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-α-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 Å of the catalytic residue Glu²⁵³ 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.