Improved purification and further characterization of an isomalto-dextranase 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. E1%1cm 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 5 HIM Hg2+, Fe3+, KMnO4, and NBS. The enzyme split dextran, retaining the α-configuration of the anomeric carbon atoms in the hydrolysis products. The Km value of the enzyme for dextran was 0.038%. Isomaltitol was a potent competitive inhibitor (Ki=0.79 mM)