Enzymological Studies on Isomalto-dextranase from Arthrobacter globiformis.
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Isomalto-dextranase (EC 3.2.1.94) was purified from the culture of a soil bacterium, Arthrobacter globiformis T 6 by successive chromatographies on CM-cellulose and CM-sepharose to a homoge neous state as confirmed by PAGE. The molecular weight of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme hydrolyzed α-1, 6-glucosidic linkages of dextran or isomalto oligosaccharides to release exolytically α-isomaltose from the non-reducing ends . The optimum pH and temperature of the enzyme were pH 5.3 and 65°C, respectively . The enzyme showed a weak isopullulanase activity, an endo-type attack on pullulan to produce isopanose. The isomalto dextranase expressed by the recombinant E. coli cells also produced isopanose from pullulan . The enzyme hydrolyzed α-1, 4-glucosidic linkage of panose as well as α-1, 6-glucosidic linkage of isomaltotriose. The kinetic features of the experiments with the mixed substrates of isomaltotriose and panose were in good agreement with those expected for a single catalytic site mechanism. The ionization constants, pKel and pKe2, of the essential ionizable groups 1 and 2 of the enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both the substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate . Modification experiments with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), modifying carboxyl residues specifically, also indicated that the carboxyl groups were essential to the enzyme activity . The subsite affinities of the enzyme were culculated to be >7.3, <-7.2, 6.7, 0.74 and 0.18 kcal/mol for subsites 1, 2, 3, 4 and 5, respectively, from the rate parameters (Km and 10) for the hydrolysis of isomaltooligosaccharides. Subsites 1 and 3, showing large affinity values, were thought to attract the substrates and form the productive bindings. A new method for preparation of isomaltose was developed by using the enzyme and an acid-treated dextran. The branch points of dextran were selectively hydrolyzed by a mild acid pretreatment. When the acid-treated dextran was acted on by the enzyme, the maximal degree of hydrolysis went up to over 90%.
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