Enzymatic synthesis of 3-O-α-maltosyl-L-ascorbate using an engineered cyclodextrin glucanotransferase

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 a-maltosyl fluoride (alpha G2F) and L-ascorbic acid as the donor and acceptor, respectively, yielding 3-O-alpha-maltosyl-L-ascorbate (AA3 alpha G2). The optimum conditions were determined by high-performance liquid chromatography analysis with 20 mM alpha G2F and 40 mM L-ascorbic acid as the substrates at pH 7.5 and 25 degrees C with 1 mg/ml of the enzyme for 24 h. Calcium ions bound in CGT-E284A played an important role in the transglycosylation. CGT-E284A exhibited typical saturation kinetic behaviour for aG2F at a fixed acceptor concentration (40 mM), and substrate inhibition by L-ascorbic acid was observed at high L-ascorbic acid concentrations (>60 mM). AA3 alpha G2 was isolated from a preparative scale reaction with a yield of 29%, and it showed extremely high stability under oxidative conditions. (C) 2014 Elsevier Ltd. All rights reserved.