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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(duì)己有的基因重組轉(zhuǎn)化子進(jìn)行篩選試驗(yàn)，獲得了6個(gè)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(zhǎng)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(duì)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(hào)：TQ920文獻(xiàn)標(biāo)志碼：A文章編號(hào)：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
