Abstract
BackgroundCyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly γ-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble γ-CGTase by Escherichia coli were investigated.ResultsThe yield of soluble γ-CGTase in shake-flask culture approximately doubled when β-cyclodextrin was added to the culture medium as a chemical chaperone.When a modified two-stage feeding strategy incorporating 7.5 mM β-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L− 1 was achieved. Using this cultivation approach, the total yield of γ-CGTase activity (50.29 U·mL− 1) was 1.71-fold greater than that observed in the absence of β-cyclodextrin (29.33 U·mL− 1).ConclusionsSince β-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble γ-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.
Highlights
Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others
Effect of chemical chaperones on γ-CGTase production by E. coli In this set of experiments, we assessed the efficacy of Ca2+, proline, betaine, sorbitol, and cyclodextrins as chemical chaperones in shake-flask culture
No inducer was added to the TB medium, but 0.5% (w/v) glycine was added to enhance the secretion of γ-CGTase
Summary
Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, γ-CGTase, requires their efficient production. Cyclodextrin glycosyltransferases (EC 2.4.1.19, CGTase) catalyze the transglycosylation (cyclization, disproportionation, coupling) and hydrolysis of linear α-(1,4)-linked glucans [1]. These enzymes can synthesize cyclodextrins from low-cost starch resources by cyclization. Inclusion bodies generally accumulate in the crowded cytoplasm or periplasmic milieu of E. coli during the protein overexpression process [9].Several strategies have been exploited to reduce the formation of inclusion bodies in vivo, including co-expression of chaperone genes, addition of “chemical chaperones”, and lowering the culture temperature and
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