酿酒酵母脂肪酶基因TGL3的克隆及其在毕赤酵母中的高效表达

Cloning and High Expression of a Lipase Gene TGL3 from Saccharomyces cerevisiae in Pichia pastoris

目的:克隆Saccharomy cescerevisiae脂肪酶基因,并实现其在Pichia pastoris中高效表达。方法:根据NCBIGenBank中已公布的Saccharomyces cerevisiae脂肪酶基因TGL3设计引物,以Saccharomyces cerevisiae总DNA为模板,PCR扩增目的片段,构建重组子pPIC9K-TGL3,转化到Pichia pastoris GS115。结果:扩增得到1929bp的基因片段,与GenBank中公布的序列氨基酸同源性达99.7%,编码的643个氨基酸中有脂肪酶的保守序列GXSXG,位于235～239位。重组子摇瓶发酵120h发酵液上清脂肪酶酶活达到60U/mL,酶学性质研究表明:该酶的最适作用温度为40℃,在25℃～40℃之间能保持60%以上酶活力。最适pH值为8.5,在pH6.5～9.0之间能保持50%以上的酶活力。除Al3+和Fe2+外,该酶不受Ca2+、Mg2+、Zn2+、Cu2+、Mn2+、Ni+、Sr2+等多种金属离子的影响。结论:发酵液上清脂肪酶酶活达到60U/mL,是原菌株的10倍,成功实现了该基因的高效表达。

Objective：The lipase gene TGL3 from Saccharomyces cerevisiae was cloned and achieved high expression in Pichia pastoris.Method：According to the published lipase gene TGL3 of Saccharomyces cerevisiae in NCBI GenBank,a pair of primers were designed and synthesized.Using the Saccharomyces cerevisiae total DNA as template,the target gene was cloned into expression vector pPIC9K to construct the recombinant lipase expression vector,then transformed into Pichia pastoris GS115.Result：The lipase gene of 1 929bp was amplified,which was 99.7% identical to the amino acids sequence published in GenBank.The sequence that encoding 643 amino acids had conserved sequence GXSXG of lipase,which was located in 235-239.After induction for 120h in shake flasks,the yield of enzyme activity achieved to 60U/mL in the supernatant.Enzymatic properties showed that the lipase exhibited maximum activity at 40℃ and pH8.5.It still showed more than 60% enzyme activity between 25℃-40℃ and more than 50% enzyme activity between pH 6.5-pH 9.0.Except for Al3 ＋ and Fe2 ＋,others metal ion such as Mg2 ＋,Zn2 ＋,Cu2 ＋,Mn2 ＋,Ni ＋,Sr2 ＋ had no affect to the enzyme activity.Conclusion：After induction in shake flasks,the yield of enzyme activity achieved to 60U/mL in the supernatant,which was 10 times of the original strain.The lipase achieved successfully high expression in Pichia pastoris.