Position 228 in Paenibacillus macerans cyclodextrin glycosyltransferase is critical for 2-O-d-glucopyranosyl-l-ascorbic acid synthesis

The markedly stable l-ascorbic acid (L-AA) derivative 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) has been widely used in the fields of food, medicine, cosmetics, and husbandry. Cyclodextrin glycosyltransferase (CGTase) is considered suitable for the large-scale production of AA-2G. In this work, Paenibacillus macerans CGTase was used to produce AA-2G and the production was 13.5 g/l. An amino-acid sequence alignment of α-, β-, and α⁄β-CGTase indicated that the Phe at position 228 of P. macerans CGTase was different from the amino acids at this position in other CGTases (Met, Val, or Ile). In addition, the CGTases from Anaerobranca gottschalkii and Bacillus circulans 251, which have Val and Met at position 228, were shown to produce 28.9 and 35.7 g/l AA-2G, respectively, which verified the importance of this position for AA-2G synthesis. Subsequently, P. macerans CGTase mutants F228M and F228V were constructed and shown to produce 24.8 g/l and 24.0 g/l AA-2G, respectively, which are 84% and 78% higher than that of wild-type P. macerans CGTase, respectively. Kinetic analysis of AA-2G synthesis showed that affinities of the two mutants for L-AA and the catalytic efficiencies increased. Meanwhile, the mutants had lower cyclization activity but higher disproportionation activities, which is beneficial for AA-2G synthesis. All these results indicated that amino acid at position 228 of P. macerans CGTase is crucial to AA-2G synthesis.