College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, China; Hunan Flag Bio-tech Co. Ltd, Changsha 410100, China.
Hunan Flag Bio-tech Co. Ltd, Changsha 410100, China.
Enzyme Microb Technol. 2018 Dec;119:65-70. doi: 10.1016/j.enzmictec.2018.08.006. Epub 2018 Aug 24.
Penicillin acylase is commonly used to produce the medical intermediates of 6-Aminopenicillanic acid (6-APA) and 7-Aminodesacetoxycephalosporanic acid (7-ADCA) in industrial process. Nowadays, Penicillin G acylase (PGA) has been widely applied for making pharmaceutical intermediates, while penicillin V acylase (PVA) has been less used for that due to its low activity and poor conversion. In this study, a PVA from Bacillus sphaericus (BspPVA) was employed for directed evolution study with hoping to increase its catalytic efficiency. Finally, a triple mutant BspPVA-3 (T63S/N198Y/S110C) was obtained with 12.4-fold specific activity and 11.3-fold catalytic efficiency higher than BspPVA-wt (wild type of BspPVA). Moreover, the conversion yields of 6-APA catalyzed by BspPVA-3 reached 98% with 20% (w/v) penicillin V as substrate, which was significantly higher than that of the BspPVA-wt (85%). Based on the analysis of modeling, the enhancement of specific activity of mutant BspPVA-3 was probably attributed to the changes in the number of hydrogen bonds within the molecules. The triple mutant PVA developed in this study has a potential for large-scale industrial application for 6-APA production.
青霉素酰化酶通常用于工业生产中 6-氨基青霉烷酸（6-APA）和 7-氨基脱乙酰氧基头孢烷酸（7-ADCA）的医药中间体。如今，青霉素 G 酰化酶（PGA）已广泛应用于制药中间体的生产，而青霉素 V 酰化酶（PVA）由于活性低、转化率差，应用较少。本研究采用来源于球形芽孢杆菌的青霉素 V 酰化酶（BspPVA）进行定向进化研究，希望提高其催化效率。最终获得了一个三重突变体 BspPVA-3（T63S/N198Y/S110C），其比活和催化效率分别比野生型 BspPVA 提高了 12.4 倍和 11.3 倍。此外，BspPVA-3 催化 6-APA 的转化率达到 98%，而以 20%（w/v）青霉素 V 为底物时，BspPVA-3 的转化率明显高于野生型 BspPVA（85%）。基于建模分析，突变体 BspPVA-3 比活的提高可能归因于分子内氢键数量的变化。本研究开发的三重突变体 PVA 具有大规模工业生产 6-APA 的潜力。
