门多萨假单胞菌Pseudomonas mendocina P10脂肪酶基因的克隆及其在酵母中的表达

Cloning of the lipase gene of Pseudomonas mendocina P10 and its expression in Pichia pastoris

摘要: 采用同源克隆法获得了门多萨假单胞菌Pseudomonas mendocina P10脂肪酶基因的全长序列,DNA测序结果表明,该基因的DNA全长序列为801bp,编码267个氨基酸,推测蛋白相对分子量为29.95ku。将该脂肪酶基因连接到pPICZαA载体上得到重组表达质粒,转化Pichia pastoris X-33。脂肪酶基因在Pichia pastoris X-33中实现了分泌性表达,摇瓶发酵液上清酶活最大可达8U/mL。

Abstract: The genomic sequence of P. mendocina P10 lipase (pml) was isolated by homologous cloning method. The result showed that full-length DNA sequence of P. mendocina P10 lipase (pml) was 801bp in size, encoding a protein of 267 amino acids with a calculated molecular mass of 29.95ku.The DNA fragment coding lipase was subcloned into the pPICZαA expression vector and the reconstructed expression plasmid pPICZαA -pml was then transformed into Pichia pastoris X33. The pml was actively expressed in the Pichia pastoris X33 and the recombinant lipase protein was secreted into the cultural broth, the maximum activity of the culture supernatant was 8U/mL, when the recombinant strain was cultured on a rotary shaker.