重组环糊精葡萄糖基转移酶温控型工程菌的构建及其培养条件的优化

Construction of Temperature-Regulated Recombinant Escherichia coli for β-CGTase Expression and Optimization of Culture Conditions

采用聚合酶链式反应(polymerase chain reaction,PCR)法从蜡状芽孢杆菌(Bacillus cereus)中扩增了β-环糊精葡萄糖基转移酶(β-cyclodextrin glucanotransferase,β-CGTase)基因,将该基因克隆到pBV220质粒中,转化E.coli DH5α,经氨苄青霉素抗性筛选和酶切验证后,得到能表达β-CGTase的重组大肠杆菌E.coli DH5α/pBVcgt。通过对工程菌产酶条件的优化,得到最佳产酶条件为:OD600 nm值达到1.0、初始培养温度30℃、发酵培养基初始pH 8.0、温度梯度诱导39℃培养0.5 h,40℃培养0.5 h,41℃培养1 h,42℃培养2 h。酶活力由优化前的445 U/mL提高到956 U/mL,酶活力提高了1.15倍。温度梯度诱导比直接诱导酶活力提高20%。该酶的克隆表达以及发酵条件的研究表明,该酶能在大肠杆菌原核表达体系中高效表达。

The β-CGTase gene from Bacillus cereus was cloned in pBV220 plasmid after PCR amplification. The recombinant plasmid was transformed into E. coli DH5α. After ampicillin-resistance screening and restriction enzyme digestion analysis, we acquired recombinant E. coli DH5α/pBVcgt. The optimum fermentation conditions in shaking flask were acquired as follows: OD600 nm = 1.0, initial culture temperature 30 ℃, initial medium pH 8.0, and gradient-temperature induction at 39 ℃ for 0.5 h followed by 40 ℃ for 0.5 h, 41 ℃ for 1 h and 42 ℃ for 2 h. The activity of β-CGTase under these fermentation conditions was increased from 445 to 956 U/mL, representing a 2.15-fold increase compared to that obtained before optimization. Cloning and expression of the β-CGTase gene showed that this enzyme could be expressed highly in E. coli expression system. Therefore, this study proves the possibility of protein engineering of β-CGTase for largescale production of β-CD.