TY - JOUR
T1 - Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production
AU - Jiang, Yujia
AU - Zhou, Jie
AU - Wu, Ruofan
AU - Xin, Fengxue
AU - Zhang, Wenming
AU - Fang, Yan
AU - Ma, Jiangfeng
AU - Dong, Weiliang
AU - Jiang, Min
N1 - Publisher Copyright:
© 2018 The Author(s).
PY - 2018/8/31
Y1 - 2018/8/31
N2 - Background: Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-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 α-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 Ni 2+ -NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75U.mg -1 . In addition, the enzyme was adopted to transform L-AA into 9.1g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5g/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 Ni 2+ -NTA resin is appreciably enhanced. α-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.
AB - Background: Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-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 α-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 Ni 2+ -NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75U.mg -1 . In addition, the enzyme was adopted to transform L-AA into 9.1g/L of AA-2G. Finally, more economic substrates, including β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5g/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 Ni 2+ -NTA resin is appreciably enhanced. α-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.
KW - Cyclodextrin glucanotransferase
KW - Glycosyl donors, 2-O-α-D-glucopyranosyl-L-ascorbic acid
KW - Optimized codons
UR - http://www.scopus.com/inward/record.url?scp=85052619322&partnerID=8YFLogxK
U2 - 10.1186/s12896-018-0463-9
DO - 10.1186/s12896-018-0463-9
M3 - 文章
C2 - 30170578
AN - SCOPUS:85052619322
SN - 1472-6750
VL - 18
JO - BMC Biotechnology
JF - BMC Biotechnology
IS - 1
M1 - 53
ER -