耐热羧肽酶Taq基因在毕赤酵母中的表达

Expression of the thermostable carboxypeptidase Taq gene in Pichia pastoris GS115

摘要:

为了获取表达羧肽酶Taq毕赤酵母工程菌，通过密码子优化，依据毕赤酵母密码子偏爱性，在体外合成了栖热水生菌的耐热羧肽酶Taq基因。将该基因克隆到毕赤酵母表达载体pHBM905A上并引入6×His标签，构建了重组质粒pHBM905A-Cpase Taq。将该重组质粒转化毕赤酵母GS115，经1%甲醇诱导表达72 h，酶产量达0.1 mg/mL。对纯化的重组酶进行酶活性分析表明在75 ℃，pH为7.5时，该酶比酶活性为80 U/mg。本研究首次证明了羧肽酶Taq能在毕赤酵母中有效分泌表达，可以被大量制备，进而为多肽水解为氨基酸奠定工业基础。

Abstract:

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6×His?tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 ℃ and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because?of?its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.