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1、灰綠曲霉肛葡笱糖苷酶分離純化及性質(zhì)表明較低濃度SDS即可對BG活力有較強(qiáng)的抑制作用。以熒光發(fā)射光譜研究酶經(jīng)不同濃度鹽酸胍和SDS變性后的分子構(gòu)象變化,結(jié)果顯示,酶經(jīng)變性后,338nm熒光發(fā)射峰逐漸紅移,說明酶分子充分伸展,變性完全。比較酶經(jīng)鹽酸胍和SDS變性后的活力與構(gòu)象變化的關(guān)系,表明酶在鹽酸胍和SDS溶液中的失活均先于其去折疊的過程,這種活力喪失快于酶分子整體構(gòu)象變化的現(xiàn)象表明酶活性中心處于對變性劑較為敏感的區(qū)域。優(yōu)化灰綠曲霉纖維素酶水解稻草粉產(chǎn)糖條件發(fā)現(xiàn),在100ml含3%稻草粉的酶解體系中發(fā)酵產(chǎn)糖最適條件為:溫度55℃,酶解體系pH5.0,酶液用量12ml。在此條件下,
2、發(fā)酵24h后,還原糖濃度為13.1mg/ml,稻草粉糖化率為39.1%。關(guān)鍵詞t灰綠曲霉p.葡萄糖苷酶分離純化酶學(xué)性質(zhì)糖化2灰綠曲霉p葡萄糖苷酶分離純化及性質(zhì)AbstractA13-1,4-glucosidase(BG)waspurifiedfromfermentationliquorofAspergillusgtaucusbyfollowingprocedures:ammoniumsulfateprecipitation,gelfiltrationonSephadexG·100,andhydrophobicchromatographyonPhenylSepharoseFast
3、Flow.ThespecificactivityonsalicinWasdeterminedtobe1747.4U/mg.ThemolecularweightofthisenzymeWasdeterminedtobe23.5kDaand92.5kDawithSDS—PAGEandgelfiltrationonSephadexG-100,respectively,indicatingthatBGisatetramer.TheenzymewasstableatthepHrangingfrom3.0to7.5,anditsmaximumactivitywasobtainedatpH3
4、.6.Theenzymaticactivitygraduallyincreasedintherangefrom40to600C,butasharpdecreaseoccurredat650C.ItWasstablebelow650C.EnzymatickineticsindicatedthatMichaelis—Mentencontant(‰)ofthehydrolysisforsalicinbyBGWas2.58mmol/L(pH3.6,60oC).Chemicalmodificationshowedthattheenzymeactivitywasnotinhibitedby
5、mercaptoethanol(ME),dithiothreitol(DTT),p-chloromercuribenzoate(pCMB),acetylacetone(ACAC),andPhenyimethanesulfonylfluoride(PMSF),suggestingthattheresiduesofdisulfidebond,mercapto,L-arginineguanidine,serinedeformitywerenotnecessaryfortheenzymeactivity.WhiletheresiduesofHis,Trp,LysandAsp/Gluwe
6、reessentialtotheactivesitesofBG.Fermentationproductsethonal,aceticacidandlaticacidshowednon—competitiveinhibitiontoBG.Theeffectsofseveralions011theenzymeactivityhadbeenresearched.Theresultsshowthat:CI‘、S042。、N03‘、Li+、Nd、K+、M92+、ca+、Ba2+andZn2+hadnoeffectsonBGactivity,itwasactivatedbyMn2+,F(xiàn)e2
7、+,whilestronglyinhibitedbyCu2+,ab2+,Cd2+andH92+.Theinactivationeffectsinionssolvents(Cu+,Pb2+,Cd2+,H礦)havebeenstudiedusingthekineticmethodofLineweaver-Burkplot.Cu2+,Pb2+,andCd2+showednon.competitiveinhibitiontoBGwhiletheinactivationoftheenzymeinH礦+