Several mutants of chitinase A1 from Bacillus circulans WL-12 have been prepared by site-directed mutagenesis of tryptophan 433. The hydrolytic activity of these mutants toward p-nitrophenyl chitobioside was lower than that of wild-type chitinase A1. The mutants were found to catalyze the transglycosylation reaction of the 1,2-oxazoline derivative of N-acetyllactosamine (Gal-GlcNAc-oxa) to N,N′-diacetylchitobiose (GlcNAc-Glc-NAc), giving rise to a transglycosylated tetrasaccharide product (Gal-GlcNAc-GlcNAc-GlcNAc) in high yield. Based on these results, a one-pot synthesis of chitooligosaccharides by combined use of mutant W433Y and β-galactosidase has been demonstrated. By controlling the reaction conditions, chitotriose and chitotetraose were obtained in 74% and 40% yield (determined by HPLC), respectively, without isolating any intermediate products.
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