少根根霉(Rhizopus arrhizus)脂肪酶基因在毕赤酵母中的分泌表达

Expression of Rhizopus arrhizus lipase in P.pastoris

利用PCR技术从少根根霉中扩增出脂肪酶基因(包括前导序列和成熟肽),并将其连接到酵母分泌表达载体pPIC9K中,转化毕赤酵母GS115。利用抗生素G418从重组阳性克隆中筛选得到高拷贝的转化子。在5 L的发酵罐中,当碳源耗尽后开始流加甲醇诱导脂肪酶的表达,经过96 h培养后发酵液上清液中重组脂肪酶(rRAL)的表达量约为90 mg/L。rRAL经过超滤,SP-Sepharose离子交换层析和Butyl-Sepharose疏水层析纯化。纯化后的蛋白在SDS-PAGE上为单一条带,表观分子量为32 kDa,比酶活为1 543 U/mg。N-端序列分析表明rRAL是经过加工后的产物。同时没有发现全长的Rhizopus arrhizus脂肪酶(RAL)被分泌表达。

The lipase gene（ including prosequence and mature lipase）was cloned from Rhizopus arrhizus L-03- R-1. The extracellularly active production of recombinant Rhizopus arrhizus lipase （rRAL） was carried out by the expression the lipase gene using pPIC9K vector. A transformed Pichia pastoris clone containing the multicopy lipase gene was isolated through G418 screening. In a fed-batch cultivation, where, methanol feeding was controlled by on-line methanol analyzer, the supematant contained 90 rng/L rRAL after 96 h of cultivation. rRAL was purified by uhrafihration, SP-Sepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. The molecular weight of rRAL is 32 kDa. The specific lipase activity of rRAL was 1 543 U/ mg. The N-terminal analysis showed that rRAL was the processed production. The complete lipase was not expressed by pichia pastoris.