Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35

The gene encoding an intracellular isoamylase from the Pectobacterium chrys anthemi PY35 was cloned in Escherichia coli DH5 alpha and sequenced. The is oamylase gene (amyX) had an open reading frame of 1974 bp encoding 657 amin o acid residues with a calculated molecular weight of 74,151 Da. The molecu lar weight of the enzyme was also estimated to be 74 kDa by activity staini ng of a SDS-PA gel. Isoamylase from P. chrysanthemi PY35 had 59% pairwise a mino acid identity with glycogen debranching enzyme from E. coli and contai ned the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7 and 40 degreesC. AmyX hydrolyzed alpha -1-6-g lycosidic linkages of amylopectin, while did not hydrolyze alpha -1,4-glyco sidic linkages of amylose. (C) 2001 Academic Press.