毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域

血管紧张素转化酶（ACE, EC3.4.15.1）在调节血压方面具有重要作用。研究证实，ACE的C结构域（ACE-C）是使血管紧张素I (AngI)分解的主要活性位点。在5 L 发酵罐中, 对重组毕赤酵母表达ACE C-结构域的发酵工艺进行优化，探讨温度、pH、甲醇浓度等主要因素对重组蛋白表达量和酶活力的影响。结果表明,当工业培养基添加2%蛋白胨为氮源时, ACE C-结构域的降解现象得到了有效控制;采用诱导温度为26℃,pH5.5,甲醇含量为1.5%的表达条件，ACE C-结构域表达量和酶活力分别达到446 mg/L和38.2U/ml，比活力达到86U/mg，是Sigma公司ACE标准品比活力的2倍，为大规模制备ACE C-结构域蛋白，筛选专一性更强的ACE C-结构域抑制剂奠定了基础。

Optimization of Fermentation for ACE C-Domain from Pichia pastoris

Angiotensin I-converting enzyme(ACE,EC3.4.15.1) plays an important role in regulating blood pressure.Now,ACE C-domain is identified to be the main site of angiotensin I cleavage in vivo.The high expression recombinant Pichia pastoris was constructed and the screening tests was performed in 5 L bio-reactor to obtain the optimal values of several key fermentation parameters.Based on effects on the expression level,the optimal values for the temperature,the concentrations of methanol and the pH were 26℃,1.5%(V/V) and 5.5,respectively.Addition of 2% polypeptone to substrate would effectively repress proteolysis.The application of these optimal parameters successfully achieved high-throughput production:the cell density(OD_(600)) of recombinant Pichia pastoris and the yield of crude target protein were respectively 397 mg/L and 446 mg/ L.After the purification with Ni-NTA columns,ACE C-domain was collected with a purity of 98.6%,and the specific activity of it was reached 86 U/mg,which is double fold than that of ACE purchased from Sigma.This provided a zymolytic condition to be used for ACE C-domain in industrial scale production,and provided a high specific activity enzyme for screening specific inhibitor to ACE C-domain.