栓菌漆酶在毕赤酵母中高效表达及重组酶的性质  

High Output of a Trametes Laccase in Pichia pastoris and Characterization of Recombinant Enzymes

作　　者：周宏敏[1] 洪宇植[1] 肖亚中[1] CUI Teng-Jiao WANG Xiao-Tang 蒲春蕾[3] 

机构地区：[1]安徽大学生命科学学院＆现代实验技术中心,合肥230039 [2]佛罗里达国际大学化学与生物化学系,迈阿密FL33199,美国 [3]中国科学技术大学生命科学学院,合肥230026

出　　处：《生物工程学报》2007年第6期1055-1059,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos30370045;30470056和30670069);安徽省优秀青年基金(No04043048);安徽省教育厅自然基金重点(No2006KJ049A);国家863高技术研究与发展计划项目(No2007AA09Z421);安徽大学211创新团队计划(No02203109)资助~~

摘　　要：栓菌420（Trametes sp．420）漆酶基因lacD以两种方式在巴斯德毕赤酵母（Pichiapastoris）进行异源表达，产生两种重组漆酶：rLacDx（具有天然N-末端）和rLacDe（N-末端带有8个额外的氨基酸残基）。摇瓶发酵18d，rLacDx和rLacDe的产量分别为1．21×10^5u／L、7、38×10^4u／L[以2，2＇-连氮-3-乙苯-二噻唑-6磺酸（ABTS）为底物]。在高密度发酵条件下，rLacDx的产量增加到2．39×10^5u／L，同时其生产周期降至7．5d。两种重组酶对愈创木酚底物的氧化特性相似，且在50℃和pH3～10的范围内均稳定。然而，rLacDx对底物ABTS的比活力（1761u／mg）高于rLacDe（1122u／mg），其表观Km值（427μmol／L）低于rLacDe（604μmol／L）。A laccase gene （lacD） from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 × 10^5u/L and 7.38 × 10^4 u/L, respectively, as determined with 2,2＇-azinobis（3-ethylbenzothia-zoline- 6-sulfonic acid） （ABTS） as substrate. The yield of rLacDx was further increased to 2.39 × 10^5u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50℃ and at a pH range from 3 to 10. However, the specific activity of rLacDx （1,761u/mg） for ABTS was higher than that of rLaeDe （1,122u/mg）, and the apparent Km value of rLaeDx （427 μM） was less than that of rLaeDe （604 μM）.

关 键 词：异源表达 高密度发酵 漆酶 毕赤酵母 栓菌420 

分 类 号：Q78[生物学—分子生物学]