Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution
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Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-d-glucopyranosyl-l-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.
KeywordsCyclodextrin glycosyltransferase High-efficient production Directed evolution Negative charge High-cell-density fermentation CGTase family
This study was financially supported by the National Natural Science Foundation of China (31730067, 31771916), the Science and Technology Project of Jiangsu Province - Modern Agriculture (BE2018305), the Natural Science Foundation of Jiangsu Province (BK20180082), National first-class discipline program of Light Industry Technology and Engineering (LITE2018-03), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_1832).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
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