植物乳杆菌ZS2058的亚油酸异构酶基因在乳酸克鲁维酵母中的克隆表达  

Cloning and Expression of the Linoleate Isomerase Gene from Lactobacillus plantarum ZS2058 in Kluyveromyces lactis GG799

作　　者：朱敬华[1] 陈海琴[1] 张白曦[1] 田丰伟[1] 赵建新[1] 陈卫[1] 张灏[1] 

机构地区：[1]江南大学食品学院食品科学与技术国家重点实验室,无锡214122

出　　处：《中国生物工程杂志》2010年第4期65-70,共6页China Biotechnology

基　　金：国家"863"计划(2007AA100402);"十一五"国家科技支撑计划重点项目(2006BAD27B08)资助项目

摘　　要：目的:将植物乳杆菌ZS2058(Lactobacillus plantarum ZS2058)的亚油酸异构酶基因在乳酸克鲁维酵母(Kluyveromyces lactis)中进行克隆表达。方法:根据NCBI中已报道亚油酸异构酶(linoleate isomerase,LAI)基因的序列特征,设计引物对筛得的植物乳杆菌ZS2058进行PCR扩增,得到亚油酸异构酶全基因序列,克隆至乳酸克鲁维酵母表达载体pKLAC1,电转化得重组菌pKLAC1-LAI/Kluyveromyces lactis GG799。结果:SDS-PAGE检测,重组菌进行分泌表达获得目的蛋白,大小约为67kDa;气相色谱(Gas Chromatogram,GC)检测到共轭亚油酸(conjugated linoleic acids,CLA)典型峰。结论:植物乳杆菌ZS2058中的亚油酸异构酶基因在乳酸克鲁维酵母中得到分泌表达,重组酶转化效率约为26%。Objective：To express the Linoleate isomerase gene of Lactobacillus plantarum in Kluyveromyces lactis.Method：Linoleate isomerase （LAI）gene was amplified by PCR from chromosome of Lactobacillus plantarum ZS2058,then the gene was cloned into Kluyveromyces lactis expression vector pKLAC1,the recombinant plasmid pKLAC1-LAI was transformed into Kluyveromyces lactis GG799 by electroporation,The expression level and the activity of recombinant enzyme were detected by SDS-PAGE and Gas Chromatogram.Result：It was demonstrated that a 67 kDa protein which was equal to LAI in molecular weight was secreted in supernatant culture.The analysis of gas chromatography showed that there was a noticeable peak in the retention time of 30.304 min in recombinant broth,the peak was conjugated linoleic acid.Conclusion：It showed that the linoleate isomerase gene from L.plantarum ZS2058 had been expressed and secreted by Kluyveromyces lactis GG799.Enzyme activity experiments showed that the recombinant enzyme can converse about 26% LA into CLA.

关 键 词：共轭亚油酸 亚油酸异构酶 乳酸克鲁维酵母 分泌表达 气相检测 

分 类 号：Q78[生物学—分子生物学]