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

Abstract

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 Glu 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 values of the mutant K228R/M230L decreased by 35%, whereas the k/ K 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, 624 mM) reported to date after optimization of the reaction conditions.

Heterologous Expression of Cyclodextrin Glycosyltransferase my20 in Escherichia coli and Its Application in 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid Production

“…As shown in Figure 7, the effects of L-AA and β-CD concentrations on the synthesis of AA-2G by recombinant CGTase were explored. The experimental data were fitted to a ping-pong mechanism, which is consistent with the experimental results of Tao et al (2018). The kinetic parameters of the recombinant enzyme were calculated as follows: V max = 21.6 ± 0.13 U/mg, K mA = 9.2 ± 0.06 g/L, K mB = 6.9 ± 0.04 g/L, and K cat = 99,105 h −1 .…”

“…The enzymatic synthesis of AA-2G followed the method described in Tao et al (2018). Briefly, 0.5 g L-AA and β-CD were accurately weighed and dissolved into 5 ml water.…”

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes

“…substrate scope ISM C3-epimerization of d-fructose and l-sorbose [126] Cyclodextrin Glycosyltransferase (Bacillus stearothermophilus NO2) activity ISM ISM on nine residues in the binding pocket [127] ADH-A from Rhodococcus ruber DSM 44 541 stereo-and regioselectivity ISM ISM combinedw ith activity screening [128] approaches was application of the Sloning technique of solidphase gene synthesis,i nw hich we compared ac onventional SM library with the Sloning counterpart. [145] TheS loning library appeared to have ah igher quality,b ut at the time we neglected to perform massive DNAs equencing for reliable assessment.…”

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

“…Enzymatic synthesis is a preferred method for AA‐2G production over alternative chemical synthesis owing to the regioselective glucosylation reaction. [ 10 ] Up to now, AA‐2G has mainly been synthesized by transglucosylation of L‐AA catalyzed by several enzymes, including α ‐glucosidase, [ 12,13 ] cyclodextrin glycosyltransferase (CGTase), [ 14 ] amylase, [ 15 ] sucrose phosphorylase, [ 16,17 ] and α ‐isomaltosylglucosaccharide synthase. [ 18 ] Specially, it should be noted that the synthesis of AA‐2G by α ‐glucosidase from higher eukaryote such as rice seed and mammals has the advantages of less intermediates and by‐products.…”