Improved Purification and Further Characterization of an Isomaltodextranase from Arthrobacter globiformis T6

Abstract

A highly purified isomalto-dextranase (EC 3.2.1.94) was simply separated from the cell-free culture broth of Arthrobacter globiformis T6 by CM-cellulose column chromatography and characterized further. The purified enzyme was essentially homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. The enzyme was an acidic protein with pI 5.15. at 280 nm was 24.6. The molecular weight of the enzyme was estimated to be about 69,000 by SDS-PAGE. No carbohydrate moiety seemed to be associated with the enzyme protein.

The optimum pH and temperature of the enzyme was pH 5.3 and 65°C, respectively. The enzyme was completely stable over the range of pH 3.0 ~ 8.0 at 4°C for 24 hr, and retained about 90% of its original activity after heating at 60°C for 10 min. Inactivation of the enzyme was found to be partial with 5mm Ag ⁺, and complete with 5mm Hg²⁺, Fe³⁺, KMnO₄, and NBS. The enzyme split dextran, retaining the α-configuration of the anomeric carbon atoms in the hydrolysis products. The K_m value of the enzyme for dextran was 0.038%. Isomaltitol was a potent competitive inhibitor (K_i= 0.79 mm).