白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达

Cloning and overexpression of lipase gene from Geotrichum candidum Y162

摘要:

借助生物信息学, 对已克隆的地霉属脂肪酶全长基因序列进行同源比对, 根据保守序列设计引物, 在基因组DNA和cDNA水平上, 于国内首次克隆了Geotrichum candidum Y162脂肪酶基因。G. candidum Y162脂肪酶基因全长1692bp, 不含内含子, 编码包括19个氨基酸信号肽在内的563个氨基酸。与NCBI GenBank中已报道的地霉属脂肪酶氨基酸序列有86％的一致性。将该基因克隆到pPIC9K表达载体上, 转化毕赤酵母GS115, 摇瓶发酵96h后毕赤酵母分泌表达55 U/mL重组脂肪酶, 实现了脂肪酶的高效表达。酶学性质研究表明, 该重组脂肪酶对C9位顺式双键的甘油酯具有明显的底物特异性; 对甲醇、甘油等有机溶剂呈现耐受性; 最适温度和最适pH分别为50℃和8.0, 在pH 6.0~10.0及60℃以下能保持60%以上的酶活力。底物特异性、有机溶剂、温度及pH耐受性赋予该重组酶良好工业应用潜力。

关键词:白地霉Y162; 脂肪酶; 基因; 克隆; 表达

Abstract:

By means of bioinformatics, we aligned nucleotide sequence of reported lipase gene from Geotrichum. Primers were designed based on the conservative nucleotide sequence, and the lipase gene of G. candidum Y162 was cloned for the first time in China. Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides without any introns, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues, which is 86% identical to lipase I of G. fermentans. Subsequently, we cloned the lipase gene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 55 U/mL after induction for 96 hours in shake flasks. The purified lipase exhibited maximum activity at 50°C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60°C. Lipase activity was compatible with the presence of organic solvents such as methanol, n-heptane, hexane, cyclohexane, glycerol, benzene and diethyl ether. Lipase showed hydrolysis prefer-ence for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.

Key words:Geotrichum candidum Y162; lipase; gene; cloning; expression