Abstract
The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.
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