期刊
BMC BIOTECHNOLOGY
卷 18, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s12896-018-0463-9
关键词
Cyclodextrin glucanotransferase; Optimized codons; Glycosyl donors; 2-O-alpha-D-glucopyranosyl-L-ascorbic acid
资金
- National Natural Science Foundation of China [31700092, 21706125, 21727818, 21706124]
- Postgraduate Research & Practice Innovation Program of Jiangsu Province [KYCX18_1109]
- Jiangsu Province Natural Science Foundation for Youths [BK20170993, BK20170997]
- Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture [XTE1834]
- Project of State Key Laboratory of Materials-Oriented Chemical Engineering [KL17-09]
Background: Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries. Results: In this study, the cgt gene encoding alpha-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U.mg(-1). In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including beta-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively. Conclusions: N-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His(6)-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. alpha-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.
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