Cloning and Expression of Chaetomium Thermophilum Xylanase ll-A gene in Pichia pastoris

Abstract

T'lie tliermopliific fungi liave consiaera6Ce importance 6ecause of tlie tliermosta6{e nature of tlieir enzymes, wliicli is mostfy an inaustriaC requirement, sucli as in teJ(JiCe aenim ana 6iofinisliing, puCp ana paper ana pouCtry feea peCCeting, etc. }lmong tlie various tliermopliiCic fungi, Cliaetomium tliermopliiCe lias a potentiaC source of vCanase ana ceCCuCase enzymes, 60tli requirea in tlie treatment of fi6er in tlie pouCtry feea. In spite of tlie fact tliat a num6er of microorganisms are gooa proaucers of vCanase, stiCC tlie titer of tlie enzyme neea to 6e enliancea 6y using (]YN}l recom6inant teclinoCogy. P.fforts were maae to fulfiCC tlie requirement of tlie inaustries. Por enliancea enzyme proauction we construct a proRgryotic ana euk.,aryotic expression cassette tliat can 6e donea unaer specific strong promoters ana enliancer eCements to get maxjmum gene expresswn. In tlie present stuay prok.,aryotic expression system i.e. pP.T' ana euk.,aryotic eJ(pression system Picliia pastoris was usea to express VCanase gene. In proRgryotic ana eURgryotic expression system 1£.. cofi (]3L21 strain ana Picliia pastoris (]Sl15 strain were usea as moaeC organisms ana pP.T' 32a(+) ana pPIC3.5k., vectors, respectivefy were usea. In tliese expression systems tlie VCanase gene was inaucea 6y using 1 m?1 IPT(], 1 m?1 Cactose ana 1 00% metlianoC up to finaC concentration of 30%, respectivefy. In case of proRgryotic expresswn system tlie confirmea PC1\. target vCanase gene fragment from pSSZ810(a) approx:.: 810 6p was figatea into pP.T' 32a(+) vector ana transformea into (]3L21 strain of P..cofi for expression. 11ie recom6inant done pSSZ 810(6) resuCtea in maxjmum VCanase activity of 6.02 V/mL in tlie presence of 1 m?1 Cactose inaucer ana 2 % VCose as a car60n energy source. T'lie maxjmum VCanase Vlll Abstract actrl)ity in case of 1 m?d factose im{lIcer mu{ 2% x;y[ose '(vas o6serf/el{ after incu6a.tio-n '!! 10 m£lls (It 40 °C 'wfiercas in wse vf Im:M I¥['r;; inl{ucer tfie I1w:x:imum (lc/,.i·vity 4.62 'UlmL was 06sel'vea after illcll6atioll of 2 firs at 40 °C. Xy[ose fOmlea was aCso aeteeted 6y J{iPLC anarysis, wnien snowed [arBer peali.)n case of 1 111* [actase as comparea to 1 11194. IPTr,; ina liceI'. S{])S-PllqE ana 'western 6[ot allarysis snowea appro" 43 fi..{])a mo[ecu[arweiBnt of ""uwase a[ollB witnfllsion prot":11 of p'£/1' 32a(+) tlector. 'Witn ana 'fvitli()1I.t /lI.,ion prote,:1/. tlie XJ'(alla.<;e act£vity wa.~ 4. 0 a1llfO.387 V/I1l[., respecti:vefy as comparea to controC 'l1ie acti",ity of tota[ protein of'lieafi !JJL21 tra1lsfol'med 1vitn pSSZ810(6) 1vas 06semed in tne presence of 1m* IPTq alia 1 '//tOIl. [octose sllppfemell.tea 'f,vitli 2% XJ(ose ana 100mg/m[ In case of 111l:M J(PTq illauce r tfie maxjmum ana minimum acti'()ity '(Vas 0.66 ana 0.37 mg/m{ 'Ivfiereas in case oj 1 miftf. (aetose inaucerwas 0.6 alia 0.35 11Ig/m( l'espectl:veEy. In case of yeast tJ.Kpression system the ",,[anase gelle from pSSZ81 0(6) 'Was c,gated into Picnia pas tons PPIeJ.51i.. vector. CO/finllea recomGillallat cfolle pSSZ810(c) was trallsformea into tfie genome of P.pastoris 95115 strain tfirougfi cfectroporation. rfransjormants were sewctea on 'YPJ) (yeast peptone aeJ(J:.7'Ose meaium) pfates containi1lg antiGiotic gelleticin (100 mBlmL) lip to fina[ concentratioll of 0.75 mBltlLL. 'l1i, maximum ~ry[allase activity of 2.04 V/ml was o6servca in tnc presence of 100% metfiano( as ilU[ucer alia after illcu6ation of 2 firs at 50 ·C as compal'ca to control JiPLC anarysis represents farBer peali.. of ",,[as' as comparea to prol0ryotic ex:pressiolL cassette. S{])S-PllqE itufi'cates appro" 281i..{])a proteilL.