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1、第65卷第8期化工學(xué)報(bào)V_01.65No.82014年8月CIESCJournalAugust2014菱3≥03j;:;3j)jjjj外切-p一葡聚糖酶基因重組里氏木霉的篩選及其產(chǎn)酶性能孔芹,方浩,夏黎明(浙江大學(xué)生物質(zhì)化工教育部重點(diǎn)實(shí)驗(yàn)室,化學(xué)工程與生物工程學(xué)系,浙江杭州310027)摘要:外切.D一葡聚糖酶是纖維素酶的重要組分之一,提高該組分的活力是增強(qiáng)纖維素酶協(xié)同降解性能、降低纖維素水解成本的關(guān)鍵。分別采用微晶纖維素瓊脂平板法和濾紙崩解法,對己有的基因重組轉(zhuǎn)化子進(jìn)行篩選試驗(yàn),獲得了6個優(yōu)良
2、轉(zhuǎn)化子,其濾紙崩解速率和微晶纖維素瓊脂平板上的生長速率都較大。進(jìn)一步在搖瓶條件下進(jìn)行復(fù)篩試驗(yàn),獲得了外切.p.葡聚糖酶(C1)高產(chǎn)轉(zhuǎn)化子TrichodermareeseiZU.101,液體培養(yǎng)48h,其C1酶活力可達(dá)18.24U·ml~,是出發(fā)菌株的2.16倍;分析結(jié)果表明:重組轉(zhuǎn)化子的纖維素酶體系中內(nèi)切.D.葡聚糖酶和纖維二糖酶的活力與出發(fā)菌株相比變化不大,但由于外切.D一葡聚糖酶活力得到了大幅度提高,纖維素酶的總活力(濾紙酶活力FPA)也提高了61.9%。采用纖維素酶對堿預(yù)處理玉米秸稈進(jìn)行酶
3、解試驗(yàn),當(dāng)酶用量為20FPIU·(g底物,水解48h,重組轉(zhuǎn)化子T.reeseiZU.101纖維素酶的酶解得率高達(dá)94.4%。本文的研究結(jié)果在可再生纖維素資源的生物轉(zhuǎn)化與利用方面具有廣闊的應(yīng)用前景。關(guān)鍵詞:重組里氏木霉;外切.D.葡聚糖酶;轉(zhuǎn)化子篩選;玉米秸稈;酶水解DOI:10.3969~.issn.0438-1t57.2014.08.037中圖分類號:TQ920文獻(xiàn)標(biāo)志碼:A文章編號:0438—1157(2014)O8—3l22—06Screeningandcellulaseproducti
4、onofrecombinantTrichodermareeseiwithhighactivityexo-p-glucanasesKONGQin,F(xiàn)ANGHao,XIALiming(KeyLaboratoryofBiomassChemicalEngineeringofMinistryofEducation,DepartmentofChemicalEngineeringandBioengineeringZhejiangUniversity,Hangzhou310027,Zh~iang,China)A
5、bstract:Increasingtheactivitiesofexo—p—glucanases,oneoftheimportantcomponentsofcellulase,isakeytostrengtheningthesynergisticdegradationofcellulaseandreducingthecostofcellulolytichydrolysis.ThemethodsofMCC—agarplatescreeningandliquidculturemediumoffil
6、terpaperwereusedinthisworkrespectivelytoscreenTrichodermareeseitransformants,obtaining6superiortransformantswithhigherfilterpapercollapsingrateandgrowingrateonMCC-agarplates.Furtherscreeningexperimentwasconductedundershakingflaskcondition,obtainingth
7、ehighlyexo一~-glucanase(co-producingtransformantreeseiZU一101whoseC1activitywasl8.24U·ml—after48hliquidculture.2.16一foldhigherthantheoriginalstrain.Analysisresultsdemonstratedthatfilterpaperactivity(FPA)ofthecellulasesystemofthetransformant.representin
8、gtotalactivity,increasedby61.9%asexo—p—glucanaseactivityascendedsignificantly,althoughendo一13-glucanaseandcellobiaseactivitiesvariedinsignificantlyfromtheoriginalstrain.TheenzymatichydrolysisyieldofreeseiZU一101’Scellulaseinthehydrolysisofalkalipretre