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A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The
deduced amino acid sequence showed a high degree of identity with ChBP
(chitobiose phosphorylase) from another species, Vibrio furnissii. The
recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2
(chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate
[GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that
the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction,
the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as
well as GlcNAc-1-P to produce
beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the
acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the
acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of
(GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were
determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic
reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the
phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently
observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was
observed in the synthetic reaction. The enzyme was considered a unique
biocatalyst for glycosidation.
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