title: Enzymatic synthesis of 3-O-α-maltosyl-l-ascorbate using an engineered cyclodextrin glucanotransferase.
abstract: A mutant derived from a cyclodextrin glucantransferase with an alanine residue as its acid/base catalyst residue (CGT-E284A) catalyzed regioselective glycosylation at 3-OH of l-ascorbic acid using α-maltosyl fluoride (αG2F) and l-ascorbic acid as the donor and acceptor, respectively, yielding 3-O-α-maltosyl-l-ascorbate (AA3αG2). The optimum conditions were determined by high-performance liquid chromatography analysis with 20mM αG2F and 40mM l-ascorbic acid as the substrates at pH 7.5 and 25°C with 1mg/ml of the enzyme for 24h. Calcium ions bound in CGT-E284A played an important role in the transglycosylation. CGT-E284A exhibited typical saturation kinetic behaviour for αG2F at a fixed acceptor concentration (40mM), and substrate inhibition by l-ascorbic acid was observed at high l-ascorbic acid concentrations (>60mM). AA3αG2 was isolated from a preparative scale reaction with a yield of 29%, and it showed extremely high stability under oxidative conditions.
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Republic of Korea.
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Ahn
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Republic of Korea.
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Republic of Korea.
authors.forename: Hye-Bin
authors.initials: HB
authors.lastname: Cho
authors.affiliation: Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon 406-840, Republic of Korea.
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Park
authors.affiliation: Center for Food and Bioconvergence, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea; Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: pschang@snu.ac.kr.
authors.forename: Pahn-Shick
authors.initials: PS
authors.lastname: Chang
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Republic of Korea. Electronic address: ywankim@korea.ac.kr.
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D008320:Maltose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid
keywords: 3-O-α-Maltosyl-l-ascorbate; Acid/base mutant; Cyclodextrin glucanotransferase; Transglycosylation; l-Ascorbic acid