Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-alpha-d-Glucopyranosyl-L-Ascorbic Acid Production
文献类型： 外文期刊
作者： Song, Kai 1 ; Sun, Jingjing 2 ; Wang, Wei 2 ; Hao, Jianhua 2 ;
作者机构： 1.Shanghai Ocean Univ, Coll Food Sci & Technol, Shanghai, Peoples R China
2.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Key Lab Sustainable Dev Polar Fishery, Minist Agr & Rural Affairs, Qingdao, Peoples R China
3.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Drugs & Bioprod, Qingdao, Peoples R China
4.Jiangsu Collaborat Innovat Ctr Exploitat & Utiliz, Lianyungang, Peoples R China
关键词： 2-O-alpha-D-glucopyranosyl-L-ascorbic acid; cyclodextrin glucosyltransferase; E. coli; heterologous expression; I-ascorbic acid
期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：5.64； 五年影响因子：6.32 ）
ISSN： 1664-302X
年卷期： 2021 年 12 卷
页码：
收录情况： SCI
摘要： 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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