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

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.