State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Bioresour Technol. 2013 Oct;146:379-385. doi: 10.1016/j.biortech.2013.07.074. Epub 2013 Jul 26.
In this study, the effects of temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration, and osmolytes (proline, K-glutamate, and betaine) on cell growth and soluble pullulanase productivity of recombinant Escherichia coli were investigated. The yield of soluble pullulanase was found to be enhanced with decrease in cultivation temperature, lower IPTG concentration, and betaine supplementation in a shake flask. In addition, a modified two-stage feeding strategy was proposed and applied in a 3-L fermentor supplied with 20mM betaine, which achieved a dry cell weight of 59.3 g L(-1). Through this cultivation approach at 25 °C, the total soluble activity of pullulanase reached 963.9 U mL(-1), which was 8.3-fold higher than that observed without addition of betaine at 30 °C (115.8 U mL(-1)). The higher expression of soluble pullulanase in a scalable semisynthetic medium showed the potential of the proposed process for the industrial production of soluble enzyme.
在这项研究中，考察了温度、IPTG（异丙基 β-D-1-硫代半乳糖苷）浓度和渗透剂（脯氨酸、K-谷氨酸盐和甜菜碱）对重组大肠杆菌细胞生长和可溶普鲁兰酶生产能力的影响。结果发现，在摇瓶中降低培养温度、降低 IPTG 浓度和添加甜菜碱可以提高可溶普鲁兰酶的产量。此外，提出并应用了一种改良的两阶段补料策略，在 3-L 发酵罐中补加 20mM 甜菜碱，实现了 59.3 g/L 的干细胞重量。通过在 25°C 下进行这种培养方法，普鲁兰酶的总可溶性活性达到 963.9 U/mL，比在 30°C 下不加甜菜碱时观察到的活性（115.8 U/mL）高 8.3 倍。在可扩展的半合成培养基中可溶普鲁兰酶的高表达表明，所提出的工艺具有用于工业生产可溶性酶的潜力。
