Abstract
We found a new enzyme, neopullulanase, and proved that the enzyme catalyzes both hydrolysis and transglycosylation at α-(1→4) - and α-(1→6)-glucosidic linkages by one active center. A series of experimental results using neopullulanase indicated that the four reactions described above could be catalyzed in the same mechanism. On the basis of the common catalytic mechanisms and the structural similarities among the enzymes which catalyze the four reactions, we proposed a general concept for an enzyme family, α-amylase family. The substrate specificity and the transglycosylation activity of neopullulanase were altered by site-directed mutagenesis on the basis of information from a threedimesionalstructure predicted by computer-aided molecular modeling. From the standpoint of industrial application, we developed a new way of producing isomalto-oligosaccharide syrup using the transglycosylation reaction of neopullulanase. We also expanded the concept of α-amylase family into branching enzymes and constructed chimeric enzymes of starch branching enzymes I and II isof orms from maize endosperm. The results indicated that the N- and C-terminuses may be involved in determining substrate preference, catalytic capacity, and chain length transfer.
