灰绿曲霉β葡萄糖苷酶分离纯化与性质

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1、灰绿曲霉肛葡笱糖苷酶分离纯化及性质表明较低浓度SDS即可对BG活力有较强的抑制作用。以荧光发射光谱研究酶经不同浓度盐酸胍和SDS变性后的分子构象变化，结果显示，酶经变性后，338nm荧光发射峰逐渐红移，说明酶分子充分伸展，变性完全。比较酶经盐酸胍和SDS变性后的活力与构象变化的关系，表明酶在盐酸胍和SDS溶液中的失活均先于其去折叠的过程，这种活力丧失快于酶分子整体构象变化的现象表明酶活性中心处于对变性剂较为敏感的区域。优化灰绿曲霉纤维素酶水解稻草粉产糖条件发现，在100ml含3％稻草粉的酶解体系中发酵产糖最适条件为：温度55℃，酶解体系pH5．0，酶液用量12ml。在此条件下，

2、发酵24h后，还原糖浓度为13．1mg／ml，稻草粉糖化率为39．1％。关键词t灰绿曲霉p．葡萄糖苷酶分离纯化酶学性质糖化2灰绿曲霉p葡萄糖苷酶分离纯化及性质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+，Fe2

7、+,whilestronglyinhibitedbyCu2+，ab2+，Cd2+andH92+．Theinactivationeffectsinionssolvents(Cu+，Pb2+，Cd2+，H矿)havebeenstudiedusingthekineticmethodofLineweaver-Burkplot．Cu2+，Pb2+，andCd2+showednon．competitiveinhibitiontoBGwhiletheinactivationoftheenzymeinH矿+