利用Sed1p锚定蛋白在毕赤酵母表面展示米黑根毛霉脂肪酶及其应用

Surface Display of Rhizomucor Miehei Lipase in Pichia pastoris Using Sed1p as an Anchor Protein and Its Application

摘要:

通过PCR扩增米黑根毛霉脂肪酶基因, 在米黑根毛霉脂肪酶N端加入Flag标签。将米黑根毛霉脂肪酶基因与酿酒酵母细胞壁蛋白Sed1p基因的N端融合构建质粒pPIC9K-Flag- RML-Sed1, 转化毕赤酵母GS115获得重组菌GS115/pPIC9K-Flag-RML-Sed1。重组菌经过甲醇诱导表达后, 显微镜免疫荧光分析与流式细胞仪检测结果均证实米黑根毛霉脂肪酶已经成功展示在毕赤酵母上。该重组菌水解活力达到169.6 U/g (Dry cell weight), 在非水相中催化脂肪酸甲酯的合成, 72 h后脂肪酸甲酯的产率达82.36%。

Abstract:

We constructed a Rhizomucor Miehei Lipase(RML)-Displaying yeast whole-cell biocatalyst and applied it to methylesters synthesized from triglyceride and methanol. RML was fused with the Sed1p cloned from Saccharomyces cerevisiae to constructed plasmid pPIC9K-Flag-RML-Sed1. The plasmid was linearized and transformed into Pichia pastoris GS115 and Pichia pastoris recombinant strain GS115/pPIC9K-Flag-RML-Sed1 was obtained. Cell-surface display of the RML via Sed1p was confirmed by flow Fluorescence micrograph and flow cytometer. After incubated at 28°C for 48 h the hydrolytic activity of the GS115/pPIC9K-Flag-RML-Sed1 reached a plateau, 169.6 U/g (Dry cell weight). In nonaqeous media, the yield of 82.36% methylesters was obtained from triglyceride and methanol after 72 h using lyophilized RML displaying yeast whole cells.