State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Laboratory of Biopesticide and Chemical Biology (Ministry of Education), College of Life Sciences & College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China.
Fuzhou Center for Disease Control and Prevention, Fuzhou, 350004, Fujian, China.
World J Microbiol Biotechnol. 2019 Jan 21;35(2):23. doi: 10.1007/s11274-019-2595-z.
A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5 µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20 mg/L. The purified BtCspB showed a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.
一种新型苏云金芽孢杆菌（Bt）细菌素 BtCspB 被鉴定并纯化，其活性针对食源性病原体蜡状芽孢杆菌，这是我们实验室之前通过传统的四步色谱法实现的，产量低（44.5µg/L）。本研究的目的是通过异源表达 BtCspB 显著提高其产量。Bt BRC-ZYR2 中的 BtCspB 基因成功在大肠杆菌 BL21 (DE3) 中异源表达。亲和层析可获得高达 20mg/L 的纯 BtCspB。纯化的 BtCspB 对蜡状芽孢杆菌 ATCC 10987 的 MIC 值为 12.5µg/mL，MBC 值为 50.0µg/mL。BtCspB 对 ATCC 10987 的抑菌活性在凝胶覆盖实验中直接检测。然而，抗蜡状芽孢杆菌活性低于传统四步色谱法纯化的细菌素，可能是由于结构修饰所致。与传统方法相比，异源表达的细菌素产量增加了 449 倍，且纯化步骤大大简化，为该新型针对蜡状芽孢杆菌的冷休克蛋白样细菌素 BtCspB 的工业化生产奠定了基础。
