串珠镰孢菌来源的角质酶基因的克隆表达及其酶学性质  

Expression a Cutinase from Fusarium oxysporum in Pichia pastoris and Characterization

作　　者：陈志琳 林青君 王鹏[1] 叶秀云[1] 李仁宽[1] CHEN Zhi-lin;LIN Qing-jun;WANG Peng;YE Xiu-yun;LI Ren-kuan(Fujian Provincial Key Laboratory of Marine Enzyme Engineering,Fuzhou University,Fuzhou 350116,China)

机构地区：[1]福州大学福建省海洋酶工程重点实验室,福建福州350116

出　　处：《食品工业科技》2018年第18期87-92,共6页Science and Technology of Food Industry

基　　金：福建省海洋高新产业发展专项项目(闽海洋高新[2013]20号);福建省企业技术创新项目(海洋藻类资源高值化利用技术创新平台建设);福州市科技计划项目(2016X0005)

摘　　要：克隆一个来源于串珠镰孢菌(Fusarium moniliforme Sheld)的角质酶基因,该基因全长693 bp,编码231个成熟的氨基酸。克隆的角质酶基因构建到p PIC9K质粒,获得重组表达载体,转入毕赤酵母(Pichia pastoris Gs115)中进行高效表达。经甲醇诱导96 h,测得重组酶酶活为71.68 U/m L,经纯化获得比活力为2490.1 U/mg。对纯化后的角质酶进行酶学性质分析结果表明,其最适反应p H为9.0,在p H5.0~9.0范围内之间重组角质酶的酶活相对稳定;最适反应温度为35℃,在40℃条件下保温1 h酶活力保持70%以上。KCl、Triton X-100、Mn Cl_2、SDS对该酶活有促进作用,Na Cl、Ba Cl_2、Cu SO_4、Tween-20、Fe SO_4、Zn SO_4、Ni Cl_2、EDTA、Tween-80对该酶活有抑制作用。A 693 bp gene and encodes 231 mature amino acids for cutinase from Fusarium moniliforme Sheld was cloned and constructed into p PIC9K plasmid.The recombinant cutinase was hyper-produced extracellularly using Pichia pastoris GS115 as the host.After induction in a shake flask for 96 h,the yield of recombinant cutinase reached 71.68 U/m L,and the enzyme purified displayed a specific activity of 2490.1 U/mg.The optimum pH was 9.0,and the activity of the recombinant cutinase was relatively stable within the range of pH5.0-9.0.The optimal temperature of this enzyme was 35℃,and more than 70%of relative activity was retained after incubation for 1 h at 40℃.The recombinant enzyme activity could be stimulated by concentration of KCl,Triton X-100,MnCl2,SDS,but inhibited by Na Cl,Ba Cl2,Cu SO4,Tween-20,Fe SO4,Zn SO4,NiCl2,EDTA,Tween-80.

关 键 词：角质酶 克隆 分离纯化 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]