Abstract
The effects of temperature on extracellular secretion of the α-cyclodextrin glucosyltransferase (α-CGTase) from Paenibacillus macerans JFB05-01 by Escherichia coli were investigated. When protein expression was induced at constant temperature, the greatest amount of extracellular recombinant α-CGTase was produced at 25 °C. Higher or lower induction temperatures were not conducive to extracellular secretion of recombinant α-CGTase. To enhance extracellular secretion of α-CGTase by E. coli, a two-stage temperature control strategy was adopted. When expression was induced at 25 °C for 32 h, and then the temperature was shifted to 30 °C, the extracellular α-CGTase activity at 90 h was 45% higher than that observed when induction was performed at a constant temperature of 25 °C. Further experiments suggested that raising the induction temperature can benefit the transport of recombinant enzyme and compensate for the decreased rate of recombinant enzyme synthesis during the later stage of expression. This report provides a new method of optimizing the secretory expression of recombinant enzymes by E. coli.
Highlights
The cyclic oligosaccharides α, β, and γ-cyclodextrin consist of 6, 7, and 8 glucose units, respectively, linked by α-1, 4-glycosidic bonds
Shifting the temperature to 34 °C at 24 h further accelerated the increase in extracellular α-CGTase activity when comparing with the increase seen at 30 °C, but in this culture, the plateau was reached earlier and the activity at 90 h was not a significant improvement over the level seen in the control culture
High initial induction temperature hinders the α‐CGTase translocation Extracellular α-CGTase production occurs in a series of steps
Summary
The cyclic oligosaccharides α-, β-, and γ-cyclodextrin consist of 6, 7, and 8 glucose units, respectively, linked by α-1, 4-glycosidic bonds. Cyclodextrins form inclusion complexes with many different small, hydrophobic guest molecules, improving their solubility and stability in aqueous environments. This property makes it have many applications in scientific, medical and industrial fields (Roy et al 2017). The industrial use of α-cyclodextrin is in its infancy, yet is still expanding because of its small internal cavity, high water solubility, and resistance to enzymatic hydrolysis. Work focused on CGTase production in Bacillus strains (Gawande et al 1998; Rosso et al 2002), and efforts were made to improve CGTase yield by manipulating environmental factors (Arce-Vazquez et al 2016; Es et al 2016). The strict regulatory mechanisms present in wild-type strains have limited productivity enhancements, resulting in high costs and low yields
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