期刊
FRONTIERS IN MICROBIOLOGY
卷 12, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2021.664339
关键词
2-O-alpha-D-glucopyranosyl-L-ascorbic acid; cyclodextrin glucosyltransferase; E. coli; heterologous expression; I-ascorbic acid
类别
资金
- National Key Research and Development Program of China [2018YFC0311106]
- Provincial Key Research and Development Program of Shandong [2019GHY112030]
- Central Public-interest Scientific Institution Basal Research Fund, CAFS [2020TD67]
- Shinan Science and Technology Plan of Qingdao [2018-4002-ZH]
This study successfully obtained the CGT gene from marine microorganisms, optimized its expression and purification, and applied it for the synthesis of AA-2G, achieving a high production yield.
In this study, the cgt gene my20, which encodes cyclodextrin glycosyltransferase (CGTase) and was obtained by the metagenome sequencing of marine microorganisms from the Mariana Trench, was codon optimized and connected to pET-24a for heterologous expression in Escherichia coli BL21(DE3). Through shaking flask fermentation, the optimized condition for recombinant CGTase expression was identified as 20 degrees C for 18 h with 0.4 mM of isopropyl beta-D-L-thiogalactopyranoside. The recombinant CGTase was purified by Ni2+-NTA resin, and the optimum pH and temperature were identified as pH 7 and 80 degrees C, respectively. Activity was stable over wide temperature and pH ranges. After purification by Ni2+-NTA resin, the specific activity of the CGTase was 63.3 U/mg after 67.3-fold purification, with a final yield of 43.7%. In addition, the enzyme was used to transform L-ascorbic acid into 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G). The maximal AA-2G production reached 28 g/L, at 40 degrees C, pH 4, 24 h reaction time, 50 g/L donor concentration, and 50 U/g enzyme dosage. The superior properties of recombinant CGTase strongly facilitate the industrial production of AA-2G.
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