Enhanced 2-O-α-D-Glucopyranosyl-L-ascorbic Acid Synthesis through Iterative Saturation Mutagenesis of Acceptor Subsite Residues in Bacillus stearothermophilus NO2 Cyclodextrin Glycosyltransferase

Low synthesis yields of the L-ascorbic acid (L-AA) derivative 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA -2G) limit its application in the food industry. In this work, the AA -2G synthesis yield of Bacillus stearothermophilus NO2 cyclodextrin glycosyltransferase (CGTase) was improved. Nine residues within 10 A of the catalytic residue Glu(253) displaying <= 30% conservation and located in the acceptor subsite were selected for iterative saturation mutagenesis. The best mutant, K228R/M230L, produced a higher AA -2G yield with maltodextrin as the glucosyl donor than that produced by its parent wild-type. The L-AA K-m values of the mutant K228R/M230L decreased by 35%, whereas the k(cat)/K-m increased by 2.69-fold. Kinetic analysis indicated that K228R/M230L displayed enhanced L-AA specificity. These results demonstrate that acceptor subsite residues play an important role in acceptor substrate specificity. Mutant K228R/M230L afforded the highest AA -2G concentration (211 g L-1, 624 mM) reported to date after optimization of the reaction conditions.