外切-β-葡聚糖酶基因重组里氏木霉的筛选及其产酶性能孔芹，方浩，夏黎明（浙江大学生物质化工教育部重点实验室，化学工程与生物工程学系,浙江，杭州310027）摘要：外切-β-葡聚糖酶是纤维素酶的重要组分之一，提高该组分的活力是增强纤维素酶协同降解性能、降低纤维素水解成本的关键。本文分别采用微晶纤维素琼脂平板法和滤纸崩解法，对已有的基因重组转化子进行筛选试验，获得了6个优良转化子，其滤纸崩解速率和微晶纤维素琼脂平板上的生长速率都较大。进一步在摇瓶条件下进行复筛试验，获得了外切-β-葡聚糖酶（C1）高产转化子TrichodermareeseiZU-101,液体培养48h，其C1酶活力可达18.24U·mL-1，是出发菌株的2.16倍；分析结果表明：重组转化子的纤维素酶体系中内切-β-葡聚糖酶和纤维二糖酶的活力与出发菌株相比变化不大，但由于外切-β-葡聚糖酶活力得到了大幅度提高，纤维素酶的总活力（滤纸酶活力FPA）也提高了61.9%。采用纤维素酶对碱预处理玉米秸秆进行酶解试验，当酶用量为20FPIU·g-1底物，水解48h，重组转化子T.reeseiZU-101纤维素酶的酶解得率高达94.4%。本文的研究结果在可再生纤维素资源的生物转化与利用方面具有广阔的应用前景。关键词：重组里氏木霉；外切-β-葡聚糖酶；转化子筛选；玉米秸秆；酶水解中图分类号：TQ920文献标志码：A文章编号：0438-1157（2013）00-0000-00ScreeningandcellulaseproductionofrecombinantTrichodermareeseiwithhighactivityexo-β-glucanasesKONGQin,FANGHao,XIALi-ming(KeyLaborotaryofBiomassChemicalEngineeringofMinistryofEducation,DepartmentofChemicalEngineeringandBioengineering,ZhejiangUniversity,Hangzhou310027,Zhejiang,China)Abstract:Increasingtheactivitiesofexo-β-glucanases,oneoftheimportantcomponentsofcellulase,isakeytostrengtheningthesynergisticdegradationofcellulaseandreducingthecostofcellulolytichydrolysis.ThemethodsofMCC-agarplatescreeningandliquidculturemediumoffilterpaperwereusedinthisworkrespectivelytoscreenTrichodermareeseitransformants,obtaining6superiortransformantswithhigherfilterpapercollapsingrateandgrowingrateonMCC-agarplates.Furtherscreeningexperimentwasconductedundershakingflaskcondition,obtiningthehighlyxo-β-glucanase(C1)-producingtransformantT.reeseiZU-101whoseC1activitywas18.24U·mL-1after48hliquidculture,2.16-foldhigherthantheoriginalstrain.Analysisresultsdemonstratedthatfilterpaperactivity(FPA)ofthecellulasesystemofthetransformant,representingtotalactivity,increasedby61.9%asexo-β-glucanaseactivityascendedsignificantly,althoughendo-β-glucanaseandcellobiaseactivitiesvariedinsignificantlyfromtheoriginalstrain.TheenzymatichydrolysisyieldofT.reeseiZU-101’scellulaseinthehydrolysisofalkalipretreatedcornstoverwas94.4%whentheenzymedosageandhydrolysistimewere20FPIU·g-1substrateand48h,respectively.Theresultsofthepresentworkhavepromisingapplicationprospectsinthebioconversionandutilizationofrenewablecellulosicmaterials.Keywords:recombinantTrichodermareesei;exo-β-glucanases;screeningoftransformants;cornstover;enzymatichydrolysis引言2013-00-00收到初稿，2013-00-00收到修改稿。联系人：夏黎明。第一作者：孔芹（1988—），女，硕士研究生。基金项目：浙江省重点科技创新团队计划资助（2011R50002）。Receiveddate:2013-00-00.纤维素是一种由D-葡萄糖通过β-1，4-糖苷键结合而成的葡聚糖大分子链，作为植物细胞壁的重Correspondingauthor:Prof.XIALiming,xialm@zju.edu.cnFoundationitem:SupportedbytheProgramforZhejiangLeadingTeamofS&TInnovation(2011R50002)网络出版时间：2014-01-1515:23网络出版地址：http://www.cnki.net/kcms/detail/11.1946.TQ.20140115.1523.009.html要成分，是世界上最丰富、最廉价且未得到充分利用的可再生性资源。纤维素生物质如农林业废...
