mill showed a rcduclion of 0 3 % of chlonne us€ in ihe pulp 

htned Nnl! th€ enzymes, resuhing in incrcascd biShtncs, conparcd b 

coilcntioDal bleachiie. Tne cnzlmes vere norc cflicicnr if applied before lhe 

'nltralbldching 

seqlcnc€, in a non-pre-oxye€nared pulp. 

Ri'r d d/. (2001) inv€srigared rhe condirions of the biosylrhcsis of 

pccrinolyts. cellulol),1cs ard henicolluldcs by rhe filamenrous fungus ,.1 nis€l 

IBT-90 by ! mathenadca! mcthod offactorialplanning.nd sradicnt opiimization. 

Thc prcccss of oFimizarions led ro a rhrcefold increde in rhe adivity of 

pcctinolyric cnz)imcs and doubles rhe aclivily of ccttuloltaic 

xyhnac. Octario and Cordola (2001) isolared a diploid nnin (D4) 

bei*en t\!o /. ,ig.r xylan.sc oleQroducing 

tems ofcnzyne prcduction and carabolirc repression by 2-dcoxy- 

D-glucor (2DC). This srdin increased rylanasc prodnion (60? nka/ml). which 

\rere neallt 100 % higher $ai titcts lchievcd by $c s,ild rt?e srain (305 

nkrl/nl)lid 28 %higherth.n $ebestmutant uscd ro inducepanscxual cycte.

]E 

Seyis aad Aksoz (2001) studied diffe.enr Inarod"raa slrains and 

Trichodentt hazianunt 1073 Dl was folnd to bc lhe mosr potent xylanM 

producer.'fhci sone cultuFl paramcreB, nanely, incubation iine, substralc 

concentation. inilial culrure pH and remp.Eturc wcrc optimized in order ro 

inc.ear xllanase production froh T. haEianuht 1013 Dl. The opdnufr 

ircubation rime w6 found lo be 13 days. It wd con.luded that 1.0 % xylan 

co.ctrtratioi is sunable for hieb xylanase prcd&rion rate. Rahmatr e, a/. (2001) 

studied thdl renewdble natural resouEes such N xylan arc abundanl in many 

agricuflu.dl wasles. Penicilliun sp. AHT-I is a strcng prcducq of xylanolydc 

cnz)m€s. Optimun ploducrion of the cizynes was found in cultue conniniig 

oat spelr xylan ar 30'c and initialpH ?.0antr6ilays. Dos erar (2003) studiedrhc 

xyla.ase product'on by soliddare leincnhtion usinS rhe totrsaE arnntia.u'. 

Maximun prcduction (500 U e ) brSasse) was lchicved on the sixlh day of 

cuhiv.tion on slid susarcane bagls medium supplcmencd with I 5 % (v/*) ricc 

b6n extrdcl. The fungal biom6s, d.temined flon iri glucosrmin€ contcnt, 

reachcd 28 nggCl) on thc Erh dayofcultivatio.. 

cdffdcho and Aguilar (2003) worked on cx1n cellular xylande produccd 

by a Mcxican ,.1p?€'['6 strain. ,{yer8il/rs sp. FP-470 was ablc ro g.ow and 

prcduce extra cellular x/anas on birch wood xylan. @l spclt xylan, whcal 

straw. and cofrcob. wilh highcr produclion obsened on comcob, The stnin also

l9 

prcduced cnzymes wirh cellulasc, adylase and pectinase activilics on ftis 

subsrtule. Opritulm t€mpe.arulc sd pH wee 60"C and 5,5, resp€cdvely. 

Biki sral. (2003) rcponed that lllamentous fungi h!!cbeen widelyuscdro 

producc hirclrtic enzymcs for i.dusfial applicarions, includiiS xylanascs. 

shos lcvcls in fungi are gcicrally fruch highcr rhan those in yoasi .nd bacreria. 

Influencc ofcatuoD sources. niircgen $urc6 and moisorc conlenl w6 evalualed 

on xylandse production by Pe"killiun.z,.i..,r I(!l0c in solid-sute 

femenotion Among agricultuml sasrcs t6red (shear bbn, unlrcarcd whcal 

stnr. rrcuted wheal straw, bccr pulp. and soja m$l), unlrcaled wheat straw Save 

th€ biehcn producrion oi xylanase. Tle addnion of 0.4 I of xylan o. €asily 

melabo|/lble sngar, such as glucose and xylose. at a concenimtion of 2.0 % to 

wheal s(&rs enhanced lhe xylanase p.oduclion. Ycdsr extdct was rhc bestnitogcn 

souEc anong lhe nitrcgcn sources itrvcstigated: pcptonc, amnonium nndle. 

sod'u nrtEre,0nnonium chloride, and amnonium sulphdre. 

Coelho and Camona 12003) isolated Arpersi 16 sisa,/els stFin as an 

cxcellenr producc. of rylanasc ds@ialed with los levcls of cellulasc. oprinrol 

xylanasc produdion $as obtained in liquid Vogel medium conraining xylan as 

carton source, pH 6.5 ro 7.0, at 25"C al 120 rrn dunDe E4 h. Amotrg lhe sevchl 

carbon sources restcd! highc! xylanmc producrion xas verificd rn xytan, xytose, 

sugnF.dnc bagassc. lvhear bran and com.ob cuhures Narayln and Belaluddin 

(2004) isolatcd an aerobic, alkaliphilicj xylanolytic ,a.r//rs strains fron lo.al

40 

Soda Ldke on xylan agar mcdiun and screened by xylanolysis hethod. Alkaline 

Xylandse produced by four dirsen $6iN of Btr.,/,rr rp. weE chaacGnzed 

aier l0 nrin in rhc presencc of 0.7 % o6t spell xylan. The adivilies of thc four 

\rd,is {e,( 102.2ll, l8s.nd 20/ U mlc, Fspec|ncly. 

Krosh c, a/. (2004) investisat€d enzlmatic hydrclysh of lign@cllulosic 

mareirl by collulol,,tic and xylanolylic €.zldcs. Aboul t2 filan€.lous tungi 

from genus /zxt./1u,, werc invesiigatcd and compared with rh^t of frichode,na 

feerci. Eiftq Solka-Floc ccllulose or oat spelt xylar vd used as ca.bo. sourc€ ii 

shakc fiask culiilation. All the fungi invsti8ared showcd co induction of 

cellulol',tic a.d xylboltaic enz'ftes during srcwth on cellulose as wcll as on 

xylan. Tne highesl filler paper activily wd measurcd aftd cultivation of 

Peaicillittu bftsilianu on ccllulose. Narayan and Belaluddin (2004) isolared rhe 

four &{1/,j sibins frcn soil and wattr at RaFhahr region. The strains {ere 

isolucd on xylan agar nedium and screcned by xylanolysis method. A?.!//rs 

smins Nerecapable ofgrcwingin xylan medium ar pH 7-0 and produced 55lU of 

xylanlse when cultivarcd in neutnl fr€diufr dt pH 7.0. Maxin.l .nzyme 

pmduclion $a obtained by crltivation on whesl bran alpH 7.0. 

Kang e/ ol. (2004) inveslieared ihe prcduction of cellulases and 

hemicellulascs frcm ,.r. ,,acf KKz, by elid{larc femenEtion using differe.r 

mhos of dce slrdw and whcal bnn Whch,.{. ,rgef KK2 was growi on rice sm{ 

alo.c is a solid suppoa in SSF, the m{ximnm FPdse activity was 19 5 lU 9r 4 

'n

4l 

days. Aho. CMCase (129 IU e ), bcta-slucosidasc 1100 lli gr). xylan6se (50?0 

tL C rl..d beb-xylosi

42 

inhibnoB ofrhe enzyme. DTT and Na'aclivated xylanase K ll by 24 and 13 %, 

respcdirely. Enzyme K II used as addnive to 0our idprovcd dough PrcPenies. 

inccascd rhe volume ot shealjye and whole mcal bread, and jnc.eased tbe 

porosiry ofcrumb and thc moisrure ofrhe frnal prcduct. consequdtly cxtendins 

Jeya ?ral (2005) oprimized thc mediacompoienrs for xylanasc produclion 

bt Asperyiltus w6icotot MKUS i. solid{rare iemenlation (SSF). Optimiation 

was cdried out usins De Moe's iiacrioial factorialdesign wilh seven componenrs. 

Maxinrm produ.tion of xylarac 1249.9 U/g wis obiaincd in SSF wnh an 

oprrniled nrednrn conraining (g rl: NdNOr 20, KrHPOr z0, MeSOa 10, fesor 

0.001. KCI I, p.ptone l0 aid yc6t qinct 10. Four componen$ namely NaNO(r). 

MgSO(r), pepronc and K,HPOrsignilicanrly inc.eased rhe xylanse production by 

,4. v.Attz./o. MK(J3. The optimized mcdia incrcascd xylane produclion by 3.+ 

fotd. Alpeteillrs ,erci.olor MKUI p@dnc€d naxinum xyl..asc afl€. lwo st€ps 

of mcdia optimiation underalftaline condnion. 

Kotrkiekolo e/ a/. (2005) usd Clortdnn .e/lzlovolarr, an anaerobic 

b.clcrilm- to dcgrade naiivc substares efficienily by prcducing ad exlacellular 

en7ytre .omplcx called rhe cellulosom€. All c€llulosomal enzyme subunns 

coniain d@kcrin domains that can bind b hydrophobic domains temed cohesiis, 

which arc repeuted ninc times in CbpA, the noncnzymatic scafioldiig proleii ol 

Cnn,nonas te ulowrans cellulcomcs, In this slurly. the synergistic i.teractions

4l 

ofccllulases (endoelncane E, EnsE; endoslu€nasc L, Engl,) and hemicellulas 

(ardbinofunnosidase A, AfAr xylanase A. xynA) $ere dctemi.ed on the 

dcaradarion ofcom fiber, a natuol subs16re contrining maitrly xylan. ambinan. 

and cellulose. The degiadation by X)hA and AfA of ceuulosc/arabinoxylff was 

grcntcr ihan rhlt ofcom fiber and rBuhed in 2.6-fold and 1.4-fold incrscs in 

syn*gy. respcdively. Sync.gistic €ffeds were obscNed in inclefrenh in bolh 

simultaneous and squenli.l reactions wilh ArfA srd XtnA. Thes€ syrergistic 

enzyhcs appea. to rcprcsenl porenri.l raie,limiri.g enzymes foi efficient 

hemiccllulos dcgradation. When niii-cellulo$mcs $ere constructed f.om the 

ccuulosomal eizymes (XlnA and EngL) a.d ninicbpA wirh cohesins I .nd 2 

{mrDr-CbpAl&2) and minicbpA wiih cohesins 5 and 6 (nini-CbpA5&6), hisher 

acrn ity $as obscned dan thal for the coftsponding enz)des alonc. Based or rhe 

dcgladation of direrenr rypcs of cclhloses and hcnicelluloscs, rhe intcraction 

between cellulosomal enzymes (xlnA and EngL) and minicbpA disptay€d a 

drlcreity thal suggesls llul dockenn{ohesin inteddion lrom (. cellulovorans 

may be nore selcclirelhan Endon. 

Knshna (2005) rcvicwed bnef hisrory ol solid{hre fcmenration (SsF), 

mator aspects or SSF rvere Biewe4 vhich iicluded lacroB areting SSF. 

bionra$. fenrenloE, modcli.g. Dduslrial microbial cnzymcs, organic dcids, 

secondary melaboliles and biorcnediation Physico-chenical and envibnncntat 

lirclors such rs inoculum t)?e, mohurc and wurer activily, pH, tenpcratu.e,

44 

subsrr.le, pariicle sizc, aeniion and agiBrion, nurrirional factori and oxygcn a.d 

calboi dioxidc arectiig SSF were reviered. The advanlages of SSF ovor 

Submcrged Femenlation (SmF) werc indicated, and $e diffeFnt lr?es of 

femcitos used in SSF desc.ibed. Thc eco.omi. ieasibiliti€s of adopring ssF 

lechnolog) in ihc comder.ial prcdncdon ofindusirial enzyncs such 6 amyl6cs, 

ccllulNes, xylanase, prcreases, ph)lases and lipasca were highlighr€d. 

Yang e/ d/. (2005) amplified ftc Sene mxynB(64) by thc dethod of PCR 

wnh lhc renrplate of thc genomic DNA or Thentutasd,u4?ma MSB8, and 

cfoncd inb l|rc cxp.ession vetors of Es.heri.l'ia toli and I'ichia pastoris. 

€spediv€ly. xylanase B(40kD) w6 succe$illly exp.escd by thc two 

helcrclogons prorein expr*ion synchs {ith hiShlevel pioduclion. The 

.ecombinair prctein of xlitB expressed in Pi(hia pastoris showed €xlrcme 

thcmoslability and pH stabiliry, which ws optimally active at 90'C and quite 

erblc over lhc pH nnge oi pH 5.0-10.8 ai 70 "C, After incubation of lhc .nzyme 

al l00oc aor 30 min, X)nB reraincd 70 oZ hisher residual aciiviiy. The 

iecombinanr X)nB expressed ;n Pichia pqiloris is of grear use in a va.i€ry of 

rnduslrial and.Aricultuml dpplicaiions. 

Tandka sr a/. (2005) purified an exlncellullr cndo'1,4'beh-xylanasc fron 

rhe curtuE supehatant of the snycare Awobqsnliun' pullula"s AICC 20524 

groNn on xylan. The purified cnzymo was honogeneous as judged by sodilm 

dodccyl sulphalc-polyacrylamide gel cletrophorcsis and isoeleclnc focusing,

45 

which showcd an apparcnl M G) ofSt lDa and a pl of8.9, rcspeiilely. Xylrnase 

acrirn) Nls oprioalar pH 60and?0'C. Thc senonic DNA andcDNAs encodins 

this protein were cloncd and sequenc€d. The xylanase genc (x)mll) €ncodcd d 26 

amiro acid siSnrl pcptidc and a ll5 adi.o acid marurc prc1ein. DNA rcgions 

cncoding rh e signal scquence and thc matu.c prolein wcrc inlerupled by inlrcns of 

56 and 7l bp..especti!€ly. The xiill s''noncoding ngion had two conscnsus 

binding sites for the transc.iption factoi PacC ncdialing pH .egulstion. 

Quantitatir€ eal'rimc polynemse chlin rcacrion .nalysis revealcd thal lhc 

lunrdprion lelcls at pH 6.0 and 8.0 were 6-fold and 22-fold higher rhatr lhat at 

pH 2.?. respeclivcly A cloned xynll CDNA ws expresscd and sedeled in lhe 

ytrsr Pichia pastoris. Scqnence alignmcit and phylogcnclic analysis suggcsied 

rhat thc xlnll belones to glycosyl hydrclasc family I0 aid thar it is evolutionarily 

dislont from lwo cluslere tomcd by oihc! family-l0xyl.ndscs. 

Rnller e, ./. (2005) pu.ified lhc XrdA io homogeneity fiom ,a.,?6 

sr,,tri nmi. 168 culture supcmarants by erhanol prccipilalion and cation- 

cxchanec chromaloglaphy Thc DNA fnemcnr encoding lhe XynA togcthc! with 

thc ttsxA promoter rcgion was anplificd by PCR from 8d.r116 r/r/i/a 168 

gcnomic D\,^, andcloned inlo rhe plasmid pT7Il to givc rhe plasnid pTTBSXA. 

Ancr thnsfomarion of Efheri.hid coli DH5alpha wilh pTTBsxA, l9-ibld 

in*clse ii rhc lcvcls of $e secretcd XynA sd deledcd ,n the suPematanr as 

comparcd ro ihe ,. r,rrtla culturc. Coftct pcr translation modificaion of the

46 

rccombinont pbtein was confimed by N tcninal anino ,cid sequencing lnd 

mass spcchonetry andlyses. The pH aid l€bp€rature dependence of ihe natile 

and rccombinot prcreins wft idenrical, indicaring thar rhe pTTB5XA @y be 

uscful lbr rhe consritutive expre$ion ofhelerologous prclcin in E coli, 

\4inirc, c, a/. r200t ampl,fied BhVlRl2 \)trllA gene. cncoding an 

cxt€cellul.r endoxylane of potential inleresl in bicbleaching appli.atio6. frcm 

Ba.illts hdladurarc MtRlz genonic DNA. Thc protein cncoded is an endo'I,4- 

b.ra-xyl.nrsc bolonging ro f.mily ll of glycGyl hydrclas. lts nuclcoridc 

seqrence was analysed and rhe nature peplido w6 subcloned inio pET22b' 

cipEssion vector. The enzyme was ovecexprcssed in a high dcnsily Es.rernrk 

.o/i culrurc as a soluble and aclivc prciein, and punficd in a sitrgle step by 

rndobrhscd nelal ion afliniry chrcmatogaphy wilh a specific acriviiy of 30?3 

lll/mg. 

Siha e/ a/. (2006) evaluated pccdnase. amylase. xylanase, and cellulase 

product;o. by Ltophi u,' songr'lophotu in labohrory culurcs and fornd rhat 

poltslccharidass arc prcduced during fungal growrh on pccrin, srarch, celhlose, 

xylan, or glucose but nol ccllulas€. whos€ p.oducrion is iahibited d!.ins fungal 

8tu\ft on xylan. Pectin sas the cadon source ihat h.sr srimrlared fie poduciion 

ofcnzyms, which showed that p{tinase had rhe highesl productioi activityofatl 

or the c..bon sources lesled. indicaring rhar rhe pEsence of rtrd. and the 

produclion ofpoctinase are kcy featurcs for symbiori. nul.nion on plant malcrial.

47 

During sro*th on starch ard c.llulor., pol,sacchandasc prcduction levcl was 

inl.mcdiare, .llhouSh dunnS 8b*tl or xylan and glucose, enzyme productid 

w6 lery low. They pbpos€d a possible prcfflc of polysaccharide degradalion 

iiside $e.6t, wher. thc firgr$ is cultur.d on th. folid $bst at , 

Oshim "t a/. (2006) investiglcd on th. slnth6is of ayldec fron 

Asperyi rt sojae. Th. st@turcs of XPs werc anallzd by HPLC to dclcmine 

sugar compotition and dolecula. m33, by mclhylation analysis Bing GC-MS to 

dctcmine liikssE, and by (l)H, dnd (I3)C,NMR s?ectomeni€s ro obtaitr thc 

anomeric configwalion ofglycosidic linkago. By chcmical analysis, it was found 

lh.r rhe smctuB of XPs e Xylpbcrat-4Psip, Xytpbcrat-sPsip, Xytpbeo t- I psif

APPLICATION OF XYI.ANA]SES

48 

APPLICATIONS OF XYLANASES 

lnt€r$t in xylan.scs frcm diflerent soudcs hs incrded markedly in th€ pasl 

decade, in pan b€causc of rhe application of th6e enzynes in rhe pllp and papc. 

indusr,r-. biobl€achine, wine industry, imprcvin-q a.imal fccd, production of 

elhcnol and ncihdne, The edliest U.S patent for a melhod of xyldnase prcduclion 

w6 issued in 1992 for an enzyme mixturc used d tn a.imal f.cd addilive for 

dairy cattlc (Ptasensatr and Oi, 1992). Xylaiase has sincc provcn u*tu| in @.y 

Bioblerchingpap€rpulp 

Paper producc.s need b Elain ellulosc while remo'i.g thc lignin frcm pap.. 

p!lp. thc classic way lo perfom rhis opcBlion c ro add chlodnc-ba*d blechcs 

lo thc pulp. Xylanase blcaks the hcmic.llulose chains that arc lcaponsible for the 

close adherence oflignin to rhe cellulose nctwork. There is thus a reduced need lbr 

blcach to rcnove lhe l@n€d lignin. whcn lhe bl@ch used i5 chlonrc-bsed, rhe 

use ofxylaiNc leads ro a redudion in organcchloine pollutails such as dioxin 

liom the papq malinS prcces. In addition. chlorine'fr€c bleaching Guch as 

psoxide or ozonc bleaching) can achicle brighler rcsulls with the addition of 

xylane (Jackson e, al. 1998). Bccauw xylande dcs not ham cellulos, the 

strength ol rhc pap.r product is not advcBely affecred.

49 

Jackson er a/. (1998) studied thai pulps wirh chemical propeni€s similar ro 

a dissolving pulp .an b. prcduced fiom once-d.ied. comnerci.lly lvailablc 

bleachcd hard*@d knli fib€. and frcm high qualiry rccov€rcd paper rich i. 

hmdwood fiber. Xyhndse improves thc rertinc of flax fibe6. Rexing is the 

dsomposnion of thc outer steD flax pl. n6estary before thc fibcu a€ 

p@c$ed into linen. Jing er al (199E) snrdied rhe molccularand biotchtrological 

aspoct ofxylanase. They fouid thal xylsn.se cat.lyz€d tlrc hydrolysisofxylan, rhe 

frajor constitueni of hemicollnlose. C.llulo$-liec xylanose pla)s an inpod6.1 

rcle in the papc. and pulp induty. The propedies ofxylanas Fom extramophilic 

ors0nisnN have bcen evaluated in teims ofbiolechnoloeical lpplicarions. 

Ronald {2001) nudied thc xylan.sc obhined frcm a gcnetically modifred 

Asryr(illus otfzae contînin9 the xylaDasc gene fioh Therhonrces Iatueirosis. 

Thc u$ of xylan.se bcnefrls the consumcr through thc greare. availdbility ol 

qualily-bak.d soods. Addirion ofxylanNe c.n reduc€ rhc vi$osity oflh. juice, 

imprcving its filt@bility. st'mutates grcwth btes by imprcving digesiibility. 

wllich lko improves ihc quality of ih. aDimal lilcr. xylanas€ imprcvcs lhe 

clc.ning abiliry of detergents that ac cspccially ereclive in cleanins frun and 

rcscbble, soils and gra$ slains. This .mcndment also benefits the i.d!s$y by 

provjding a new xylanase fo! use in lhe production of bread. nour and whoh- 

$heal nonr and fa€ihsting fie prodnction ofqualily bated 8oods.

50 

Sudha erol. (2001) reponed thar rheemue.t fon pulp bleaching prcceses 

conraining.hlorinated lie.i. and d€goded polwhenolic intem.diatcs rcmains as 

a najor sorrec ofwater pollutior from th€ pulp and p.per indusLies. Altemarive 

€lemcnlul chlori.e fiee bleaching methods based on the nsasc ofchlonnc djoxide, 

ozoic dnd hydrosen pcrcxidc ae pornrinl subsritutcs Bio-blcaching mcftods. 

which in\olle pre'tletrnenr ofpulp vnh nicrcbial enzymes such as xyhissi 

halt cnc,gcd as viable opio.s. lnv6tigations ieponed rin ar exploring rhe 

suitability ofcohnercial bacterialxyldiosc en4me prepa.dtions fo! bio-blcaching 

of baga$c pulps. Saha (2003) studied rhdr coiv€rion of h€micelluloscs to fuds 

lnd chcnli.sls arc prcblmatic. Extnclins fforc femenrable sugar frcm barley for 

making bc$. ar wcll a prccesing of thc spe.t barley for aninal fecd. ln both 

cases, xylaMse has ability lo brcal( hemicellulNs down into suga6. Addcd 

xylffosc rcdu.cd rhe viscosity of brewins liquid. inprovi.sils fiherabiliry. 

Rodnd./ a/. (2001) used thc,{ ,rAcr to produce thc fcruloyl csterase 

(FA0A) bl lonologous overcxpresioi for pulp bleachi.g applicrlior. Chrnsiig 

the nlrud rDd concctrLalion ofihe cnbon so!rce.atue (nahose toghcoscifiom 

2.5 ro 60 s l r. improved FAEA acdviry 24.5-told and. yield of I g l r of rhc 

corespondine prcrein in the culrure n.diln was achieved. Th€ .ecombinant 

FAEA was rested for wheal smw pulp bleechiie. Besr €sulis we.e obrained usins 

a lilscquctrlul p.ocess with . sequcnce including xylanase. FAEA and laclase, 

and yicldcd !€ry efficient delignificarion-clos€ to 75 % and a kappa numb€r of

5l 

3.') This is the fi6r rQon on the potenlial application of.donbinant FAEA in 

thc pulplnd paper seclor. 

lmproviDg f€ed 

Adding x)lanase stinulatg srcs1h 6ics by imprcvins digesliba|ny, which aho 

imprcv$ the qualiry of the a.imll litl.r. for example, chickcn f@d bascd on 

whcar, ryc. and many oth6. grains is incomplelely dieested *ithout added 

enzymes. lhese Ctains tend lo be too viscous in the chicken's intninc for 

conplclc digestion. Xylause thins olt the gut contents and allows inofcused 

nulieDl absorplion and incrcased diffusion ofpanoealic e.zymes inthcdigcsia.lt 

ako chanSes hemicellulN lo su8a.s so rh.r nurienb fomerly rdppcd wiihin rhc 

cellsalls arc rcleased. The chicke.s 8ct sufllcienttrergy frcm le$ feed (Oksanen 

?r tr/., 20{D). The bam is clea!€r bccau$ the fecd is nore Ihoroughly digested so 

th€ chick.n wdte is dde. and le$ sticky. ln addirio., chicken egSs a.e clcaner 

bccause $e excrement in rhe l.ying are6 is drier. In a sense, the addition of 

xylanasc toanimalfeedpre digcsts ihat feed. 

[1c6 e/ d/ (2003) slaied ihat cndorylanasc rclatile aciivirylowld wat€r- 

uncxracbbl€ anbinoxylan and Narcr+xtrgctable adbinoxylan substrarcs was 

inpona fo. ils tunctionahy jn biorcchnolosical prc{*s such as brcad mkinS 

rnd glutcn shrch separ.tion. Xyl.nrs€ us€d in separation of wheal o. olher ccreal 

gluien from starch. xylanase improvcs ihe exrnclion ot oil fron oil nch plsnr

52 

daterial such s con oil from embryos. A scrccning mclhod for npidly 

detemiiing said subrtrarc sclcctiviiy wd d.v.lopcd. 

M0klng brerd nufiier with freshener 

Addcd xyldnrse modifics wheat flour mbinoiylans and catr resuh ii t lolf{ith 

moc than I0 % 8rcalcr volumc. Crumb softtrcss aftcr sio68e L ale imPrcved 

(asez rDd Rodrislez. 2001 ). 

Aidirg in sep$.tior ofwhert or olher cer€.|gluten from strrch 

The cnzymc aho hclps itr thc scpaElion of whcai or lhe dulen fM som. oiher 

cercals frcn shrch paniclcs (Chdstov er ol. I 999). 

l rerslng julc€ yicld fmm frulls or vege|lbles 

Xylanase rids in ihe maccElion (chcwinSup) ptr6. In addilioi. Edded rylanase 

can cdu.e the visccny of lhc juicc, impmvinS is nhenbi|ily (Sicdenb.rg er a/.. 

t998). 

Ertr:cting ftore fern€ntrble sug.l 

Thc cnzymc h€lps extncting morc femmlabl€ sugar from bfilcy for making beer, 

as sell as pr@cssing lhc spent barlcy for animal fe€d.ln bolh ces. xylande bas 

thc abihty ro breal hcmicellulos. down into suga6. The added xylan.se can 

rcduccth€ liscosity of brcwing liquid (Sudha c,./..2003).

lmprovlng slhge (or enhrnced fernent.tlve composting) 

5l 

TM|mcnt of fonge with xylane (.long wilh ccllulase) €sults in bcner qualily 

silage and improv€s the subs.qucnr otc of pl.nt @ll wall diS6tion by ruminlnts. 

Thcrc is r consideEble amount ofsus.rsequestcEd in thc xylan ofpla bionast. 

ln addnion ro convcning hcmiccllulo* to nurririve sug.r th.i rhc cow or othd 

ruminanl can diges! xylan.s. also prcducer compounds thal may bc a nutridve 

source for thc ruminal rnicroflom { Pmscllsan and Oi, l9q2). 

Improv€d degr.drbiliay olpl.nt w.sle mctcrhl 

Xylanase aho itnprovc thc dcgladabiliry of plani wastc |notcrial (for instance, 

agriculruEl wrsteq fierlby €ducing orgrnic wdt€ disp6al (Nunez a dr, 2001 ). 

Inprove th€ cl€. ng rbiliay of deteqents 

n !|$ improvcs lh. clening abilily of d.l€rgents thar aG csp.cillly efTccdve in 

olcaninS fruit and vegetablc soilsa gnss slains (Nco a, a/., 1986). 

Fuel-.lcohol productiot 

Xylanasc dccrc0ses fte viscosity of the mash lnd prcvc.rs foulinS Prcblcms in 

distillinS cquipmcnt ( Ronald 2001). 

lmprove the €xtr.cliotr ofoil 

Thc cnzymc imprcvcs the cxtmdiotr ofoil frcm oil-rich plant maidialsuch.s 

com-oil frcm corn cmbryc (Chcn .t dl.. 1999).

Improv€ r€ttitrg ofllir llbers 

54 

Reding is the d€compdition of thc outcr slem of the fld plant neessary bcforc 

the fibes aE prcce$ed into linen. Choi snd won ( t998) sludied tbat the rccrclins 

of pulp gdtrates a lot of fln6 6rd caus.s lhc homificaliot of fibd- xyla.a* 

k€tment showed some refining.ffcci ai a small d$age, wbile the fines ed fibrils 

w{e reduced at higher d6ag. as shown in lhe codps.ative teatnenl with 

cellulase'hemicelhlase. Br€akin8 l€n8th ard i.ar index of recycled fibd treated 

wirh xylana$ was highd.

MATERIALS 

AND 

METHODS

IelatioD ofo.grnism 

55 

IATERIAIS AIID IETHODE 

O.e hundred and four cultures ot Asperyillrs njget \|erc isolalcd from ditferenr 

soil sanples of Labore by serial diludon method afier Clark e/ a1. (1958). The soil 

samples were collecied in sterile polythcne bags. On€ gnn of soil smplc was 

dissolved in 100 ml ofsbrilized distilled wate.. The soil suspension was turth€r 

diluled up to 10110' tim€s. One millilitrc of lhis dilute suspension was then 

transfefted to individnal peripl.tes containin! potato dexrose aear modiun 

hble l. Compcrt,on ofpor.ro dcrtbsc Ngai mediun, pH 5 6 

:19.0 

l000nl 

Pot.to dexlrose agai medium was prcpared by dissolving 390 g of poiaio 

dextrosc agar in approximately 700-800 mlofdistillcd wale..nd tben made up lhe 

volume lo IOOO ml. The medium was hcat d for about l0 nin to obiarn 

honogenNDs mixture. The pH ofth. mediufr was mainlained al5 6 bv 0 lN HCI 

or0 lN NaOH.

56 

Approxinalely t0.0 ml of tbis porab dextrN a8.r medium wd pouEd 

iito individual lesl ubcs. The tubes w.re colton plugged and tubclaved ai 15.0 

lbtinr prc$urc (l2l'C) fo. 15 min. Thc contcnts of tubcs wcre tnnsfded lo the 

$cilized petri plales (180"C for 2 h) and allowed lo solidify.l room temperature 

for abo l h- After sil suspensid addition, rhe Plales werc robt€d clck*is. and 

counrer clockwiser so that tho suspensior would sprad unifofrly- The periPlaics 

were placed in an incubalo. (Model: MIR-153, Sanyo. Japan) .t 30"C (4.6 days) 

for culturc developmcnl. 

The..{. r€cl wcro ideniifiod acco.ding io Onioi et d/. (1986) The yolng 

coloiics of,.{. ,i8ef werc picked up dnd imnsfered lo poiaio dext ose agai danis 

and incubated al30t lor 4-6 days, for maximum spodlation. The cultures *crc 

srocd in a rehgerator ar 4'C for fudher stud'es. 

Culture improvement 

UV-irradiation 

The s.lected isolaic of ,.{. nigef was subject€d to UV ladiarion fot thc 

imprevcmcrt of thc slmin. The conidia frcm ,f6 days old slant culturc acrc 

haryesred in phosphlt buffe. (Table 2).

51 

Composition ofphosphate buffe! pH 7.2 

K:HPOl 

KH,POI 

3.5 

t.5 

1000 nl 

The bure. was peped by dissolving the insrcdicnb in apprcximal.ly 90o 

ml of dislilled water a raised the llnll volune up to l00O ml. The PH was 

The concenlEiion of @nidia ws stimaicd frcm a counl mrde on 

haemocyloneler slidc (Ncubautr Precicdor HBG, Germany) afier Shama ( I 989) 

dnd th. density was adjusted to lxl0'conidia/ml for Uv-imdiation. The 

sBpcnsion e prcparcd w6 exposed to Uv'imdianon for diffcmt rime intcmals 

(5-60 nin). The dosc givo to conidill suspcnsion was l2xld Jim:is 

Apprcximatly 0.5 ml of the iftadialed conidial suspension ras then po'rrcd on 

potab d.xrM xylgn agar plaie (Tabl. 3) a.d the pl.ts q@ incubai€d .r 30qC

58 

Compcitim ofxylan a8.r mcdiun, pH 5.5. 

KHr?O. 

KCI 

(NH4LS04 

CaCl, 

The i.erediflls werc dissolved in dpprcxiDately 900 ml 

by heating the solutior for 15-20 min and th. final volune wd 

bl. The pH was mainLined a15.5 by 0.1N HClor0.l N NaOH. 

*d used as coloDy reel.ictor. Thc medium wa3 autoclaved at 

(15.0lbs/in1) and poured (abour 10.0 ml) in srerilized pelriplales. 

5.0 

20.0 

1.0 

0.5 

0.5 

0,1 

0.5 

l00o ml 

The Triton X'100 

12l'C for 15 min

59 

Xylanasc prcdrciion wd checlcd in comp..iso. wirh co.rrcl (conidia 

{'tho!r cxposuE). Muranls were picked up frcm fte plates having l.rger der 

zones ol xylan hydrolysis.nd ar l€ast 90 % d€arh nre oforsanish (stcinc. er a/. 

l99E). Ninety-Four cultures of,{.,rgelwere isolated afier Uv-iradiation. St ains 

of ..{. ,ialf w..e examined for ryl.n.sc biosynlh€sis in 250 ml Erlenmeys flasks 

bysubmdgcd fementationtcchnique. 

Chemicrlmut!tiot 

Preprrrfi on ol veg€tative inoculum 

The best Uv iuadialed slnin was lsed in the plosenl investiaation. The slants 

were k€pt ar 4t in the rcf.igqator, A volume ol45.0 nl ot Vogcl 3 m.dium 

(Table 4) was dispensed in a 250 nl conical flsk. Chrcmic acid *.shed maftle 

chips ( 12-15 in numbcr and 2.5 mm, diander) weE tI nsferrcd lo ihc flast to 

break up rhc mycelial pellcls. The flask was st*ilized al 15.0 lbvin: prc$u.e 

(121"(') tbr 15 min. Then. 2.0 ml of sterilized 50.0 % (w/!) stock solution of 

gllcose was deptically added in the aut@laved vogel s medium as an additional 

carbon source (Roy and D6, 1977). Thc flask Ns inaulated enh 1.0 ml of 

conidial su$ensiotr u.d€r deptic conditio.s. 

nediun w.s pfparcd by dissohing all lhe ingrcdients 

ml of disliU€d water. Aft{ raising the volunc Lrp tol000 

the pH *d maintained al 5.5. The inoculum was allowed lo

60 

srow at 30eC on lobry inobalor sh0k r (2m tp!n) for 24 h. Tl. lnycclial @us 

w€re h6rBtcd, entrituged (C.nrdtugc r!ftig@t d, Modct D-37520, Ost ro& 

m HaE, G@ny) at 9,0m rpm (E,33lxt fd l5 nin.), w.lhcd twi@ with salire 

watd.rd rc.rus?cnd€d in salirc watcr (Tablo 5). 

Table 4. Corqocilid ofvogcl's n diDm, pH 5.5 

NH.|tor 

HzPO. 

(NH.ISO. 

MgSOa.THrO 

2.5 

2.0 

5.0 

4.0 

0.2 

2.0 

1.0 

l00O ml

T$le 5. Comp6irion ofsaline wat r, pH 7.2 

NaCl E.5 

Di6tilled warq 

5.0 

1000 

The mycelill susp€nsion wa lscd for inprovcmenl lhrough mtrtation usinS 

N-metbyl N'nilro N-niloso gxaridine (MNNC) as the chemical nulagmic agent. 

Abour 5.0 ml of vegclaiive nycclial suspension wd aseplically Ea.sfmed lo a 

ste.ih centrifuaetubc. MNNG wd adjusted 1o six diferenr concenlralions i.e..50, 

100, 150,200,250 aDd 300 pg/ml in phosphdte buffe! pH 7.2 (Table 2). Filc 

millilirrc of VNNC solurion of dch conccDtmtion w.s added b 5.0 ml of 

nycelial suspension. In rhe contal exp€nnent, nycelial solpension was trcalcd 

similally except thal the MNNG was rcplaced by ste.ile disiillcd waler. Ailer th€ 

Equircd tine inGrval (b€lwe. 10-40 nin) th€s tubes werc c.nEifnsed al 6,000 

rpm for 20 min. Th€ hycelial €lls werc separaed rrcm MNNG by discardins rhe 

supomaranr. The cclls wee th€f, wasbed rwice with phosphate bur€r by 

c€ntrifngation to remove the taces ofchenical reagent.

AfieNards the volume 

srerilized distilled wlrer. Thd 

dilulion sas made by sknlized saline waler. Following l*o dilutions wcre ut€d, 

62 

in the tubes was nade up to 5.0 ml wilh lhe 

the tubes *ere shaken viSorously. Beforc pl.ring, 

l0? dilution *as mad€ by dissolvi.g o.l ml of murant mycelial 

sGpensior in | 0.0 ml of strilizcd saline solutiot. 

ii) lol dilution was nade by dissolving 0.1 nl of I0r diluiion samplc in 

10.0 ml of sterilized saline solulion. 

Approximarly 0.1 ml of ach dilutio. ws iransfcftd ro each pelripl.re 

contaiiing lylan agar nedium. After t2 h of incubalion ar 30"C, the nunb* of 

colonies in each platc was counted. Tl€ you.g colonies wcrc selsted o. thc b6is 

of bigger zon€ of ryl.n hydrcllsis. Thc mutmr stains ol ,'1. ,,s?r BRClvrr with 

Iargcru..s ve.e pickcd up dd transfeEed to the poiaio dext ose agarslanu. The 

cultu.es were incubaicd at 30"C for 4-6 ddy, for maximun sporulation. One 

hudrcd d nine mura cultuG wcte picked up after trcatmenr of dilledt 

conccnlrations of MNNG al differeni iime ini€dah and screened for xylanase 

biosynthess nsi.s shak. flsk rechnique. 

Fermentation 

Pr€paralion of conidirl inoculum 

Conidia f.om 46 days old slanrculture were used for inoculalion. Ten nillihrc of 

stnlized 0.005 % diacetyl ester of sodium s fo succitic acid (Monoxal OT-)

63 

solution $as added lo thc slant having prcfrlse coridial grcwlh on its surtace, 

Slcrilized inoculating n@dle {as used for breaking lhc clumps ofconidia and lhe 

ten tube rvus shak€n vigorously to obtain homogcnons mixture of conidial 

suspensioi- The conidial cou.t *6 madc o. a hrcmeyon€ter Tte counting 

chanber is d ruled gla$ slide wirh a cover thai holds a definirc fluid volume. Thc 

nunber of.odidia in 1.0 nl of suspcnsion was cal.ulalcd ane. counling in . 

sguare (0.1 mm deplh) un

Following media {%, w/v) wer walualcd for xylandsc prcduction usii8..{. ,&e/ 

CCBT-I5i 

vl: (NHr),SOa0.2. CaClr.2HrO 0.1, NaCl 0.2, M8SO1.?H,O 0.03, wh@t bFn 

2.0 {Cawadde and Kadat. 2000). 

M2: NsNOr 0.1, NH4CI 0.15. KH,POa 0.1. MsSO4.?H2O 0.01, wheat b.d 2.0 

(Con6 er dl. 1994). 

M3r NaNO, 0.1, NHrCI 0.15, XxrPOa 0.1, MgSOa.TH:O 0.01, CaClr.2HrO 0.1, 

whedt bm 2.0 (Monica e, a/., 2002). 

M4: NaNO, 0.1, Tw€ei-80 0.2. NH.CI 0.1. KHTPOT 0.1. MgSOa.?HrO 0.01. 

caclr.2HrO 0.1. wh.al ban 2.0 (Si.dcnbere er a/.. 1998). 

b) Solid srlre 

Tcn erams of wheat bran was placed ii 250 ml conicol flask and moisiened by 

adding l0 ml of distillcd wald 6 a moistcning agerl. The fldks {crc conon 

pluggcd rd stnlized in an autml.vc at l2l'C for 15 min a.d 15.0 lbnin': 

pEsure. Aiier sionlizalio., the fl.slc were cooled to roon lempcnture dd 

inoculated walh 1.0 ml of conidial ruspcnsion and incubatcd at l0oc for ?2 h- 

Afier 72 h. 100 ml ofsodium citrate buffef (pH 4.51 was added to each fllsk. The 

fldks wcd rhen rotated at ro1ary shakd for I h ai 200 rpm for the extmction of 

dztm€. Thc fem{ted brcth was filercd lnd fittmte was used foi lhc csiinalion

65 

The diluents including 0.1 N Hcl.00l N Hcl, dislilled warcr, iap watcr and 

nineral salt solulion weie u.ed .s moisl.ning agents in solid-sub$ra€ 

c) Stirred fermentor 

Prep.mtion of vegetttlvc inmulutrl 

Two hmdted milliliFe of femdlalioo mediM cotilining (%, Y/v); NaNOr 0 l' 

NIIICI 0-1, KHTPOa o.l, MgsOa.7HzO 0 03, Caclt 2H:O 0 1, mBt exrtct 0 5. 

Tween 80 0.2, wheal bnn 2.0 in 1.0 L cotton wool pluSAed Erlenmever flsk wd 

stqiliad at l5-0 lbvinz prcss@ (l2l'C) for 15 min One millilitre of cmidial 

suspension (1.2:106 coniditmt) of ,'4 rkel wa Dscd for in@ulalion Th€ flask 

*d incubded ar 30"C in rolary incubator shaker at 200 rpm for 24 h 

F€rmentation lechniqft 

TlE produdion of xylanrse w4 canicd o in a 7.5 L N'w BMwick 8lN 

femmtor (Model: BioFlo-l l0) wih a wo.king volum"f 5 0 L The f@nentatiotr 

medium od poured in rhe f.mentor ed slerilized in e a dlaveal l2l{ for I5 

min (Model: KT 40L. ALP Co.. Lid hPan) After cooling 1o 30'C' tlie vegelaiive 

inoculum {s lransfeftd to th€ nedilm al a lwel of4 0 % (v/v) Tbe incubation 

lopenrurc wa kepl ar looc $rcugloul ihe fem.rr.don p€'iod Tle agitation 

speed of 0,e stiner was adjusted at 200 rpm while th€ ae'ation was maitrhined a1

66 

1.0 v!n. ThepH ofthc femenbtion prccesws contrelled by using ncrilied 0.1 

N HCI dnd 0.1 N Naol.l. Th. stedliz.d silicone oil was used every 24 h as.n 

d) Re-uB€ of nould mycelium 

The mould myceliun was separated fiom femenied broth lndcr ascptic 

conditio.s using sterile ce.infirse tubes. The nycelium thus obtaincd lion 

previous batch wd kansfcrcd to fresh sic.ile nedium conlain€d i. shake flasks. 

Thc femenlalion ws run for 72 h. 

e) ln'nobtlizrtion of,.{. rr?e' conidla 

An w.s renoved by vacuun fton sodiun alginale solution (3.5-8.0 %, w/v) in 

distilled wlter. various quetitic (2.5-20.0 o/") of dricd ..{. ',s.f conidia were 

suspendcd and rhe Dixlurc exhded though 0.6 mm di0neler needles into 0 s M 

calciun chlonde in distilled w.1er with mixins. Tl€ mixing wd continued for 20 

min afid bead fomation 6nd the beads wcrc fifrlly $athcd wnh waler. Tle beads 

w€re slbscquently ireatcd with 2.5 % glut r.ld€hyde in distilled wsler for I h al 

4t and $ashcd The apparot aclivily yield wd l0 lo I I %- Bead strcngrh was 

deremincd *ith tu Irstron tesrer (Linko .r a/., 1979).

D Reperted fed+rtch cultur. 

67 

xylanase fmdtarion was cdicd oui is thc atircd f.rmmtq (7.5 L crprcity) 

with working volum. of 5.0 L, continuously by r.movin8 4.0 L of fmdted brcrh 

and it wd repl&ed by stdile gluco6€-salr-Cacoj solution (4.0 L). This prcc4s 

was r€peated for 3-4 times until thc substnte was consumed mw than 90 % (w/v) 

Atralysis 

Preplntior of DNS r€rg€nt 

Th. .bove ingrEdieds w.r. disolv€d in lpprcximdely 60GE00 nl of 

disdlled watd ed gdtly h.antd in a *.tcr blth st Eo"C until a cl@ elution wa 

oblain€d. The following ch.mic.ls w4 then addEd, 

10.69 

19.58 

R(helle salr (sod'un potlssium t!ru$!.) 306 g 

Phdol (melled at 60'C) 7.5 ml 

Sodium mei.bhulfar. 8,3 s 

Afier dissolving th.6. ch.micals, lhc fiDal volume ws md€ up to t4l6 ml 

wilh distilted wats. Th€ reageni wr5 th.n lil&rcd lhrcugh a ldge c(tre gbss

68 

filte. .nd stored ar tuob l€npe.ature in an amber colour glass botlle lo avoid 

photo oxidaiiotr, li remained stablc undd these @nditions for lbout 6 monlhs. 

Standrrd curve of rylos. 

Oie gra6 of xylose was dissolved i. dpproximately 60-70 ml of disdlled waler 

and the volume w6 rai*d up to 100 ml. This st@k solulion (10.0 mgiml) wa 

used to nakc .ighr rpprcpnaie diludons hon 0.25 2-0 o8/ml. Two milliliEE or 

ech dilurion ws rlketr in individull rcsr tub6 od 2.0 ml of DNs @8enr wa 

added i.to each tube. These tubes w€n placed in a boiling water bath for 5 min 

followins Miller (1959) dd Ghde (1969), ihen cooled at rcom temperature and 

diluted ro 20.0 d wnh distill€d *atc!. A blank ws run in puallel rcplacing 2.0 ml 

of ihe sample dilution wnn distlled *at.r. The % tamit!.n.c Ms m6urd at 

546 nn usiig doubl. boam Uv/Vls.sa..ing sFct opholotnetel (Model: C..il- 

CE 7200acdes, Aquadus, UK). A graph *as plott€d raki.g the trarsmittdce at 

the o.dinar€ and sugar concfttratio. ai ihe absissa (FiCurc I ). 

Xtla trrrc srcchr rifyiDg rctiviay 

Xylanasc aclivity in cultu€ filt ale war ass.,td alier Somogyi (1952) by lating 

1.0 ml xylan (1.0 %, Vv) .s substrat€, 0.5 nl of acerale buff.r, pH 4.5 (Table 6) 

and 0.5 ml of appropriare fl)me diluiion. Reaclion mixturc ws incubated ar 

50t for l0 min and thd 2-0 nl ofDNS @gdt {d added lo stop lhe @ction. 

The tube $as pldced in a boiliag war.r barh for 5 min. Alld coolins the conlents

69 

of rcsr tub. at mn tempeFturc, rhe miatue ws dituted ro 20.0 lntwilh disrill.d 

w.ld. A bl&* wa6 run in p.r.ll.l Fpbcilg 0.5 ml ofthc cruymc dilution wirh 

distilled p!lq. The ftnsmintucc we masud ar 546 m oD rh. 

Tabl.6. Composition ofac.tare buff€., DH 4.5 

.- Dislved 2.87 r (C)of 17.4N rcedc eid in 400 ml ofdistill€d 

wdc. and tti$ h. vdMc ro 500 0t. 

b. Sodi'rn acetaE (6.89) v0! dissotved io 2()0 mt ofdislifled warer ffd 

madc the finll voluDe uplo 500 mt. 

2.871nr (C) 

Mixen 64 ml of(a) ald 36 n of(b) fd0.1M &cbte burcr (pH 4.J). 

6-E 

1000 ml

Enzymc unit 

70 

''One unir or xylanae will libenie on. millignm of Educing suSd mes!rcd rs 

xylose ircm xylan pe. hou at pH 7.0 aid 30'C. EMyne activib/ was expBsed d 

!/g for solid state and U/ml under subhc.sed conditions" (Wong, 1988). 

KINf,TIC PARAMf,TERS 

For detemining the kineric p3lder.6 of batch fmentadon prcc.$, the 

ptuedur. ofPin (1975) w6 ado ed. 

The ptuducr yield c@fficidr (Yot ) w.s dereinined by usinS rhe etarionship, ya, 

= dP/ds. 

The volumeftic lales for srbstrate utilizrdon (e,) dd prcducr fomariotr (ee) vere 

det min€d ftom rhe naximum sloper in ploi5 of subsrnle utilized and xytanase 

prcduced e.cb vs. the rime of fcmration. 

Spsilic rrte consa.ars 

Tte sp€cific Brc cdsr.nG for producr fomrion (qJ lnd subsrde uriliztion (G) 

werc detemi.ed by thc equations i.e., q! = /xYrr and qs = #!y"i rcsp€.iively.

STATISIICAL ANALYEIS 

7l 

Trrtrn at afi.cb l!& coopor.d by Se f.o@or.d lc.n siificlnt dillrcn 4 

nldhod ad orelrry ANOVA (Sp$,9, v.Ed6a) atur Snod.oor .nd Cocbrm 

(19E0). Sisnificanc. ditrdloc. e6f lh ftptio.id! brs bocn F!s!or.d d 

Dmcu'. rluftipl! rnsr, b th. fcin ofFlb.ttity (9>) v.tur.

fEure l: Stonded curvc ofrylo,c 

:40 

a 

E.o 

n 

G75 1 12t 

Xylo€. (nVmD

RESULTg

13 

RESI'LTI 

A) Icolr.i,Dn rrd sc...nirg oflt. orgl[bn 

HudEd dd four sraim of,rrp.s,?( ,&et w@ isolat d 8on difercnr hrbibrs 

md cvalualcd for xylams€ Foducrion (Table ,. Thc isold€s w€rE sub-souped 

accoditrg to lhe mg€ ofcozyme p.odlction (Tablc 7r). Iftc i€sulls show tbsr 39 

olt's prcduced enzjmc in the @9. of0-50 U/mI,37 cultures betweetr 50-t00 

U/nl, l8 cultms eeF b.tween 100.150 U/nl, 7 cultu.cs and 3 cukurcs werc in 

lh. rege of I 50-200 U/ml and 200,250 U/rnl, respecrively. 

or all $e holates investigated, mdimum .nzym€ produclion (225 U/nt) 

vas obtained by,4. ura€l CCBT-35. So. thh cutture was selecrcd for oprimizing 

the femotltiotr conditioru in 250 nl shake nasks.

74 

T.blc ?: Sc@ing of diftt€trt 3trio! of ,4. r'jsel' 

GCBT-1 

ccBT-2 

c,cBT-3 

c,cBT-4 

c,cBT-5 

GCBT-6 

c,cBT-7 

GCBT.8 

GCBT.9 

GCBTJO 

cfBT-l I 

GCBT-I2 

GCBT-I3 

ccBT-t4 

c,cBT-t5 

ccBT-16 

GCBT-17 

xylana3e scchari&ing 

aclivity (U/ml) 

15011 

l0+2 

50+3 

30jl 

100+2 

50+3 

32+2 

30d2 

3l+l 

3@2 

30+2

crcBT-18 

GCBT.I9 

GCBT.2O 

crcBT-21 

GCBT.22 

GCBT.23 

ccBT-24 

GCBT.25 

crcBT-26 

C'CBT.27 

GCBT.28 

(rcBT-29 

GCBT-30 

GCBT-3I 

c,cBT-32 

GCBT.33 

GCBT.34 

GCBT.35 

c,cBT-36 

75 

30+2 

35+2 

t7+1.5 

120+1.5 

90+2 

6&rl.l 

3@1.2 

ll5+l 

I l5+1 

lm+2 

lo0+2 

t95+3 

2t5+4 

2t0rl 

225+l

ccBT-17 

C€BT.3E 

GCBT-39 

GCBT-4t) 

ccBT-41 

c€BT-42 

GCBT-43 

ccBT-44 

ccBT-45 

GCBT-46 

GCBT-47 

GCBT.48 

ccBT-49 

GCBT-50 

GCtsT-51 

@vT-52 

GCBT.53 

GCBT.54 

ccBT-55 

5t!2.1 

170+l 

&+2 

160+2 

50r2 

35+l 

6{}}l 

65+l 

125+l 

45+5 

t l5+4 

n+t 

11212 

145+l 

lEo+2 

lqlr3

ccBT-56 

GCtsT-57 

GCBT-58 

GCBT.59 

crcBT-60 

c€BT-61 

GCBT.62 

GCBT-63 

GCBT{4 

CiCBT-65 

GCtsT-66 

GCBT.6? 

CCBT-6E 

GCBT-69 

GCBT-?O 

ccBT-71 

ccBT.72 

c,cBT-73 

GcBf-71 

17 

3545 

qlr4 

10!2 

55+5 

@+2 

155+1.5 

It5+2.5 

155+2 

60+t 

35+l 

t20*2 

r0rl 

50+1.5 

55+t.5 

95+l 

3Al

GCBT-?5 

GCBT-76 

C€BT.TI 

ccBT,78 

ccBT-79 

GCBT.8O 

GCBT-8I 

GCBT.82 

GCBT-83 

GCBT-84 

GCBT,85 

C,CBr-86 

GCBT.87 

ccBT-88 

GCBT.89 

GCBT,9O 

GCBT,gI 

ccBT-92 

ccBT-93 

7E 

55+3 

98r2 

I l04l 

135 +2 

60rt.5 

5012 

tuz 

48+2 

75!2 

lm+2 

l l0+2 

t05+l 

85+4 

65+3

'Thc 

GCBT.94 

GCBT.95 

GCElT-96 

ocBT-97 

c,cBT-98 

GCBT.99 

cctsT-100 

GCBT-l0l 

GCBT-102 

GCBT-103 

GCBT-I04 

19 

l2l+l 

l(xl!l 

lo54l 

68+l 

4?l1 

67+2 

10t+l 

10911 

+ iDdic.ld srand.dt dcvi.tid dort llc |IE p&dLl i.plic|rc.. nlc v.l6 diffa 

Fmat tion pqiod 4E h 

ltulralpH 5,0 

hobrion rdpdat@ 30.C

80 

Tsbl.h Sub€rq+iry ofdiftrlrn rbrins of,{. dlget t .odirS lo xyltna& 

Fqnrdivirr: 

39 

37 

IE 

7 

3 

' GCBT-35 8rvc tmidd ry'@lc Fldstid i.c-, 225 u/dr. 

Rdg. of xylurse prdrtivity 

(u/ml) 

0- 50 

50 - 100 

100 - 150 

150-200 

200 - 250

8l 

B) Optimizations in shake flasks 

ScreeniDg of culure medi. 

The culiure media (M-1, M'2, M-3 and M-4) {ere examined for tbc prcduclion of 

xylande by ,,1. ,€er GCBT-35 in shake ndk Gig!.e 2). Andg tll lhe nedit 

lested, medium M-4 gave bener prodrction of xyla@e (225.0 U/ml). Tte 

ploduction of xylanase was @imum in medium M-4 and vdied significanrly 

(p

82 

d.cEascd. The producdon of qzymc, howq6. w6 oFimun tt pH 4.5 ard 

v.n.d significandy (p

Figue 2: 

E 

:t 

s 

200 

i50 

100 

E3 

scr€ning of @ltuE m€dia for xylan se production bv ,4 

GCBT-35 in shake ilask' 

IniiialpH 

M-Z M-3 

48h 

5.0 

3opc 

Y

Figur€ 3: 

?2@ 

> 

€ 150 

.E 100 

E4 

Tim @'N study for ryl.t'r!. Fod&1i{ by,l. ,'l€. GCBT-15 in 

2a 

Initi.l pH 5.0 

Tin|€ (h) 

30pc 

Yff b6 idiet! st€rdsd d.vi.lion ,mi8 tlE ltc pd![el ftpliclrd.

e 20o 

€ 150 

E r0o 

85 

Eficcl of dificmt inilid pH @ prcductid of xyl&!s. by ,t. r!g.t 

CCBT-35 ir shate fl"*!. 

t5 6 6,5 

30pc 

Y{Drbd indicn st adld daidih.do8 the thE p.nlt.t r!pli.!a-.

E6 

C) Strain improvement by mutation 

i) UY radl.lions 

The pdenral st@in of,1. nisef GCBT-35 wds subj€cted to UV inadiation for 5"60 

min. Ninely four r 16 sof ,,t. nse' wft pick d o. th. bsis ofbiSgq clcd 

zones of xylan hydrclysis ftom the pehiplales having at l.!st 90 % dcath dte. The 

mutanrs wcrc evaluated for thc pNducrio. of xylans€ in 250 ml shak flask 

(T.ble 8). Of all the nuldt shins examined, ,.{. tk"r BRCuv!5, islal€d aftd 45 

min ofUv imdiation gave maximum enzyme Foduction 

(3)9 U/ml) and il vd 

scleded for rhe chemical mutagen ltdndt fo. fu her imPrcvfl€nr. The mulrnls 

were also sub SrouPed 

ii) Chemicd nutrtiotr 

(Table 8a) according to the r.nge of th.i. enzvme 

The conidi! of UV nutared st ain ,'{ ,,g"/ BRCuvrr wcre subjecled to lhe 

chemical mubtion and tr@l€d *ith diff@t d@s of N-mcthvl N-nito N'nilroso 

Buanidine ( 50'300 Atmt)- S.vcnly-four chmicallv develoP.d hutant sFai$ of '4 

rig?f were pick€d up and les1cd for xyldae Produclion 

in 250 ml shahe flsk 

(Tabl6 9). Ofall $. mltants invcstigat d, lhe nulant isolar.d aftq 200 Pglml dose 

of MNNC t.eatnenl, gave naximum enzyme Produciion 

(467 0 U/ml). This strain 

*6 slercd for tunhcr r@mots of Ml'lNG at diffeMl timc ittcnds &d 

a$icn€d thc code as .4. ,/ael GCBTMNN6 zo. ,'{. riser GCBTMTNG Ts wd th'n

81 

bcat.d with 200 &e/ml of MNNC .t diff.rcnt ti!i!€ intwdls (lG1|() min). ftc 

mui.nts wee picked up on the basis of clelr xyld hy&olrsis atr$ fion ih€ 

Fnipllte having ,0 % dcsrh !!c. Fuihd sccding for xyh.!. fcnnmtltion 

w6 .sied oDt i! 250 d snq|.c fllsk (T.bl. l0). Ir Tabl. loa tu tho$n d|. sub 

grcuping of ch€mic.Iy d.v.Lp.d mt rt3 rccoldiDg to dE tu8e of r/ee 

activity. The b€st nulart stnin of,,l. d3€. for xyl.mle bicy !6is (493.0 U/nD 

was obbined .fte. 200 rrglml do!. of MNNC fd 30 hin. This 3tain w$ $siSncd 

thc cod. GCBT me! md selectcd fo. tunher investigations.

88 

T$le 8: Scre.ning ofmubnt slrainr developcd through W inadiation ofl. 

rris€r GCBr-35 for xylrDu. bidyrthcsb in 3h.1rc fl$k.. 

Si 

No 

Dudi@ fGW XybD& r.livity (UfDl) 

23 ta.611 

2 

3 

4 

5 

6 

7 

8 

9 

l0 

tl 

t2 

t3 

t4 

t5 

l6 

t1 

IE 

l9 

55.9r2 

rro.or3 

6.2t3 

97,W3 

32.lrl 

59.5i2 

l(,6.3'j1 

l to0+2 

13.9t2 

7t.&2 

58.9i1 

52.3r1 

69jX2 

36.7t2 

52j*2 

56.1t2 

vt.t+3 

68.1+l

20 

2l 

22 

24 

25 

26 

27 

2E 

29 

30 

3t 

l3 

3/t 

35 

36 

3E 

39 

4 

t2 

tl 

t0 

t5 

E9 

t0E.l13 

l24.Or3 

n3!3 

4A.G:t 

t41.6!2 

19?.8!2 

E.613 

27.6t3 

61.7r3 

t572L1 

38.113 

109.311 

20t.3+3 

92.6t1 

98.811 

99.OH 

t7.3it 

96.4r 

33.013 

225.0+J 

ual+i

4l 

12 

45 

6 

47 

48 

49 

50 

51 

52 

53 

54 

55 

56 

58 

59 

60 

20 

25 

90 

76.2!X 

I ll.9!4 

t27.312 

2t7.O+2 

147.9t2 

at9!2 

t63j{ 

t29.1+2 

t78.5!2 

15.G2 

l86.li2 

t32.2r2 

100.012 

269.7t2 

214.3t2 

279_6t3 

109.313 

u1.5!3 

106.1€ 

2t.9t) 

1t2.1ll

62 

63 

64 

65 

66 

67 

68 

69 

70 

7l 

72 

73 

77 

78 

79 

80 

8l 

E2 

l0 30 

4{) 

9l 

78.611 

255.7t1 

2l5.6tl 

t69.Eil 

231.9t1 

261.5t1 

!02.311 

93.4r1 

t97.Otz 

t69.3t2 

2:21.7t21 

275.62 

252.7r'l 

l9l.oll 

l8?.lll 

210.614 

1o.ft3 

58.313 

75.&3 

t76.2t3 

t86.li2

83 

E4 

E5 

86 

E7 

EE 

89 

m 

9l 

92 

93 

94 

95 Nil 

45 

50 

55 

92 

3t6.4r2 

239.O!2 

u2.64 

I l6.013 

l8l.9I2 

tn.@l 

126j!2 

3l9.Ot3 

209.3!2 

106.4i2 

153.3i2 

170.4!2 

. Th€ do* givar to th. cdidilt luposio rs l,2t I d J/nzls. Md.nr! *@ picl.d d 

lhe bsk ofcLd zon6 of xylao hydnltlh having 90 % d..th F1c. 

I Indic&s dF sllrdard devi.tio lIorS 0E to!! porrllcl rcDti..L{ 

F€|r@trli@ p.riod ,18 h 

fnilid pH 4,5 

lmub.lid tnFnrG lo'C

T|bh &: 

93 

$b rryi.s of udd driE of .1. ',i!r GCBTJs d.t.lq.d 

rtoc.lrD lJv nnr$dm tu ryld.. tiatd.dr in 3[.rc lrdl 

Rrr. of rybe d!,iv (I,d) 

23 

A 

12 

,15 

7rtto 

l5l-225 

2263t0 

3ll-319 

. llB n|llet itrh of,L rSr E ..it d iL .od. ofB&Cw4 rd h.iDg t td 

F!d&.( of ryt@ *r eLc.d b( eid untdo trouib MNIIC .. . eu8-ia

S. No 

2 

3 

4 

5 

7 

9 

t0 

ll 

t2 

t3 

l4 

Sc@ing of nul!trt strrins dcv.loped dt@d chtnicd reltlnent 

(N- m.thyl N-niFo N{itsosuanidine) of l. tri€t BRCuv.45 for 

xylMe biGjdtheeis in shlke nskr 

(Fd,nl) 

Xyla.arc aclivily {U/ml) 

28 50 85.3i2 

76.7!2 

93.6t2 

77.712 

74.5!2 

50.612 

43.4!3 

26.2t3 

82.3t3 

109.0!3 

209.5x3 

43.513 

15.3i3 

26.2t33

t5 

l6 

t7 

l8 

t9 

20 

21 

22 

23 

24 

25 

26 

u 

2a 

29 

30 

3l 

95 

19 100 

n.v2 

45.@2 

98.7t2 

t26.9!2 

I t0.312 

45.2+l 

92.Oll 

96.Oll 

69.lrt 

t23.3+l 

t I l.qll 

t29.7t2 

3l6.3rl 

426.8' 

2l6.tt2 

239.82 

E6.312

14 

35 

!6 

]E 

39 

:t0 

4l 

42 

43 

44 

45 

46 

96 

4? l4 150 

a7.ot2 

196.9t2 

234.7{. 

253.6!2 

392.Or3 

267.2t4 

58.713 

o,G3 

395.2p. 

120.3+l 

58,7fl 

375.9t1 

l55.6rt 

275.ttl 

2t9.3+1 

152,3!l 

27t.1t3

49 

50 

5l 

52 

53 

54 

55 

5E 

59 

60 

6t 

62 

64 

65 

91 

E N 

315.2+3 

427.Ot3 

3299t3 

3n.7x3 

I l9.Ei3 

26r.&3 

53.5r3 

317.6+2 

367jr2 

120,0x2 

253.6t2 

225.2x2 

26,3t2 

139.0t2 

35t.lll 

217.8+l 

209,7tl

66 

68 

69 

10 

7l 

72 

NiI 

9E 

250 

300 

167.0t2 

392.5t2 

216.613 

t92.8r2 

r7]3', 

367.0!2 

32l.t13 

$.24 

59.6!2 

. &Erbatiotr rqnpqlnr 30pC, ptt 45, fanai.rid pqiod 48 L tinc of MNNG 

I hdicats th. srdd$rl deviation anqU rhe thtc peatt.l Eplic.res.

99 

Sub grouping of murdt stnint, dev.lop€d ihroud ch€mi@l 

ttqltMt (N-m.thy' N-nito N-.itro6ogumidit.) of ,{. rigef 

BRcuv{ for aylanae bi$}rlbesi6 in shrk flask' 

28 

t4 

t6 

8 

R.ngc of xylan&re activiry (U/n'l) 

l5-105 

t0G195 

l9G2a5 

2EG375 

375467 

. Th. nut nc wft pickcd d rh. b6is ofbiggd cla ryt!tr hldety.is 6B 8w rhc 

pelriplals having 90 % d4tn 6tc ed bqhg th. h}?a produar ofxyl@ wd $sign d 

nF code ,.1 da./ GCBTltEe

100 

Table l0: ScMins of nurarr staids devcloped throush diffcr.nr line 

int Fals of N-merhyl N-.it o N-nitt6oeu|nidine trea1rn.nl of,.{, 

,,ie. GCBTMNN6.u fc xylmre bio6ynth6is in shake flsk' 

S. No Duration ofMNNC Xylde. &tiviry(U/ml) 

I 

2 

3 

4 

5 

6 

7 

9 

l0 

ll 

t2 

ll 

l4 

15 

t7 

18 

tl t0 

l5 

t26.tt2 

200.2r2 

t25.r!2 

95.0r2 

60.7!2 

703r2 

| 16.911 

73.E+3 

72.tt3 

319.511 

2t7.2!3 

250.0r3 

326.5!2 

2l9.8rt 

367.5+l 

392.3!l

l9 

20 

2l 

22 

23 

24 

25 

26 

21 

2E 

29 

30 

ll 

J2 

33 

34 

35 

36 

4 

6 

I 

Nit 

2A 

25 

l0 

35 

40 

l0l 

109.9j2 

t26.0!2 

94.4r2 

93.3+3 

157.6+3 

ta7.8t2 

63.li2 

4?3.713 

357.6t3 

269.5t2 

323.313 

147.4t3 

372.0t2 

369.9+3 

493.W 

324.8t2 

2r3.1t2 

3t6.3+l 

. Incubltion tcmF.lrc 30 !C, pH 4.5, fdrdtdiotr pdiod 4a b dm. of MNNG 

it Indicat6 ihe stddard deviatid dnong th€ tbr€. pa.attct r.ptical6,

t02 

T!bl. loa $b goopirg of nur4r !t!iD! &eekDcd dFwh ditrcr. litic 

inlervah of cftmicl lrllltn.nt (N-methyl N-nito Nnitososu.nidinc) 

of /. ,nser GCBTMNGa for xylea$ 

bicyndrcaiE in shlk flrd.. 

r6 

6 

P...ng€ of xyl.lle actiMty (U/tuj) 

@160 

t6t-2@ 

26|.3@ 

361-460 

461493 

' Th. h€s nut d srrh of ,.t. ris' GCFTINIG{ br ry's|e bi6Frt6is s.! 

ai8'cd lhe @

103 

D) Subm€rged fermentrtion in shake llasks 

R te ofxyhn.se synthesis 

In Fisure 5 is shosn the hte ofxylanlse synthesis by slecied hould in shake 

fldk. The culture w6 incubarcd at 30'C for 72 h. T1E prcduction of enzyme was 

mcrc.sed wnh rhe i.cEse i. rhe incubsnon period aid @ched muimum (494 

U/Dl) 48 h aftd inoculation. Funher increde in the incubation pqiod resnlied in 

rhe d€cresed productivity of xyl&sc. 

Th€ linetic paramoEic valu€s such s Yr", Qr .nd Q, atso rcvealed rhe 

significancc of xylanae prcducriotr by rhe mua.nr sFah th& rhe pftnrd sbain of 

A. niger ('table ll) Thus, incubaiion pe.iod of 4E h ws seleted for the 

producion of xylanase by ,r. nige' cCBTMNNc.ro. 

EflectofiDiti.t pH 

Erer of dilTemr innill pH (4.0 - 8.0) on thc preduciion of xyt!.6e wrs srudied 

(Fig. 6). Maximln production ofxylatrAe (491.8 U/ml) was achieved when the 

pH of femenlation mediuh was .djusted ar 4.5. As the pH of lhe m€diub was 

Dcrcased or decrcacd ihe prodlcrion of xytse show€d a dcline. The 

prcduciion of eElmc sr pH 4.5 vdicd siSnificantly (p

Efiect of dilTerent nitrogen sources 

104 

EfT.cl of lhe addition of diflq6t nitotcn sources such d u@, ,€asl €xtrac! 

pol'?.pto.. or m@t exFact was studicd on lh€ prcductim of xylmde by ,{. rirger 

GCBTMNC I (Fisue 7). The prcduction of eMyn. wd found to be muinutn 

ed hidly sisEi6@r (p

Figw 5: 

a 40o 

i3@ 

105 

Rlt€ of xyl.nase production by ,a. ,8€r GCBTNT3o in shake 

ldat l tH 

30 /|o 

rnE (h) 

4.5 

30.c 

Y.ftr blts indic.t 3t rdlrd d.viation arnong th. $e p.!.ll.l Eplirsl€s

l(b 

Tablc ll: Coqdiron of tifttic !Im.t r! fo (y'a.. pmthclion by thc 

p[lotal .tdr of,l. dr€r CCB'35 dd ftue GCBTM$&.30 ir 

rh.Lo tr .t 

P.rdl fi:i!G@35 

GGr.{rc 30 

5.51 1_39 

o?8 t.0t 

0.14 0.la 

0.85 1.4 

q 1,08 0.19 

q 0.!5 lJ9 

. Kin ric rr.na.$: Yrd - U arrd. prodlc.d/g 6ut ed.4 Yr6 = U .nz)@ 

F.&..d/t {t-n @@.4 Yrr - | c.lk/8 ,6-e dilit 4 q = U dF. 

F!d!.d/ai!!/h, q = s.!lccw!r#44! q-r€|l to.drlit /L

Figtrc 6: 

= zl{r 

? 

E aoo 

x 

g 

s. 200 

t07 

Etrsl of difrercnt ioirisl pH of tbe culbr. nediurn otr th. pmduction 

of xyra@ by /. ,rjr€r CCBTyIF30 il sh.k fl&b+ 

5.5 6 6.5 

Inilld pfi 

,18 h 

300c 

Yfr! bs indicde fl.|rd.rd d.viadd mong dE rhFe plra .t Epticftr

E 

:> 

400 

300 

200 

t08 

Efr@t of difr.ilol rib!96 so|l'w @ drc plodetiod of rybnse by 

,,1. ria GCBTmc30 h shl6 as!k. 

r.idrl ptl 

Yeaslo(bd Pttyp.pbno 

Ni!€gdt .ourc€s (1 .O 

/ah 

4.5 

3opc 

|v/v) 

'6, 

Yfr b6 indicd. !rad.!d

Figur E: 

E 

a 

i 

t 

/rco 

m0 

2N 

t09 

Efrcd of difrdat colcdtditd of ft.t .xt d oo {Ic prod.ricn 

of xyko& by .{. rrjg"' GCtsTM3o h ltrtc flxls. 

'f.5 2 2.5 

M..t .nlad oonc, (%, wfu) 

48h 

4.5 

30.c 

Yff bd iDdic$c *od.d d.vi&d .do8 rbe rhi!. podrd rqlic.r6.

ll0 

Selection of rgriculturll by-producl rs srbstrate 

Different agricultual by-prcducis such as wheat bdn, rico bnn o. rice stmw 

(gnnded) as carbm source sere siudied for the produclion of\yt@6e by A. rig.r 

CCBT Mi\c.r Oable 12, Figure 9). Thc @ncentralion of c&h by ploducl vuied 

frcm LG4.0 % (dv) The mdinlm cMyme productron (481-4 U/nl) *.s 

obtained wher wh€t bra al a level of 2.0 % (w/v) was used ed edyme 

prcductioi signilic.ntly varied (p3.05) ih.n frcm lhe olhe. subst ates. Rice bon 

and rice slrdw g.ve conpanlively less xyldnase activily. Rice srr.w (grinded) was 

found io be the ledl effective ca$on source, TterefoE. wh@t bnn w6 opiimized 

Elfect of difierent nitmgetr sources 

Th€ effed of difiml inorg.nic nnrog.n so!rces in compaisn with thc conrol 

wus rcsred on the production of iylanase (Table l3). The inorg6ic nitrcgen 

soulces such as NaNOr, NH4CI or NHdOr were added to tbe femcnt6tion 

rnediun .l 0.05 ' 0.20 % (w/v). Among lll the nit osd sour@s tcstcd. xyhnasc 

prcduciion ws hishly significart wha NaNOr ws used 4 .trc8d souEe 

(502.4 U/ml) ar a lqol ofo.l0 % (/v). Tlc Nrlcl at.level of0.l0 % (*/v) wss 

almon equally good (500.5 U/dl). Th. cffect of nitrosen $urces fo. incrcasing 

rhe fnnsal cell activiiy ro prcduce xylanas. .sn be summanzed in the following 

orde4 NdNo, > NtLCl> NHiNor(Figure l0).

lll 

Efi.ct ofdlfistra ohod.!. rorn . 

Dfforcnt phoQhrre sol|lq .!.h !| KlltPrO4 KrI{PO. or N&HPO. wsre 

corFied eidr lb. @td f6 dxim'n rt.!|!. ..tivity (Ilblc l4). Tt 

oonc.ntation of.lch .our!. nngcd ftfrl 0.05 - 0.15 % (wto). Th. connol gave 

372 U/d ry'orrc &livity. tlowva 0.1 % (#v) KH+oi amo.r.d 'uibm 

enzym. &tivity (513.2 U/n ). Oth!. ptorphd! rorEca 8sv€ l.€s.Dzync aorivity. 

N.rHFO.w ldrd iobc th.ldroffrciE d.[ Lv.lr (Fig I l).

l12 

Table 12: Emect of difreml agricuftusl by-ptldwa q fie ptoductio of 

xyldEs. by ,{. &igal CCBTNa30 iD tht*€ flak! 

Differ€nt aSrtculfir6l by-Fodtcls & 

h.ir mc (94 wv) 

F@olrlio! Fdod 

48 h 

Initial tH 

4.5 

Incubsdonr.npdahft 30'c 

* Indicarc allldatd d.vi4ion lmong l[. rhE DdElel €9li6L3 

XylanLre &tivity (U/ml) 

t.0 314.0!6 

2! 4El.4rrl 

1.0 198.5r4 

4.0 

345.0r5 

1.0 29E.0r2 

z.o 

1.0 

4.0 

Rice suae (siDd.d) 

.t02.5r5 

290.6r5 

22t.8r7 

1.0 2t2.ztE 

2.0 

252.5t4 

1.0 220.6+5 

4.O 

190.2r5

Figur.9: Conpdison of dirwnt .Eiculturul by-prcd('. on the production 

of ryls.ase by ,1. rks CCBTN-3o in shal. flalts' 

Em 

3 

3 

&nrta tflodd P.0 rr rt 

4Eh 

4.5 

!0pc 

Y4m' b6 'ndic& shd.d d.viar@ uog rh. thrc. p.rd l€l Epli.c

l14 

Trble Il: Erel of diiT.Mt inorSeic nnroSen sdrc6 d rle pro&crion of 

xylaru€ by ,1. trri./ GCBTWNG-30 in sftatc flask. 

Ioorgdic nnrogcr source & their conc 

(%'*t") 

0.05 

0.t0 

0.l5 

ozo 

NI{CI 

0.05 

0_t0 

0.15 

0.20 

NH.N03 

0.05 

0.10 

0.15 

0.20 

. F.mdtrrion pdiod 48 n 

Initial pH 4.5 

Incubalionthp€raru. 3O.C 

t Indicar. si.nd&d dqidi@ dont rhe rllE panltet rytisl6 

Xylanas€ activity (U/ml) 

242.5t] 

4E2.Oj4 

5O2:4!4 

418:5L 

440.016 

496.{}jt2 

599:l+r1 

oeo.y.!, 

4.23.8t12 

3tr?l!! 

31gji1 

320.6t7 

296.5!9

PiSm l0l 

Ead 

I3.{ 

hiri.l pH 

ll5 

Conpai@ of diffdndtl 

produotiotr ofxylan & by,1. ,iigel CCBTliM-3o 

60Cn r*r* linb*d dldt! almiB dn 

Olric 't!or &i- P.l ta, rlu 

43h 

4.5 

30pc 

Y.mr bs rndic.rc 3redad d.vi.rio smons $. dm. prdl.l Eplicar.!

l16 

Tallo !4: Effcct of ditr@t phorph.tc 5o@3 o dte prcdu.tid of xylanase 

by,1. r&€/ GcBTNa3o in d',&e fl6Ls' 

Difrfied phosphlt. s'rc & oFir 

crnc. (vo, w/v) 

Kflfor 

0.05 

0.t0 

0.15 

K:HPO1 

. F€llmtdion paiod ,A h 

Ioirirl PH 

4.5 

Ilcub.lionLDFlr@ 30'C 

1 lndicai. ridd.rd devidtion dong rh. thN Flallel Eplic*es 

XylaIale !.rivitt (U/d ) 

312.0$ 

487.018 

513.2r8 

507.srE 

0.05 396.44 

0.10 400.5r2 

0.15 390.6!2 

N.rHPOa 

0.05 112.2!2 

0.t0 156.5r5 

0.15 80.5i4

I 

Eo 

bilial pH 

l7 

CooFrim of dift{at phcph& sdlt!€. @ dt Fo.nlciid 

xyle.!. by ,1. ri8"' ccBfrbro-3o ir st kc n$|o. 

gd.&rdffai tt dritrdq.r O.drffigtr 

d'!d- dry. *l.Aa 

Ptu.L.dB0lX,l,\, 

{ah 

,t.5 

30.c 

Y.oM bd iinnr!.brh d.yilid @lg rt lbE Fr.lLl npticc

ll8 

Effect of differedt colcentrrtiom of mlgn$ium sulphte 

The etTecl ot differenr conccnthtio.s of M8SO4.?HrO (0.01-0.05 %, w/v) was 

studied ii culture medium for xylanasc production by,.{. "ls€l CCBCX-2o (Table 

l5). Maximun p.oduction of xylanase (529.5 U/ml) wds schiev€d when 

femenlaliotr medium was supplemcntcd wirh 0.03 % MssOa.THro. Further 

ircrese or decrase in the corcetrration ofMSSOi.THrO r$ulted in th€ dec@s€d 

prcduclioi of xylanase. Xylane production .i 0.03 % of MgSO..7H,O 

concenration w6 fou.d lo bc highly sigiifidnl (P9.05) and thus used 6 I 

magnsium source for furthd studies. 

EfIe.l of dillered calciudr sources 

h Table 16 is shown rhc d.i. rcg.rding the cfidt of dilTemr concorrafon of 

CaClz.2HrO on rylana!€ prodlldion by ,.1. ,Eei Th. conce.tFlion w v$ied 

fron 0.05 0.25 % (w/v). Tt€ prcduction of cnzyne wa! folnd to be |wioum 

(532.6 U/ml) ar 0-10 dZ (Vv) dd vari.d signili.antly (P4.05) from olhq 

conco.irado.s of ihe caclr.2Hro. Funher i.cree in $e condtfarion of 

C.CI,.2HrO rcsulted i. the d€ci€as€d prcductio. of xylanase As 0 10 oZ (w/v) 

CaCl:.2HrO gave daxinum pioduction of enryme therefore. it wss used as a 

calcium source fo. turther studics.

l19 

EfT€.t ofdilfered corccdtr.dolt ofTwe.r m 

Thc efleo of diffcmt coFnll.rioc of TwecFE0 (0. 10-0.40 yo, v/v) wa sndied 

in culturc medium fo! xyldEse prcductid by,1. rtge' CCBCX-2o (Tabl. l?). 

ttaximum production of xylanase {542.2 U/nl) wd lchievad when fmdratio. 

rcdiuh was suppl@cnted wirh 0.20 % (v/v) of Twm-80. Funhcr i.@sc or 

dccMs in thc co!@tr.tiq of ln|@-Eo rGulcd in rhe dc.tused ptldetion of 

xylo6c. Xylanase p.oduction !t rhk @ncdt adon ws fomd !o be higl y 

significant (p 9.05) ard fius optimiad foi maximal xylln.s€ prcduction.

tm 

T|bb 15: Btr ct of di&..oi cdc.@ior of d.g!..i!n tllrtd. or S€ 

proddin of ry1re by,a. ''!t' C,CBTmto-3o i! d& lbb' 

WSo+ Iro (9.' wlv) Xte... rd!,ity (U/nl) 

Cdol 3lE.0ia 

0.01 

0.(I2 

. Fdadioo Fi'd ,18 i 

IliddDlt 45 

I!4lbrlion t np.(rte 30pC 

rldin i-d!d drdrio @t ti. b. !-rld ryli.rd 

(U,5r3 

gLar3 

0.03 5T).5tt2 

0.04 502.41E 

0.05 8.@

l2r 

T.Hc 16: Eftct of difrrd c.trc.ot tli6. of c&iuo cbL.ib o rt 

prdrcri(n ofxylrc byl. rlr GCBTn o"30 h .brc n .br 

Clclr.2Hro (96, w/v) Xylne .6dvity (Irtrl) 

Contol 36..(ts6 

0,05 4ft5*7 

ol0 532.&7 

0.!5 510219 

OA 495,5du 

0r5 4nst2 

Fd.ddid!.iod aEi 

bid|l pU 4.5 

bcthdi! bprdG 30.C 

1lldierdrdd6,id@fuogtt.d|!.Fdtdl!?tic.B

Trbh l7: 

t/2 

Eect of diftGrt cdo.ddioE of TS!.GEO od ft. Fo&.rim of 

ryluta by ,r. ,|&!r GCBTwo-3o i! .h& ntdc. 

T"!co.8o (%, v/v) Xy'llli. activity (U/'[l) 

Coltol 390.O!3 

ol0 326.5t4 

0-20 5422!5 

03) 52j,.M 

0,10 504.6d5 

F bdioGiod ath 

Lili.l pX {,5 

Indibdi6i.q.e! !0.c 

rLdicc cshrl d.vi&r mg 6. b. t-[.r r?|i.s

l2l 

IMMOBILIZATION OI' ,{. A{6AT FOR XYLANASE PRODUCTION 

Reoerted batch culture 

The xylanae fem€ntation ws @ricd out by fivercpeat€d b.rch dlturcs using 

immobilized conidia of,{. ,,sel in th. sodim alginde dd pol}rrcthdc form, 

rcspectively (Figur€ l2). wher,.1. ,,aer ws immobilized in th€ sodium alginat€, 

the prcduclivily of xylrnae g.dually decE.sed (f.om 514 U/ml to 306 U/ml 

only) from 3'balch on*lrd to thc 5" b.tch. Howevd, ih. prcduction of xyl&6e 

by,r. ,taer immobilizcd in lhe polyuralsc foam w6 incEas€d (al0 U/nl) in 2d 

baich, thereaftd in 3",4'" rnd 5'" batoh cllture, the production of xylease was 

sisnilicantlr d€.s.d (31G234 U/ml). Sodium alsiraie which s.ve better Esuhs 

thatr rhar ofpolyuErh.ne f@m wa sl.cted for tine course profile. 

R.ie ofxylanrsc production by Inmoblllzed,{. uig?r 

ln Fisurc ll is shown the Et€ of xylsase p'ldlchon by imnobilizd slEin of,.{. 

,,?e/ with sodium alginsle. The fennenbtion medium was incubated at l0"C for 

l2-96 b. The production ofxyleas. was in..esed wiih ih. in@ase in i&ubdion 

p..iod and found mximum 48 h after in@ulatid. ,{3 lh. incubadon p€riod wls 

tufther increred, thc substratc consumptiotr also incr.rse4 howevf the 

prcdnction ofxylan.s€ show€d a d.clinc.

figG l2: 

' 

t24 

Prcducrid of xylde fq five rcp@ted batches 

immobitizod stlain of,.4. ,i€:a CCBTMNNa.3o 

rlgbaie md polyftlbo. fom. 

. bn.F*. dr4b.fm 

Y b6 indide the lbnd,rd .rcr fiom dd vslu.. 

TmFdrrc ltrC, pH 4.5, fmdr'doo D..iod 48 h

125 

Figu! 13: Re of {ylt[|!. Fttllcdm ty imobilizcd rya.lb 6 A nigq 

CCBTMIM9 in sodium dsiD't ' 

5|I' 

:'1oo 

i 930 

! 

a2!o 

10 

o122a${tot2l,glill 

TdDdem lGrC. pg a.5 

Im€ O)

t26 

RX.USE OI'MYCELTUM FOR XYLANASE PRODUCTION 

The prcduciion ofrylnus€ by trp€lt d u* ofmycclium w{s sMi.d (Fig!rc l4). 

Tho mould mycelia of e&h f.tlMiarion wft s.pdar.d ilom ferlmtcd brcth by 

ccnEitu$tion. Th€ my@lium thB obtain€d &om pBios bllch vE lusfeftd 

to Aesh sGrile m.dim mrlined in rhake flak. Each f.mmlario wa run for 

72 h- By r€-6ing lhe nyc.lia, fisl b.1ch of xyl!6e wrs bener in tmt of 

xylsrs€ produdion eha comp.rcd to thc iwo subscq@l bttch6. Howdd, th. 

hidst xylande activity (424 U/ml) obLeined from fi6t batch wd low.r than lhe 

origitul b.t h culhrc. ThB, i. fte pl*rl study, dle F@ of mould my..liun 

did not give flcouragiDg r$ulls.

Figuc 14: 

5(h 

,r50 

.to 

350 

:. rxl 

!20 

1(r0 

50 

0 

t27 

Reuse of mix.d mould my@lia for xylanar€ production bv 

imnobiliztd ,1. ,ir.. GCBTMNGT.' 

i5 i75 a 

+H|.rt +rrd!.|n +rdb 

I ybNildic.t tb. $an Lrd .M toE n .! ttlN 

T@Dcanfr 3dC, pH 4.5, f.rn aid Frtod 43 h

E) Solid-state fermentrtion 

Selectior of substrlte 

l2E 

Different aericullural by-producrs such s rice skaw, wheat brm, rice hDsk, wheat 

st@w, sunflo$er meal, bagasse. soybm meal or lewspaper (10 8/250 dl flask) 

ws evalualed for lhe production ofazyme xylalrN (FiSure l5). Tle mdimum 

produclion of xylanrle (1850 U/g) was obbin€d in wheat brd nediun while th. 

ofier snbslrlt6 gave rclatively less producdon ofxyldase- Thus, ph@t bran w6 

selected !s subslrate for the prcduclior of xyldAe,.{. ,€ef GCBTMNNa-10. 

Depth ofsubstr.te 

D€prh of subsrmrc dd irs pociry have gred influence on rhe produclior of 

cnzymes ir aercbic iemenlalions. rt€ efecr of ditlerent depths ofwhql brb on 

xylMe productioD by ,4. ,/aer cCBTmc-10 ea srldied (Figw 16). fte 

rhicknes ot the substEte tuged fiom 0.2-l-0 cm (5-25 8) in 250 ml E.lenmeyer 

flask. T1'€ maimum enzyme producrion (1852 U/g) was obrain€d when depth of 

the subslrale e6 0.4 cm (10 g of subsrdte per flsk). The p.oducrion ol enzymc 

w6 found to be m.ximum ar 0.4 cm depth ofwheat btu. Il vdi€d sisnificanrly 

(p

t29 

U?s) ..d hiehly signilicet (p

130 

sienific.ntly de'!ed. Hae, 30.C rcmpenturc was sclected fo. rhe produclion 

of rylanae in solid-stat! fmcntatio.. 

Etfect of carbon source3 

ln F,gurc 20 are sho*n rhe ereds of the addilion of ditTerenr ca6on sources (1.0 

%. w/v) on lhe xylanase synthesis by ,4 r*e/ GCBTMNNC_$. Of all the carbo. 

souoes tesied, the prcdu.tion of @)inc wd found ro be oprimum in rhe p.es€nce 

of staEh and vaied signifi€.dy (p

l3l 

ot .b.d r. 02 % 16'.1 of (Ml.)bSO.. Futa iiq* io t .,Ftdr.' of 

(NH.)'SO. 8w. idisrifcd rld.dd of4rd5, rto|t, 6. rddirio of 02 % 

(NH.bSO. to lrk t'E D.di|!! i,* slccbd ft. Mnm F!&.ric of 

xylna!. by l, rtSrr CCBTMe{c-ro.

FiguE l5r 

g 

Z $.o 

it 

.l 

t 

t3z 

Scrc. trg of eltdtatc for d!. productior of xyl.n.l. by ,l. t'3"r 

CCBTma-3o i! solid st!t! f.rm6tition' 

Ilili.l p1, 

t .,,i.-,,j**,1,--, 

72h 

4.5 

3{pc 

Y{nor bd indic.l€ sl&dsd &vildon .nds lb. tbe Frdl€l replical$ 

* 

I 

t 

I I

Fi8lre 16: 

g 

= 

133 

Efrc.t of difftrc d.ptllg of sl|G{i b'|n or |nc p'lducrion 

xylar*c by,{. r,83r GCBTmo-3o in solid si.rc fermdarid' 

bitid pll 

D€lan ofwn€at bran (qrl) 

12h 

4.5 

30'c 

Y-@baB indictt sr.r.t&d d.vi.ri@ amng rh. th6 p.r.lLl Eptic.tcr

-g 

2500 

1500 

10@ 

134 

Eff@t of difrcMt diluent6 on the producliotr of xyla!6e by,.{. rrg.r 

CCBTmc-3o in solid stste fmenbtion. 

0.1N HCI 0.01N HCI Dislill€d Tap$ar Miner€tsal 

sol0Uon 

Initid p}| 

12tl 

4.5 

Dlluents 

300c 

Y-€mr bs indic.t. $and{d ddi.rid Imtr8 0E drd p.hltet cpticaiF.

Fignr! lE: 

q 

a 

$t* 

i 

500 

135 

Rr!. ofrvht$ Fodrdio tt ,{. ,48!t GCBT'{€-3O h lolid tt ie 

Iritid DH 

a0 48 56 64 72 80 88 S8 104 112 120 

tht O) 

45 

b.utdio!8nlqrnlr! 30.c 

Y

g 

-a 

t36 

Effcd of difrcrcnt iMb0tid temp€stuG on th. prduction 

xyhneby,1.,rlge'GcBTilrc3oiDelidsr . fenl|enirlio!. 

Fmalrlio Fiod ?2 h 

biri.l pg 4.5 

Y-@ bs iodic.r. srddlit daialion .|Mg ilE thF p.nllet EDlicrre. 

30 

of

Figlr! 20: 

: 

I E 

! 

t 

1500 

1000 

l3? 

gtrc.a of ditr€rlot citon loutcec oD ttc Fod|.tio|l of xybtte by 

.,{. rriger GCBTaNo.30 in solid Btlic fcrn€ntliid' 

Ittit'ral ps 

Fu.L.. dr6. aldl 

Ctrbd4@(1.0%,w,!) 

72h 

1.5 

3ec 

Yftr bs indic.r. sito

FiEurc 2l: 

g 

!. 

:E 

s 

25oO 

z)00 

t0(r0 

138 

Eff.ct of difrcm! conc.nFrtion of scar.h on tltc pldlcdott of 

rylam$ by,,l. d8a CCBT[lltG-3o in slid sttlc fematdion* 

Inili.l pH 

134 

Conc€ntdton of araEh (%) 

72h 

4,5 

. 3(Pc 

Y-@r bar! indicat€ str.ddd dwiario. mong thc thrc. pdal€t r.ptic.rd,

FieuF22: 

6 2000 

? 

E 15OO 

!!_ tooo 

500 

t39 

Efr..t of differcnt nitog€n 3ol|rc on th€ produotio! of xyrare by 

,r. r,i.r GCBTmaro in solid rtioc f.rmcnblioia 

Initi.l ptl 

It*t4P30a ullfl@ 

tltlgr| !our€. (0.2 %, wrv) 

72n 

1.5 

30ec 

Y

1,() 

Figurc 23: Effect of diffcmt €oncentraiion of 

Plodlctiotr of xyLlr& by ,t. ,i8€r 

fm.ntation* 

g 

; 

:E 

3000 

2m0 

1500 

1000 

bitid prf 

0.1 0.2 0.3 0.4 

Con6€ntEtion ot amlnonium sutphat€ (%) 

12h 

4.5 

30!c 

allutroniun .ubhat€ on th. 

OCBTxrr3o in ooli(kttt 

Yftr bG ndic.lc .rodad dlvi.tior .tmg rh. dlE Fdrd Epticc. 

0.5

F) F€rmentor Studi€s 

Typeoflnocrlum 

l4l 

In subncreed fementations. the ilTo of in@utum used Sready influ.nces rhe hle 

of femenlalion. Itr Figure 24 is shown rho cffeci of two inocula on lhe 

b'osrolhesis of xylanae by ,., nrs€' cCBTMmc.l in stined femtrior. Conidial 

suspensron and ves€tative c.lls (developed in shake flaskt w{e employed 

sepdately for inoculation. wlen conidial inoculum was nsed. the prodnclion of 

cnzlme was 510.3 U/ml. The prcducrion of xylane sd grearer (694.0 U/dl) 

wh€n 24 h old pre groM vegersrive ccls ofrhe moutd wcE used as an in@ulun. 

The tungal celh werc very acrive during logalirbmic gro*th phse_ 

Size ofinocultrn 

Th€ €rect of differcnt sia of veg€tative inoculum on the prcductioD of xytanas. 

by,.4 ,iser CCBTMMNC.Tj is shown in Figu,e 25. The maxibun amouit of 

xyldde (694 U/nl) was produc.d in the medium coni.inins 3.0 % veg.rarive 

iroculum. Th€ prcduction ofxyta.de w6 (339 and 509 U/nt) whcn rhe size of 

v.Setative ineulun mged l_0 and 2.0 %, @speciivety. tr ircftG.d with 

in.rcding rhe size of vegerarive in@ltun ro Lo %. but increasc in size of 

vegetairve 

'nculum beyond 3_0 % der.ced rhe xyta@e bioslnth.sis. IlErcfoE. 

1.0 % size ol in@ubn w4 oprimiz,cd for xyloase bicynthGis in rhe srired

f, ffect of rgitrtlon lnf ensity 

t42 

The cffed of diffeEnl agilstion otes (150-250 rym) on rhe xyldsc biosynrhesis 

by,4. 'rs.. GCBTMTNGIV6 canied out i. the srjred fenenror (Figure 26). Th€ 

tuximum rll.n6e acrivity (694.31 U/nl) was &hieved when rhe lsiraton 6ic of 

lhe fementor ws kepi .r 200 rpn. Funher incrase in agrration dec@r€d 

xyhnde biosynrhesis. Hence, .siraton dte of 200 rpn ws uscd for tudher 

Effe{t ofreration rrte 

Tlt biosynrhGis ofxyldase is an aercbic f.nenralio. p@ess_ ThN, th. suppty 

ofoxysen ior dissotv.d oxys€r) to 6e mould.ulturc is of grcar importdc€. In 

Figure2T isshoM the efltrr of supplyiog an to thc f.menio. ar diflftnr aedrion 

hres (1.0-.1.0 wm) on xytanase bioly.lhdis by l. /!s./ @BTMnc& Tbe 

cn7)r,c adrvny w6 maximun (781.9 U/mt) when aehtion E1e w4 kcpr at 2.0 

wfr (0.7 % dissolved oxyS€n). A d@rcase or funhd incrcae in rhc adarion Ele 

dec@ased rhc enzyoe actviry. 

Rai€ of xylanase biosynthesis 

Time course femenrarion of xyl[sse synthesis by ,{. ,igel CCBTMINC.i in 

stined femenlor wos invesiigated (Fi$rc 28). The fermdbtion ps caried ou1 

for96 h ate. inoculation. The amounr of xylanse prcduc€d,24 h afier inocutation 

sas 321.6 U/nl dd ir increaed with in.r@e in ihe incubation pqiod. The

l4l 

maximum xylahse activity (78t.4 U/ml) was achieved,60 h afler rie inoculalion. 

Furlh$ increas. in incubaiion time, did not enhance xylanase biclarb€sis. Th€ 

oprimum timc p€riod for cnzym. fmation wss hence 60 h after rhe i.@utation. 

EtT€ci of dln€reDt pH 

In Figu.e29 h shown rhe effeci of diffftnt inidalpH (3.0-5.0) on thc bioslrrhsis 

ofxylana* by,{. ri8?r GCBTMMNa ain tbe stiftd fementor. Maxinum xylanse 

actvny (El0 U/ml) was achi*ed wh€n inilialpH of rho fcmeniation mediu,n ws 

at 1.0. Wten the pH was indesed, rh. p.oduction of xytanas. ws defiea$d. 

TleEforc, iniri.t pH 3.0 *s selccred for tunhq exp€.inen$. 

Elf€ct of dillerenr redperarures 

The prcducrion of xylanas. by -.{. ,&a GCBTMMNG.h was caned oui in ihe srift€d 

fcmenror al diflercnr renpenrrc (Figure 3o). wh€n rh. tempeFtur. of 

fmem.tior hedium was kepr d 2j'C. rh. prcduct,on of xylee ws 652.6 

U/ml. Maximum xytare p.oducrion (8ll U/ml) was obrained when ihe 

tempehtuE of the nediun B majnrained ar lot. Whd thc remp€rorue of rlE 

femenr.rio. mediuD ws tcpr ahole 30"C, prcductior of xyl.rdc w.s sMrly 

dec@ed. Herce, tcmpeBtue of 3o.C ws sclered for funher experimenrs.

Filur. 24: 

EO0 

700 

, 600 

5 5oo 

Flm 

d 

I 

E roo 

g 

200 

100 

t14 

Efr..t of t)Dc of itr@ulu o rb€ bi@FftG3ir of xyl.I|t. by,{. 

&its GCBTnca i! iirrcd fan rtor. 

Ilili.l pH 

lmoium (4.0 %) 

rt8 b 

5'0 

30ec 

vcg.adiE

Figure 25: 

E 

.! 

800 

700 

600 

500 

300 

200 

100 

145 

Effet of diff*nt conc.nlElion 

bicynth.sis of ryl.nec by ,1. 

lnnial pH 

Level of v.g€talte inodjum (%) 

of vesclative ino.ulun on thc 

rtsel CCBTMNNG.a i. stirrcd 

48h 

lfc

t6 

Fige26: Efrlct of.sitdiE id.odly o S. biayd..it of:ylre by,{. 

rigr OCBTN&!. b rth.d M 

f, 

16 ro 10 2$ 

raFr r-rr ftnt 

. Fc'@id.EFid 4El 

Ilnidplr 5.0 

hcubrtidldpccts 30.C

Figure2T: 

800 

600 

500 

100 

2gJ 

0 

t47 

Effcct of aeradm nte o. the bioslnilBh 

GCBTmcjo in $iNd fcnnqioa 

InitialpH 5.0 

t0.c 

of xyl$se by ,{. r/S.r

Figure 2E: 

; 

:E 

900 

800 

700 

600 

500 

400 

300 

2W 

100 

0 

l4E 

Rale of xyhNe biosrr$dis by ,{. ,i8"r ccBT||rcro itr lticd 

.86072U96 

Rat€ of ryhn€3. biosynln€sb (h) 

48h 

5.0 

30.c 

r(lE 

1m

149 

FiguE 29: ElTecl of differeni initial pH 

biGynlhsis of xyldasc by 

E 

3 

a 

900 

800 

700 

600 

500 

400 

300 

2@ 

100 

0 

of fementltion medium on the 

,.{ ,,ser CCBTmcr in stined 

3 

ldt6lpH 

Initi.l pH 5.0 

3opc

Figure l0: 

150 

Eflect of diflerent 1enpe.atures 

,{- ,t8". GCBTimGI in srinEd 

on th€ biosyntbesis of xylanasc by 

48h 

5.0

t5l 

Pmduction ofrylrl.le by fed b.t h rylt m lo rtlrr€d f€rD.rtor 

The ,(ylalasc f.rentltion w.! c'rricd out by four-rcpe{€d f.d brlch s}st m 

uins immobilir.d,,l. rls€r nycclium in stirrcd fcincntor (Figun 3l). Aboor 90 

% of thc fcrln nied b$tl we rcpl.ccd !nd.r.!r?ric @ditiG, by ft.sh mcdium. 

TI|€ fc@ntdtioB w@ oa.dcd out for 48 h. Thc prcduction of ,(ylan$. was 

found optimum in tlt€ 2d botch. Ihcr! wt' . grldull rcduction ir d|. xyl.n.s. 

Droduclion !ff€r lhc 2'd boEh. Howcvcr. lh. atiE f.d bttch qhG Balcd 

insaSnific! r.3uh in td$ of xyl!|||lc prlductio.

Fisuc J l: 

€00 

000 

7@ 

-600 

g 500 

3 {tio 

i 300 

200 

100 

152 

P.odwiion of xylm!5! for four r€p€al.d f.d batche (in slir€d 

f.rm€ntor) by inunobiliz.d.,l.,is€' GCBTmncr.' 

lio, oltd !.4h.. 

TdpdrG 3CC. pH 4,5, f.mnlalion Friod 48 h

DISCUSSION

t5l 

DrSGUaa|or| 

A succe$ful fementation p.@.ss and bctter enzrre prcduction depend both on a 

pole.t strain dd opt'misation of f€mcntarion parameles. XylMe is an 

important enzlmo produced by the f€rnentalion wilh specific moulds, nostly 

Asperyillus niEer (Pinaga ct I994i Arinahu ei a1.,2003). !o. this purpose, 104 

^1., 

stnins of !. cdpable of hydrolysing xylan 10 xylose, wer isolated frcn 

",ael, 

dilTqst soil s.nples. These strainr w*. scrcened for the produclion of xylanase 

in shake flask. Amone all ihe strainr tcslcd,,{. riSer GCBT-]5 gav. mdimum 

prqtuction (225 U/nl) of xylanse, thcrcfore n wss slecred for tunhq 

optiniation bysubnc.ged fcmcnt.iion, 

The selecdon of suitabl. fcmenBlioD medium due to economic r@n is of 

great imponance for the production of metqbolit.s sp€cifically e.zlm6. In the 

pBenr srudy, rhe fem€nraion m.diufr {M-4} conrdininS &, wh): NaNor 0.1, 

lween'8o 0.2, NH4CI 0 l, KH,PO! 0.1, M8SOa.7H:O 0.03, CaClr.2HrO 0.1 and 

whear bnn 2.0, in dislilled waln (pH ?.0) wds s€lected for lhe maimal.nzlmatic 

aciiviry(225 U/nl). h mighrb€ duc lhat M-4 was d balanced m€diumwhich was a 

pre{equisne nor orly for fungal growlh but dko for the subsequent cnzyme 

production The prcper C/N ratio of the optirnidd medirm wd of a supeno. 

quality conparcd to all other mcdia os!d. This fidding is in accoftlancc with the 

$trk repoded by orher workcts (Anb.kar er al.. 1965i Ferieh er a/., 1999i Joya

154 

The nle of enzyn. slnlhesii in shake 0aks by the mould cultw wG aho 

cdied out- The prcduction of enzyme *as inclqed *ith ihe inq6. in 

f€menladon pe.iod and r@ched maximlm (247 U/ml), 48 h aftq in$ulalion- 

Funl€r inc.eae in the incubaiion p€riod rcsut|ed in the decffised production of 

xylame. It mighl be due to the depletion ofthe nulrienls that inhibited ine 8.o*th 

of tungi ad h€nce dec.eased xylanase produclioD. Thus, 48 h of incubdtion Period 

was selecled for lhe production of xylan 5e. Archua er a/ ( 1999) ale optimized a 

f.menridon period of 48 h for the optimal yield of xvlaMe (1285 U/dl) 

alrhotrgl the cultures used w€r€ $ermoPhilic Thercforc, the prcsenl work is dore 

signific@t conpmd to pevious workcB ln lhe prsnl sludv, xvlde 

prodNtim \6 mqimutn (250 U/ml) when innial PH oflhe mediun wd adj6ted 

to 4.5. Fuflhq incl€e or decrca!€ in pH qusd r€duction in eMvne prcdudion. 

tr nighl be due to thal orgrnism rcquiG acidic PH fo! fte 8lo*'th ed subsequdt 

xylMe prcduclion (Gomes ?t ol., 1994i Krogh et dl ,2OA4l 

The productiot ofenzyme can bc e uced by dcveloping nuranl strains of 

,.{ dg4 s seli as optimization oflhe cultuml conditions (Sleinet er al, 1998) 

Fo. the inprovement ofthe fltgus, the conidia olwild_culturc,4 riael CCBT-35 

wec subjecled to Uv inadiations for t60 min. Mdimun death rale of aboul 90 

o/r sa &hieved after 45 mit ofUV exPosure, A total otlinety-lbu mulanB were 

islaled afte. UV iradiadons and .xamined for enzyme fo.nation Among all rhe 

mutants tested, ,r. zlser BRCuv4. isolat d after 45 min of Uv imdiatic (dc€ 

l.2rlo'? tmls), gave mdimun production of xyloe (ll9 U/nn) Fudhs

155 

increas. iD th€ Uv l.estmot, rcsult€d it th€ complct d€afi of l ,Eer' me 

imprcv.ocnt in $e .nzyn€ fomslion by th. tungus may b€ duc lo thc mut0tion 

in th. gcnome of thc tungos. AnodS lh. surviving proSny, $c gcne rsponsible 

for the production of xylanele may ovd .xpr€55, r.sulling in lhe incc!!€d enzvme 

prcducaioo (chcn €/ dl, 1990: Brki ?, al., 2003)- 

To obtah ! hypo Ploducd of xyless€, lh. uV traled nulrtt sinin {ts 

tunher Elatd by MNNG (50-300 lrg/ml). Hundr.d tnd nine multnts w@ 

isolaled by obswing fie cle3r zooc ofhydiolysis of xylon in thc petriPlates- Th* 

mut rts werc ften evrlutcd fd .nzlne ptoduction. AmmS all rhe mul4rs 

rcsrcd, mutlnt ccBT MN!c.!d isol.tcd afrer 30 min of MNNG (200 !g/ml) 

tt dtne w.s foud to bc a pol.nt culturc fo. xyl t5e Production 

(493 U/nl). 

Oveall, the muaant CCBT MN6.! Ev€.lcd lboul 2.2 fold hiSn€r xylana* aclivitv 

6mped to the wild{ulhrc GCBT-3s d sbqt 1.6 fol.l ovq to BRCftr' The 

volum.tdc xylanase productivity of th. mulr (q =1.1 U/Umin) *rs highlv 

siglifi@t (p

156 

innial pH of4.5 after 48 h ofi.cubation. Similar kinds offindings havo also b@n 

reponed by Rashid (1999). The tritrogen sourc€s such 6 uEa, yeast extmct. 

polweplone or medl extracl has Br.i 

innuence on the enzyne production. Amo.g 

all th€ nitrog.n sources tested in th. pres€nr study, rhe m€at oxtract at lhe level of 

l 0 % was lound to be the best source of nitogcn for the produclion of xylanase 

(496.8 U/nD. As the level of meat cxtracr vas fnrther increased in the 

femenialion medilh, the p.oduciion of€nzyne was Sreatly inhibiied. The present 

sork is subslanliated with the findings ofCokhale er dt (1991) tud Gouda (2000). 

Agricultu.al by-Foducts, being thc cheape( and abundanrly available 

substrats have widely bccn uscd 6 subsiEtes for microbial fementation of both 

primary ed secon

157 

wheat bran resuhed in th€ d.*.ascd xylande producrion. It mighl be due lo rhe 

lhickening of rhe fement.tion mcdia. which made hindmnce in the prcper 

aeilarion and acnlion that.esulted d€c€lsc ii air supply (Palma eral, 1996). Th€ 

sumcient supply of air is very $senrial for beiler groath of mycelia as well as 

s€oetion of enzlme i. the feDenr.d brcth. Thc other worke$ like sawahchom 

(1999).nd S.hd (2003) rlso sclccicd wheat bnr as the basal nediun for rhe 

p.oductio. of xyldse. The.eforc. prcsent study is in an asrcenenl $ith the work 

.eponed by previous worle$. 

Th€ prcductivily of xyl.n.sc b greally influenced by bolh the soure and 

conccnr.aiion of nitlgcn (Kulkami er al. 1999). The .rd of di{Temt inorganic 

nihogen sourcB in conpadsn with thc control was tcst€d on rhe p6duction of 

xylanse. The inorganic nilrogen eurccs such a NaNOr, NHICI or NH.NOT weE 

added to the feDcnralion m.diun ar 0.05 - 0.20 % (v!). Among all th€ nirrosen 

souEes re$ed, xylanasc producliotr wes hiShly siSnificant (P4.05) whd N.Nor 

was used as nnrcgd source (502.4 U/ml) al a level of0.l0 % (wlvJ. ClEn et al. 

(1999) found maxinun xylanas. producrioi (157.2 U/l) wirh urca od NaNq d 

nilrogen source. The cffccl of nilrcg.n sources for increasing the tunsal cell 

aclivily to prcduce xyla.se has also bcen suPPoned by previous wo.k€rs 

(Cokhal€ '/ dl, l99l; Kdnsoh ?r al, 200lbr Silva e/ al, 2006). As the level of 

NaNOr w.s fu.the! inc.eascd in the fcrmentation medium, ihe production of 

edlme was grcaily inhibired.It mightbe du€ to the facr lhat higher concentaiion 

of fEe nitogen causes ioxiciiy and ii has.dve6e cffects on the producrion of cell

158 

mass a! well on lh. produdivity of xylanasc (Sun €/ al. 2000). Goknab er al 

(1991) stldi.d th.i ,{. rte' NCIM 1207 produced high l.v.k of xylane 

acriliries in th. subm.r8.d fcmoiation. Among lhe nihoSen sources 

inv.stigaled. amDonium solfate,.ft nonium-dihydrcgcn orthoDhosphal. &d com' 

sreep liquor werc foud to bc bcfid for lhc producion of xyldollric cnzymes by 

In rhe ptwnt study, ph6ph6r. soulc€s such as KH!PO., KTHPO. or 

NlrHPOa *erc .ampdd wilh il€ mntrol for Mximum xyl.nase aclivity. The 

@ncdtration 6ngcd from 0.05 - 0.15 % ( v). The cotlrcl Aw. 372 Vtml 

xylanase lctiviry. Howev.r, 0.1 % (v/v) KHTPO. suppon€d lnainnun cMyne 

activity (513.2 U/ml). Orher phoGphnc sourcs gav. siSnilicantly l6s cu}lnc 

.cdvny (g

159 

incrcr!€ or decreas€ in lhe coNetrtration of MgSO4.THrO rcsulled i. !h. d€creased 

prcducdon of xyl@e. H€nce, xylanase produclion w4 found to be higl'ly 

significant (p:0.05) at 0.03 % ofMgSOa.THzO concentmlion md thus oplimied 

for maxinul xylaiase prcduction. Tlr studies also showed the effsl ofdife.€nr 

concenLation of CaClz.2HrO on xylanse producriot by ,4. hi82r. T\e 

@rcotradon wa vdied lim 0.05 - 0.25 % (Vv) fld compaled with |he co rol. 

Th€ producnon of enzyme was found 10 bc ndiml (532.6 Uhl) when 

concertalio. ofCaclr.2Hrowas 0.10 % ( v) dd veied signincantly (PS0.05) 

thd oll)er colcetrrrations. Further increas€ in the concenl€lion of CaCh.2H1o 

.sulled in rhe decreased prcducrio. of xylanase. As 0.10 % (wv) cach.2Hro 

Bave Daxinum production of enzyme. Simile kinds of fitrdin8s have also been 

rcponed by Pinasa.r al (1994) a d Bi.1 at. (t999 ). 

fhe con@ntorion of trac€ elemefis (Ca*, Mg* dd Na') in 1he medium 

plays ar impo.tsnt rcle in thc 8ro*4h of A. niger ^nd subsequenlly xylansse 

production (Cai e, 41. 1997). The productivity of xylMe by ,.4. ,€sr can be 

funher imprcved by the addition of dircrent crlciom, nagnesim od ph6phale 

sourc€s |o the femdraiion medium. TIle p.oduction ofqzyme was fomd ro b€ 

mlximal (512.6 U/nl) who concentEtion ofCaCIr2HrOwd 0.10 % (*/v) and 

va.ied significan y {p:0-05)than oth ei .onc€ntrations ofthe CaCl,.2HrO. Funher 

increse in the concentration of clclr.2Hro .sulied in tbe d@redsd prcduction 

of xylonase. lr midt be du€ to lhe fact $tt CaClz.2HrO at hi€lEr concedration 

hs loxic i.hibirory eff@ts on the 8ro$,t of tulgus .s w€ll 6 xylmae synthcsis.

160 

rn a simirar study Fenim et al. (1999) obbin€d the maximum xylanGc 

production wilh 0,03 % coDccntration ofCaCl,.2HrO. 

Polyoxyethyle.€ sorbitan. nonooleate (Twccn-8o) itr .ppropriaE 

conccrrntion has a strong i.flu€nce on $e elficiency of fcmmlalion mediun to 

produce xylanase (Kennedy and Krouse, 1999). The elTect of diflere.l 

@ncentratio.s of Twcd-8o (0-1G0.40 70) w6 studied in culture mcdium fd 

xylanase production by ,,1. ,isel. Maxinum prcduction of xylanase (5a2.2 U/ml) 

wd achi.lod when feftenhdon m.diun vd suppl€nented tith 0.3 % oi 

Twed-80. 11 might be due to the fet lh.t Tw@n 80. b.ing a conpl.x source of 

nntienlr, hM a strong inductiv€ effect on en4m. synthesizing caPabilily of 

prodlcer organism (Tan€ja d zl., 2002). Tween 80 al 0 25 % level exhibited 2 0 

fold onhrncementin enzlm. prcduction (Balakrishnan 4/ o/.,2000). Any 'ncrcas 

or decrease in lhe Tw€€n-80 conceniratiotr beyond oPiimal geatly reduced the 

eEyme produclion duc lo ovcrgrcwth of mic@.emisd. Kmi .t al (1991) and 

Tahir er d/. (2002) repofted the same kind of invesliSations. 

Thc immobiliation of micrcbial cells or tungal sporcs foi imprcved 

primary ot sm.&ry metaboliies biosynth6is is of s.eat scieniific intercsr 

(Arimatsu d a/., 2003). Xylanase fementation *as canied out for fouFrep€.ted 

baah cultures aner immobiliatim of conidia of ,4 ,,8?r sl6in in he sodium 

alginate and polFrcthanc foam. The p.oduciiviry of xylan4e was gfrdully 

dsreased in each bath (excQl l"r) shc. the,r. ,tel was innobilizcd in the 

sodium altinate. The plductio of Sluconic acid was irc@ed itr Oe second

l6l 

batch as the,r. ,!g?' srrain was inmobilized in the polFrcrh.nc fdn, whilc in 

lhe 3'i and 4'i b.t h cuftures lte pioduction of xylanasc was sisnificantly 

dccrcded (Bao 2003; Mani.e e, a/., 2005)- The sodium alginatc howevs. Srvc 

bellor r€sulls thus oplimized fo. the immobiliztion of,.{. ,lael and substantiated 

wirh rhe wort rcponed by Mehnebslu (2000) dd An@tsu €r dl (2003). Sev..al 

fold higher sluconate activity w6 obiained wilh the immobili,zed ,4 niset.6dia 

Thc Bre of xylane prcduciion by imnobilized conidia ol A niger eas also 

unden ken. Tbc prcduction of xyl4ase following substrate consumpnon wa 

increased sith thc inmase in in@baiion p€riod and found mdimal 48 h aft€r th. 

inoculalion. As lbe incubatio! period wd funhcr inc@6cd, ihe subsraie 

consunplion was ino€sed. while the p.oduction of xvlanase was signific.ntly 

(p4.05) d€crced. Ho*e!er, the oplinun Potucion {6 obuined 48 h a&er 

inoculdtion. Similar,1,"€ of lork hs also bem rcported bv Hcinrich 6d Rehm 

(t9E2). 

The re-usc of nixed fungal mycelium has been rePofted bv a number of 

workes (Niazi e, al. 1969: .,@m! and Jadwisa, 1990: Sulme er zl. 1998i 

Pedrosa e, al, 2000; Mohica ?r al., 2002) Bv rc-using tho nvcclia ofprevious 

b.bh cultnrc, th€ prcducrion ofxyldasc w.s found bettd in rhe fi6r barch of the 

microbial cxpdiment. Wien the fnngal Dycelium was re-used turther fo' rwo 

suc.€sile barches, a decrcas.d mle of glucose bi@o.vdion dnd producdon of 

x'lanase sas found. lt night be due to thc fact that ihe .bihv of the organism tbr 

the po{tudion oi xyldnsse ws decreas.d afte. i|s rcp€ared usc The mehboli'

162 

behaviour of the prcduccr orgdism was allered slch thal it otered into lle 

decline or death phae of its grcwth. This fi.ding was in Sood aet€ment with the 

work rcponcd by Moycr et dl (1940) od Anbckar ?r ar (1965) who also 

discooraged the rc'usc of mould mycclia for successive batch culturcs dunng 

calcium Bluconate produciion by subn.rycd fementation, 

Pokistan being agicultuml country hd nany chsply available aSricultuol 

by producc. These agricultural by'ploducts cb be used for iheir exPloilalion as 

substratc for tuyme fomaton by solid{tate fementation (Haq er 4/. 2002) 

Tlus in th. pl* study, dirffit aSricultural by Producr such a .ice srr.w, nce 

husk, wheal straw. soybean meal, wh€at bran, bagasse, sunflowe. n€al and 

nryspap.r wc.e evalu.ted for xylde synthesis. The Prcdlction 

of enzvme wd 

foMd to be mdimal (1850 U/g) in a medium mlaining wh@l bdn. It mi8ht be 

due to thc supply of e$ential .utrienls by wheal bran (proteins 132 %. 

.arbohydrares 69.0 %. fats 1.9 %, fibre 2-6 "/o, ash l 8 %, Ca 0.05 %' 

Me 0. I I %, K 0.45 %, S o.l2 %) for the grcwth ofnicroorganitns d well 

.s for th€ production ofxylanaso. The prcduclion ofenzyme w.s geatlv inhibited 

6 rhe ncwspap€r was lsd in fmentadon medium lr might be doe to tbe low 

corceirdiion of available carbohydra&s prcsenr in lhc ncwspaPer' {hich weE 

esenrial for rhe produciion ofxylbase. In a simildr siudv, DeschmPs 5nd Huel 

(1984) studied the production ofxyl.na* by,{ ttget bv elid srare fmdution 

They gew fie organisms in column incubatoB deratcd ar looc wheE a 

conbination of wheai brao and dce husk was used as substrate. However' the

163 

naiimum xylanolfic actvny (658 U/8) w.s aboul 262 fold lowq thatr the 

Dopth ofthe m€diun in solid subrt.ate feme.tation hd grcai influence on 

th€ prcduction of enzFcs by lercbic micrcbs (Y6hida e, al , t968i Kaiaet at., 

2004). Erect of differelt depth of th€ whedr bED wd thercfore studied for the 

produclion of xylanase by noutd. ll was obsfled rhdl lhe P.oduction of enz)me 

wd mdimnh (1852 U/8) wha the d.Pth of lhe medium ws 04 m. As lhe 

deplh of th€ mediun wd funher incre6e4 thc produclion of xvlanas' wd 

decieasod sr6

164 

poduclion ofxylanasc w6 found optimal (1860 U/8) when distilled walq wd 

used fo. moistming ihe wheai bran. TiDe couBe ofenzyme prcduction plays a 

critical roh i. @yne synthcsis. The prcduction of enzyme wd incrcls€d with 

increase in the femcntation pdiod and r€&h€d m6ximum (1875 U/e), ?2 h after 

in{ulario.. Furlhd incrde in the incubation P.dod 

rcslltcd in the decreased 

prcduction of xylane. It nighl be due 10 the &cunulation of oths bv_ products 

or exhaustion of nutrienls in the fmentaiion n€diom Archtna and Satyanaryana 

(199?) srudied thc producrion of exl@ellutar themo slable cellulosFf@ 

xylanasc by Bd.i/tus lichenhotuis A99 by solid{iat€ fementaiion Tlc 

prcduction ofxylanas. Hchcd a peak in 72 h 

The incubalion rempersture was studied for the prcduction ofxllsnde The 

p.oduction of xyla.lse sd found b be ndimutn (1885 U/g) a1 30"C Fudher 

incEase in the incubdion temPeratud rcduced th. prcdlclion ol xylare' Al 

40'C, $e produdion of xylanase was gr.atlv inhibiled lt might be due 10 rhe facl 

rhar al hiSh tempdatue, the mdsturc contdl i. the solid-slale f€molaton 

conditions reduced due to evaPo..tion Wilh the reduction in moisturc conto! thc 

grcw$ of the organiso was reduce4 h4@ aficctiog enzvn. fomation (You 

and Rnnglu. 1999). The produclivity and ercwth of the o'ganism was found 

opiimum al l0'C. Thus, th; rcnlP€sturc wd select€d for the prcduction of 

rylande by solid-state femenhrion mediun Msnv workcrs hav€ also Eported 

sinilar kinds of iitdings (Deschamps 5id Huet, l9E4; Cai ar al, l998i Ring?f il 

1999i Judilh md Jlnior,2OO2i Romnowska er,/ 2005)

165 

The cffst of diifercnt carbon and nitrcs€n sources was investiSated on lh. 

production of xylanaso by fungus. Ofall ihe carbon and niircsen sources lesrcd, 

s|arch ar rhc level of 2.0 % (2150 U/g) as ca$on source and (Nr!)rSO1 at lho levcl 

ofo.2 % (2480 U/g) as nitog€n source gave haximum production of xvlandse. 

Fu.rhd incrce in thc level of carbon and nitrogen sou@s (C/N dtio) reduced 

ihc produclion of enzyme. Ii miSht be dll€ to incrcase in the mycelial grc*.ln at a 

higher lelcl of carbon sourccs {itb significdt derede i. the Prcductid of 

xylanasc at reponed by Cho (199?). Similarlv, a hiSher .oncentFtio of itrcgen 

ha! inhibiloly elTect on tho producliviiy of xylanase Therofore' stdch at ihe levcl 

of 2.0 % dnd (NH.)rSOr at ihe level of 0-2 % *erc sel@red for the Prcduction of 

Tlc sales up studi€s for eDyme production s€re cam€d olt itr a glss 

femenlor of7.5 L capaciiy wilh a working volumc of5 L bv'f is"r OCBTMNIC 

nr Oliinization of inoculum fot tn. p.oduclion ofxvlanascs wd caricd onr and 

rhc mximum xylanasc Production (6940 U/ml) ws ob*ryed wilh l0 % (*/v) 

vegetative inocnlum when thc siz€ of inoculum was 8reaLt 

or smlhr ihan 3 0 % 

(w/v), it rcsulbd in the reducrion of avlalse prcduction lt Disht bc due b rhe 

fad that at a lowe. concenlralio., dount ofmvceliun fomed was not sufficieni 

to collen Dore subslrate in to xyldse At a highcr conccDtation of vegebrivc 

inoculun, $e vi$osily of fem.ntttion medium inc'eded which causcd dimaion 

ploblems (o. reduc€d oxvgen supplv) and hence, decreased xvlana6e prodnclrotr 

(Sicdenberg "t al., l99s)- Techapun et 4/ (2002) us'd 5 0 % vegelarive in@llm

166 

for betler xylanase prcductid, bul i. pBe study 4 0 % vegeraivc inoculum w6 

Thc elfccr of differeni agitalio. €le was also caried out Th€ mexinal 

xylan.se produclion (694.31 u/nl) wa! dchieved whd ag'talnn .ate wd 

mainraiied dl 200 rpm. The agitation rate les tha. 200 am rcsuhed in the 

sE tific.rion of my@liun which inhibned the tungnl melabolism and hence 

dsF6€d thc hre of xyl@ production (Reddy er 41 2002) Ttc agitadon 

intensity abovc 2oo rpn mighl cau6e intcmPiion in the f€nenltion Pb6c and 

rhus resuliing in a lower xylanase p.oduclion (llias and Eoq, 1998i Rashid 1999; 

Silva c/ d/.. 2006). However, Liu er al (1999) achieved mdimun xvlanse 

producdvny.r a shaking sped of moE ihan 3zr0 +n which obviolslv dcmanded 

moE .nc.ey and rhus affected rhc cosl of the resea.ch work 

ln the presdt study, the a.!5tion rate of 20 vvm was optiniz.d fd 

maximal xylanise producliotr (781.9 Ulml) by A riSet GCAT'35 in sliftd 

fementor sith an agilalion intensity of200 An. Il night be due to th8t Prcpor 

aeniion and agitatio. a.€ n@sry to cnsure 'n apprcPriate oxygcn suPPly and lo 

maintain lcnrilarion md prevar conhmimdon by prcviding a Posilive p€$urc 

i.sidc rhc slired fementor (Pal6. .r 4/., 1996). Chen e, a/ ( 1 990) and R€ddv e/ 

al. (2002) have also rcponed sinilar kinds of iindings und€i submerged 

fedeniaiion condilions.

167 

The mte of rytanNe prcducion wd aho studi€d and the naxinal 

prcduction ofaylande (?E1.4 U/nl) ws achicvcd,60 h tner i.@lttim- Funler 

incrce in the incubation p€riod did not enh.nce xvla.N Prcdnction 

lt mighr LE 

due to lhe d€crcased dnou.t ofavailable nut ienc i. the femenlaiid mdinn' lhc 

as€ offungi, the presmce of itrhibitols produc€d by fungi ilselfor tbe dopletion of 

suga! contenls. simila. ty?e offindings has been rcponed bv Ilias and Hoq (19981 

Tecbapun cr al. (2003) obilined optimal xylaiase acrivitv.fter 144 h of 

inoculatioi. so our *olk is economically more tisnificdt bec.use reduction in 

incubalion penod took place which Fduced th€ con of xvlanse prcducdon 

Similar, kinds of fildi.8s have alto b€.. rcPoned bv Cam.cbo and Aguilar 

(2003). 

T1r tempedture of femenlalioo medium is onc of tbe cnii@l facloB lhat 

have profound effed on the prcduclior of xylanase (Merchant ?t al, 1988; sevis 

and Aksoz, 2003). In the present sludy. the effocr of diff.mt i.cub.rio' 

temperatures was i.vestigaicd for xylanase Produclion bv,{ ,i8er GCBT-35 'n 

siired fedenlor. Maxinun cnzymc s)hihesis was found to be al30'C i e, 652 6 

U/ml. When lhe temperature was furlher increased or demded liom 30'C. th€ 

enzlme production wd g.eally !edu4d. lt mi8h1 be due to rhe f&t thal bigher 

tenp€mture advesely .lTers ihe 8rcwih of tunsus and ddatunng of enzlme 

(Youn lnd RungF 1999i Rahhan ?t d/, 2001) A number of previou workeF 

have optinazed a rcnpdatu.e of lo'C for optimal xvlanase vield iD fmcnrou of

168 

diffemt working cq,lcitis (Amb€kff e, aa, 1965; Jain d al.,1995., ZIN .t al., 

1999; Gdda 2000j Oshim er at, 2006). 

The fed hlch oulture hs bccohe very importart for the co.tinuous 

production of vdi@s @yhcs ieluditrg xylde. In tht om@rion, the 

fmrarion was canicd oul fd four EDcared brtchcs, lt ws mt d rtEr 0E 

productiviry of xyl.ms€ pas sigrificaltly d€crcd€d aftd 2d rcFated batch. It 

mighl be due io ile tu t that the potency of the nycclium fo. th€ production of 

xylare wd sig.ifica ly d€cMs.d wirh the .epetilion of the bdrch (Liu ?, al, 

1999). Howevd, lhh sNdy n@ds tu $cr invesiigation for its exploitation ar r 

conhercial scale. Kasoh sd Garnmal (2001) €xploitcd fed-balch culturc for 

imprcved xyld& prodctivity but th@ wortes $ggetcd th.t A. niAu tnay not 

bc a suitable o.Sanisrn. JteloD)€er ,vtda,r' w6s, nowcv.r opdEizd for such

CONCLUSION

CONCLUSION 

In the present sludy. 104 stEits oI Asperyillw niser,.ap ble ofhydrolysing xylan 

to xylosc, were isolated frcm different soil samples. These stra'ns wcr. scFened 

for lhe prcdu.tion of xylgnae i. shrk€ flaks. Anone all the sftins tcsied, ,,1. 

r,?€r CCBT-15 gave n.ainub p.oduction (225 U/nl) of xylanac. In thc P'!trt 

wort the femenolion nediun M-a containing (%, Vv) N.NOr.o.li T*en-80, 

0.2; NHjCl, 0.1; KH,?O$ 0.1; MgSO4.7H?O. 0.03i Caclr.2 H,O ad wheal bmn. 

2.0. in distillcd water (pH 7.0) wa! selecled for maxinum enzymaiic activity 

(Figu.e 2). The xyluse p.oduction wes maximum (250 U/nl) whe. initialpH of 

the fenenralion medium was kept ai 4.5. 

For the imprcvement of th€ tungus, th. co.idia of,.{. ,iaer GCBT-15 werc 

subj€cr€d ro Uv imdiations fo. 5-60 nin. Mdimum doath r.t€ was achi€ved 45 

min after UV exposur, Ninety-fouf mulants were isolaled afte! UV iradialions 

and examined for mzyme fomation. Amone all mutants l€sted. the ,.{. rrsel 

B&C!vrr. isolaled after 45 nin of UV iradiatios, Save mdinum prcduction of 

xylanasc. To oblain the b?er prcducd of rybnase, the Lrv Ee.tcd fluunr $rain 

was filnher rrealed by MNNC (50-300 pglml). Hudrcd dd nide mutlntt vtre 

isolatcd by obsedng the clear zorc of hyd.ol'sis of xyla. i. thepet.iplates. Ttese 

mulanis wcrc thcn evaluared for enzlme p.oduclion. Among dll the mut.nts

170 

resrql. mutant GCBTM,rab isolated aftft 30 min of MNNG (200 4/mD 

tredtnent was lbuDd lo be m6t potnt for xylanase p.odudion. 

The prcduciion of xylanse was increased in the secoid batch by ,4. 4ig4l 

st6in indobili2ed h the polFrclhsnc flm. The conidi. of A. niger nrttnl 

GCBTM"Na r were immobilied and cultivated fd xylanasc production for 24 and 

48 h. The production ofxylanase was si8nificantly increased as,4. ,,9"/ was pre- 

Seminaled fo.24 h and Eached maximun 48 h after inoculstio.. Effecr ofre{sc 

of myceliun of,4. ,,8"a obtained by ascpric cmtitugaion of femdted brorh 

fron the previous ba&h, for lhe production ofxylanase was studied. The rale ol 

substraie utilization was enhmced in rhe fist batch of ihis experimeni. When 

mycelia sqe ftrther €-used itr .ext t*o batches, a decr€.sed Ete of subsrde 

bioconveBion and producdon of xylaMs w6 noticed. 

Diilercnl agriclltu.alby-product such d rice straw, rice husk, wheat sraw. 

soybcan meal, wh6i bhn, bag6sc, sunflower m@l dd ncwspap€r w@ ev.lu.tcd 

for iylanae sy hesis. Tle prcducdon of cnzFe wa lound lo bc maxinal(1E50 

U/s) in wheat bran mediud. Tle supplyofadequate amount of waie., during solid 

subsbal€ fe@entation is very essentid since excesive anount of water affects 

potusityi hencei acrcbic conditions dudng fmdlrlion. The prcductivity of 

xylanase by rhe fungus can be tunh€. imprcved by rhe .ddition of difforcnt cdbon 

and nilrogen sources in to the fementation medium. Ofallrhe carbon and niirogen 

sources resred, starch at th€ levcl of 2.0 o/r (2350 U/g) as carbon sou@ and

t7l 

(NHrrsor .t rhc ldel of0.2 % (2480 U/g) s nitoseD s(rc g.v€ muimum 

pmdu.tion of xylane. 

Th. scale up studi€s otr mzyh. production was cadcd dt in a glas 

femenlor of 7.5 L cap&iry wi$ a working volume of 5 L by .4. ,i8"r CCBTMNNC. 

ro. Optimiation of in@ulm fd thc productid of xylos€s was camcd out md 

dE nuximum xylll'e psdktion (694.0 U/n ) wa ob6cFcd silh 3.0 o/o (e/v) 

vog.tativ. inoculum. The '@tid 6G of 2.0 tm w6 optimizGd fo Mimat 

xylanas€ (783.9 U/ml) Foduction by,l. ,18"r GCBT-35 in sti@d fcnncntor wirh 

an agiialion inrcDsity of 200 rym. Th. production ofxylaase wai also cdicd out 

by fed-baKh system in fie strncd f.nrentor, which ra d.cu!.d shltply ator 

th.ld conrinuoc balch.

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Prcduction of xylose fiom xvle with intracelluld ehzvme svstem of 

Aspq4ills niseL J Fettunt Biodgitl T(4r: 153-t5a 

veluz. C-H., H6gling, H ttaiwd ed G Hongahi 1999 Xvlanase producl'on 

and its appli@tion it degddation of hemicetlulose. natsia\s Int Cong 

BiotechML PulP PaP.Ihd 7.65'67. 

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rl(3):305-309. 

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9:80'83. 

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influence on xylmtles Eed in baterv afflications Jowtul of cerfut 

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200 

tuchqicl'i

LISTOF 

REI'EARCH PI'BL|GATIONS

201 

LIST OF PUBLICATIONS 

l . wasecm Ahm.d B!n, Iknm-ul H!q, Sik nder Ali, Qadecr M A. and Jdved 

2. 

l. 

5. 

Iqbal. 200r. Production ofxylanase by solid{la& fetuentari'onbv AsPeryi us 

niser. PaL. J AoL,33:58l'585. 

Haq, l.,AycshaKlEn, w. A. Buq S A1' and M A Qadd. 2002 Enhanced 5- 

produdion ofxylanase by muta strain of /{rp?€rl16 ,i8?r under solid stlte 

femenration conditions. /rd.I Piutl t tu, I(I):5_E 

lknm-u]-tlaq, Ayesha K}lan, Wase.m Abmad Butt Sikander Ali And M A' 

Qads.. 2002. Effect olcarhon dd nitrogm sources on xvlane produclion bv 

nuranr strain of ?{ysrs !s,iSet ACRMX4' Onlia. J. Biol S.it 2(2): 143' 

4. Wasecn Ahmad Butt, Iinm-ul Haq and Javed lqbal 2002 Biosvnthsis of 

xylanase by Uv-treated nuta strai. of Aspergillus hiSet CCBMX'45' 

Riot.dtbot . \(t): to-14. 

Amna Ehsan, Ikam-ul-Haq, 

Ali and M.A. Qade.r. 2003. 

submcrsed femenrarion from Aspet4ilhs hi4e. Ind J Plo'? S.ir" l(4): 372- 

175. 

Wase€m Anrnad Butt, Hamad Asl6f. Sikander 

Nutritional studies for xytanese biosvnthesis bv 

6. lkran-ul-Haq, Am Ehsan, Wase€b Ahnad Buti and Sikddcr Ali 200l 

Stndics on rhe biosytrth6is of dyme xvlanase bv subm'rged fcmentation 

tom A!pe/8il6 ni4.r GCBMX-45. Pat I Atol $r',5(12): 1309-1310'

WASEEM AHMAD BUTT - Higher Education Commission

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