米黑根毛霉脂肪酶在毕赤酵母中的高效表达及酶学性质研究

根据毕赤酵母(Pichia pastoris)密码子使用偏爱性,对米黑根毛霉脂肪酶(RML)成熟肽基因进行全面密码子优化,以提高RML基因在酵母细胞中表达的水平,并对重组脂肪酶的酶学性质进行了研究。运用重叠延伸PCR合成优化后的RML成熟肽基因,并进一步构建毕赤酵母表达载体p PIC9K-RML,通过电击法转化毕赤酵母GS115菌株,经G418抗性和PCR筛选获得高拷贝转化重组子。重组酵母菌用0.5%甲醇诱导异源基因表达,在28℃摇瓶培养168 h后酶活力达到122 U/m L,较未优化的原始菌株的表达酶活提高了10倍。重组脂肪酶最适温度为45℃,最适p H值为7,甲醇体积分数为40%以下时重组RML具有较好的稳定性。

Expression of Lipase Gene from Rhizomucor miehei in Pichia pastoris and Properties of Lipase

Rhizomucor miehei lipase ( RML ) gene was designed using yeast ’s prefered codon to improve its expression level in Pichia pastoris Vector and the properties of RML was studied. According to the bias in codon choice of the high expression gene in P. pastoris, the mature portion of RML gene with high specific activity was optimized and artificially synthesized by overlap extension PCR without changing the amino acid sequence. The RML gene was subcloned into expression vector pPIC9K to yield the recombinant expression vector pPIC9K-RML. The recombinant plasmid was transformed into P. pastoris GS115 by electroporation. Subsequently, the positive clone was selected by G418 and PCR and the high expression recombinant P. pastoris GS115-RML was obtained. After induced by 0. 5% methanol for 168 h at 28℃, the activity of lipase achieved 122 U/mL and was 10 times higher than that of the unoptimized wild type R. miehei. The expression of RML gene in P. pastoris could be successfully improved by using codon optimization. The optimum reaction conditions of recombinant RML were temperature 45℃and pH value 7. At these conditions, when RML reacted in 40% methanol for 100 min,the remaining activity was 96. 9%. This showed that RML displayed a good stability in methanol.