Cloning and Characterization of an Intracellular Isoamylase Gene from Pectobacterium chrysanthemi PY35

Abstract

The gene encoding an intracellular isoamylase from the Pectobacterium chrysanthemi PY35 was cloned in Escherichia coli DH5α and sequenced. The isoamylase gene (amyX) had an open reading frame of 1974 bp encoding 657 amino acid residues with a calculated molecular weight of 74,151 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS–PA gel. Isoamylase from P. chrysanthemi PY35 had 59% pairwise amino acid identity with glycogen debranching enzyme from E. coli and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7 and 40°C. AmyX hydrolyzed α-1,6-glycosidic linkages of amylopectin, while did not hydrolyze α-1,4-glycosidic linkages of amylose.