猪肝酯酶在甲醇酵母中的克隆与表达

Cloning and Expression of Pig Liver Esterase in Pichia pastoris

猪肝酯酶(Pig Liver Esterase,PLE)是手性合成工业中最常用的三种生物催化剂之一,它的 生化提取物是多种同工酶(α、β和γ亚基)的混合物,而它的单一组分(γ亚基)有很高的手性拆分 活性。为获得重组PLE,从猪肝提取总RNA,经RT-PCR得到γ亚基目的基因,并利用pPIC9K质 粒构建成pPIC9K-PLE表达载体,转化到Pichia pastoris中,转化细胞株摇瓶发酵表达重组PLE达 450mg／L,活性染色证明分子量略低于天然酶。

Pig Liver Esterase （PLE） is one of the best catalysts using in the chiral industry, and the pure enzyme （containing only γsubunit ） has better catalytic ability. To get pure protein, total RNA of pig hver was extracted and the aimed gene was obtained by RT-PCR. The expressing plasmid, pPIC9K-PLE, was constructed successfully and transformed into Pichia pastoris afterwards. The recombinants were fermented and induced. The amount of protein expressed was up to 450mg/L. The MW of product was smaller than the natural one as shown by active staining.