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| Optimization of Fermentation for ACE C- Domain from Pichia pastoris |
| XU Jue,XU Chuan-lian,DU Fang-yao |
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Abstract 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. In this study, 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 con- centrations 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 (OD600) 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.
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Received: 08 January 2010
Published: 29 April 2010
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