Lactobacillus reuteri PYR8亚油酸异构酶基因在毕赤酵母中的表达

Expression of linoleate isomerase gene from Lactobacillus reuteri PYR8 in Pichia pastoris

以Lactobacillus reuteriPYR8菌株基因组DNA为模板,利用PCR方法扩增出亚油酸异构酶(linoleate isomerase,LI)基因,将其克隆至酵母表达载体pPIC9K中,电转化至毕赤酵母GS115中.重组菌株GS115/pPIC9K-LI经1%甲醇诱导后,SDS-PAGE电泳表明该基因在酵母中得到表达,即分子量约为67 kD的重组LI蛋白.经SP-Sepharose Fast Flow阳离子交换层析、Phenyl Sepharose Fast Flow疏水层析纯化获得了纯度为95%的重组LI蛋白样品.气相色谱分析表明,纯化的重组LI具有亚油酸异构酶的活性,酶的比活力为6.8 U?mg-1.

Linoleate isomerase gene was amplified by PCR from chromosome of Lactobacillus reuteri PYR8, then the gene was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-LI was transformed into P. pastoris GS115 by electroporation and was induced with 1% methanol, it was demonstrated by SDS-PAGE that a 67 kD protein which was equall to LI in molecular weight was expressed in supernatant culture. The recombined LI protein with purity up to 95% was finally obtained after purification through two-step chromatography：SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. The analysis of gas chromatography showed that the recombinant LI protein exhibited the specific activity of conveting linoleic acid to conjugated linoleic acid, which was 6.8 U. mg^-1.