Abstract: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.
