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

Xiumei TaoTian WangLingqia SuJing Wu
Published in: Journal of agricultural and food chemistry (2018)
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 Km values of the mutant K228R/M230L decreased by 35%, whereas the kcat/ Km 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.
Keyphrases
  • wild type
  • solar cells
  • energy transfer
  • magnetic resonance imaging
  • ionic liquid
  • image quality
  • computed tomography
  • mass spectrometry
  • climate change
  • water soluble