Abstract
BackgroundCyclodextrin 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.ResultsIn 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 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 β-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively.ConclusionsN-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-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.
Highlights
Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-Dglucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries
The CGTase from Bacillus genus belonging to glycoside hydrolases family 13 (GH13) is the most commonly used one, which can catalyze three kinds of reactions: cyclization, intramolecular transglycosylation and low hydrolytic reaction [11,12,13]
Cloning and expression of the opt-cgt gene The Opt-CGT was successfully expressed in E. coli BL21 (DE3) according to the results of SDS-PAGE and the
Summary
Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-Dglucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries. CGTase is an industrially important enzyme for α-, βor γ-cyclodextrins (CDs) production, which are extensively used in agriculture, chemicals, cosmetics, foods and pharmaceuticals [7]. The CGTase from Bacillus genus belonging to glycoside hydrolases family 13 (GH13) is the most commonly used one, which can catalyze three kinds of reactions: cyclization, intramolecular transglycosylation and low hydrolytic reaction [11,12,13]. Intramolecular transglycosylation reaction includes disproportionation reaction (transfer the donor substrate to acceptor substrate) and coupling reaction (cleave cyclodextrins and transfer the resulting maltooligosaccharide to acceptor substrate) [11]. The productivities of CGTases from wild strains are relatively low, partly resulting in high production costs for industrial applications. Typical yield of purified α-CG Tas was at a relatively low level by one-step affinity chromatography on Ni2+-NTA resin, and > 95% of the recombinant enzyme was left in the flow-through fraction because of the C-terminal His6-tag was partially inaccessible [14]
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