环糊精葡萄糖基转移酶(Cyclodextrin glycosyltransferase，简称CGT酶)能够通过环化反应生成环糊精，但天然菌株发酵产酶的水平较低，使得环糊精的生产成本居高不下，因此旨在通过异源表达以及发酵优化策略来提高CGT酶的表达量。首先将来源于Bacillus xiaoxiensis STB08的cgt基因插入质粒pET-20b(+)中，在宿主菌Escherichia coli BL21(DE3)中进行分泌表达，在适宜的培养基中发酵96 h后，胞外酶活为34.66 U/mL。然后对发酵条件进行优化，改变溶氧以及添加一定终浓度的Ca2+可使酶活提高到66.86 U/mL及83.15 U/mL，且溶氧与Ca2+协同作用可使酶活提高到105.69 U/mL，相比于优化发酵条件之前提高了204.93%。最后通过蛋白质定位分析及流式细胞分析对溶氧及添加Ca2+影响发酵水平的机理进行了研究。结果表明，较低的溶氧能够减少重组大肠杆菌包涵体的形成，且溶氧较低时细胞活性较高，但溶氧过低时菌体浓度太低，不能满足菌体正常生长及代谢的需求。而添加Ca2+能够减少包涵体的形成，同时Ca2+对细胞有较好的保护作用，因此，在溶氧以及Ca2+的协同作用下，能够保证菌体的高生长浓度，且Ca2+能够增加细胞透性并保护细胞，使得活细胞数目显著增多。该研究提高了CGT酶在大肠杆菌中的可溶性表达量，为该酶的工业化生产提供了新的策略及方法，也可为相关酶的发酵优化提供参考。
Cyclodextrin glycosyltransferase (CGTase) can generate cyclodextrin by cyclization reaction; however, the production is limited in natural bacterial strains, which results in high costs for cyclodextrin production. Thus, the authors aimed to enhance the expression of CGTase by heterologous expression and fermentation optimization strategies. First, the cgt gene derived from Bacillus xiaoxiensis STB08 was inserted into the plasmid pET-20b(+) and expressed in the host strain Escherichia coli BL21(DE3). After 96 h of fermentation in a suitable culture medium, the extracellular enzyme activity reached 34.66 U/mL. Subsequently, the fermentation conditions were optimized by changing the dissolved oxygen and adding a certain final concentration of Ca2+, which led to an increase in enzyme activity to 66.86 U/mL and 83.15 U/mL, respectively. Furthermore, the synergistic effect of dissolved oxygen and Ca2+ increased the enzyme activity to 105.69 U/mL, representing a 204.93% improvement compared to that before fermentation optimization. Finally, the mechanism of dissolved oxygen and Ca2+ addition on the fermentation level was studied by protein localization analysis and flow cytometry. The results showed that the lower dissolved oxygen could reduce the formation of inclusion bodies in recombinant E. coli, with higher cellular activity observed at lower dissolved oxygen levels. However, the excessively low dissolved oxygen resulted in insufficient cell density to meet the needs of normal bacterial growth and metabolism. The addition of Ca2+ could reduce the formation of inclusion bodies and provide good cellular protection. Therefore, the synergistic effect of dissolved oxygen and Ca2+ ensured a high growth concentration of bacteria, and Ca2+ increased cell permeability and protected the cells, resulting in a significant increase in viable cell count. This study improved the soluble expression of CGTase in E. coli, providing a new strategy and method for its industrial production, as well as serving a reference for the fermentation optimization of the related enzymes.
ZHOU Yuanyuan, LI Zhaofeng, GU Zhengbiao, BAN Xiaofeng, HONG Yan, CHENG Li, LI Caiming. Ca2+ Coordinated Dissolved Oxygen to Improve Soluble Expression of Recombinant β-CGTase[J]. Journal of Food Science and Biotechnology,2023,42(10):16-23.