Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz ( Me PUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and r Me PUL was biochemically characterised. Me PUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca 2+ ions. Pullulan is the most favourable substrate for r Me PUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of r Me PUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when r Me PUL was mixed with cassava isoamylase 3 (r Me ISA3), compared to that of each single enzyme reaction. This suggests that Me PUL and Me ISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of Me PUL in starch catabolism, which normally takes place in plastids.