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1、網(wǎng)絡(luò)出版時(shí)間：2014-01-1515:23網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/11.1946.TQ.20140115.1523.009.html外切-β-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-β-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。本文分別采用微晶纖維素瓊脂平板法和濾紙崩解法，對(duì)已有的基因重組轉(zhuǎn)化子進(jìn)行篩選試驗(yàn)，獲得了6個(gè)優(yōu)良轉(zhuǎn)化子，其濾紙崩解速率和微晶纖維素瓊脂平
2、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-β-葡-1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-β-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-β-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-1理玉米秸稈進(jìn)行酶解試驗(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
3、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-β-葡聚糖酶；轉(zhuǎn)化子篩選；玉米秸稈；酶水解中圖分類號(hào)：TQ920文獻(xiàn)標(biāo)志碼：A文章編號(hào)：0438-1157（2013）00-0000-00ScreeningandcellulaseproductionofrecombinantTrichodermareeseiwithhighactivityexo-β-glucanasesKONGQin,FANGHao,XIALi-ming(KeyLaborotaryofBiomassChemicalEngineeringofMinistryofEducation,Dep
4、artmentofChemicalEngineeringandBioengineering,ZhejiangUniversity,Hangzhou310027,Zhejiang,China)Abstract:Increasingtheactivitiesofexo-β-glucanases,oneoftheimportantcomponentsofcellulase,isakeytostrengtheningthesynergisticdegradationofcellulaseandreducingthecostofcellulolytichydrolysis.ThemethodsofMCC
5、-agarplatescreeningandliquidculturemediumoffilterpaperwereusedinthisworkrespectivelytoscreenTrichodermareeseitransformants,obtaining6superiortransformantswithhigherfilterpapercollapsingrateandgrowingrateonMCC-agarplates.Furtherscreeningexperimentwasconductedundershakingflaskcondition,obtiningthehigh
6、lyxo-β-glucanase(C1)-producingtransformantT.reeseiZU-101whoseC1activitywas18.24-1U·mLafter48hliquidculture,2.16-foldhigherthantheoriginalstrain.Analysisresultsdemonstratedthatfilterpaperactivity(FPA)ofthecellulasesystemofthetransformant,representingtotalactivity,increasedby61.9%asexo-β-glucanaseacti
7、vityascendedsignificantly,althoughendo-β-glucanaseandcellobiaseactivitiesvariedinsignificantlyfromtheoriginalstrain.TheenzymatichydrolysisyieldofT.reeseiZU-101’scellulaseinthehydrolysisofalkalipretrea
