AUTHOR=Wangpaiboon Karan , Charoenwongpaiboon Thanapon , Klaewkla Methus , Field Robert A. , Panpetch Pawinee 

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

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1114215

DOI=10.3389/fpls.2023.1114215

ISSN=1664-462X

ABSTRACT=<p>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 <italic>Manihot esculenta</italic> Crantz (<italic>Me</italic>PUL) tubers, an important economic crop. The <italic>Mepul</italic> gene was successfully cloned and expressed in <italic>E. coli</italic> and r<italic>Me</italic>PUL was biochemically characterised. <italic>Me</italic>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<sup>2+</sup> ions. Pullulan is the most favourable substrate for r<italic>Me</italic>PUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of r<italic>Me</italic>PUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when r<italic>Me</italic>PUL was mixed with cassava isoamylase 3 (r<italic>Me</italic>ISA3), compared to that of each single enzyme reaction. This suggests that <italic>Me</italic>PUL and <italic>Me</italic>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 <italic>Me</italic>PUL in starch catabolism, which normally takes place in plastids.</p>