Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhi...Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.展开更多
利用脂肪酶把餐厨废弃油脂转化成为生物柴油能够达到绿色化、资源化处理餐厨垃圾的目的。从米根霉CICC3005 c DNA文库扩增得到脂肪酶基因(proROL),并克隆到毕赤酵母组成型表达载体p GAPZαA中,电转入毕赤酵母X-33中构建重组毕赤酵母,SDS...利用脂肪酶把餐厨废弃油脂转化成为生物柴油能够达到绿色化、资源化处理餐厨垃圾的目的。从米根霉CICC3005 c DNA文库扩增得到脂肪酶基因(proROL),并克隆到毕赤酵母组成型表达载体p GAPZαA中,电转入毕赤酵母X-33中构建重组毕赤酵母,SDS-PAGE电泳发酵液上清,结果显示重组酶的相对分子质量约为35 000。以橄榄油为底物测得脂肪酶活性为(426.6±0.8)U/m L。利用重组的米根霉脂肪酶对餐厨废弃油脂进行转酯化反应,以乙醇为酰基受体制备脂肪酸乙酯,在水含量为5%,醇油摩尔比为4∶1,酶添加量为10%的条件下得到脂肪酸乙酯的最高得率为49%。展开更多
基金Project supported by the National High-Tech R & D Program (863) of China (No. 2006AA10Z308)the National Science Foundation of China (No. 20776130)+1 种基金the Zhejiang Provincial Natural Science Foundation of China (No. Y4090309)the Zhejiang Provincial Science and Technology Program of China (No. 2009C32009)
文摘Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.
