耐有机溶剂脂肪酶基因lipI的克隆及其在毕赤酵母中的高效表达

Cloning and High-level Expression of Methanol-tolerant Lipase Gene lipI in Pichia pastoris

为了实现耐有机溶剂脂肪酶基因的真核表达,参照GenBank公布的脂肪酶基因序列设计简并引物,通过PCR扩增出G.geotrichumSXL-107的全长1 692bp、编码563个氨基酸的脂肪酶基因lipI,基因登录号为JX074060。利用Signal P 3.0软件对lipI基因序列进行分析,将去除自身信号肽的成熟lipI基因克隆到诱导型表达载体pPIC9K上,构建胞外重组表达载体pPIC9K li-pI,转化毕赤酵母KM71,发酵培养72h,发酵上清液中脂肪酶活力达到70U/mL,与野生型脂肪酶产生菌Galactomyces geotrichum SXL 107相比,脂肪酶活力提高7倍。获得的重组脂肪酶的最适温度40℃、pH值为6.5,在10%的甲醇溶液中作用48h后仍然保持60%以上的酶活力。

For the expression of lipase gene in Pichia pastori,the lipI gene of G.geotrichumSXL-107 was cloned,which was 1 692 nucleotides long and encoded 563 amino acid residues.The mature lipase gene lipI without the signal peptide was cloned into inducible expression vector pPIC9K.The extracellular expression vector pPIC9K-lipI was constructed and transformed into Pichia pastoris KM71.Cultures of recombinant P.pastoris accumulated active enzyme in the supernatant up to 70 U/mL after induction for 72 h,which was 7 folds of lipase activity compared to the wild-type lipase.The optimum temperature and pH value for obtaining recombinant lipase were 40 ℃ and pH6.5,and the enzyme activity remained more than 60% in 10% methanol solution for 48 h.