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The transglycosylation reaction catalyzed by neopullulanase was analyzed.
Radioactive oligosaccharides were produced when the enzyme acted on maltotriose
in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had
only alpha-(1----4)-glucosidic linkages, but others were suggested to have
alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic
linkages in the products from maltotriose with neopullulanase was proven by
proton NMR spectroscopy and methylation analysis. We previously reported that
the one active center of neopullulanase catalyzes the hydrolysis of
alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H.,
Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts
proved that neopullulanase catalyzed all four types of reactions: hydrolysis of
alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic
linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and
transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions
are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin
glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These
four enzymes have some structural similarities to one other, but reactions
catalyzed by the enzymes are considered to be distinctive: the four reactions
are individually catalyzed by each of the enzymes. The experimental results
obtained from the analysis of the reaction of the neopullulanase exhibited that
the four reactions can be catalyzed in the same mechanism.
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