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

Karan Wangpaiboon; Thanapon Charoenwongpaiboon; Methus Klaewkla; Robert A Field; Pawinee Panpetch

doi:10.3389/fpls.2023.1114215

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

Front Plant Sci. 2023 Jan 27:14:1114215. doi: 10.3389/fpls.2023.1114215. eCollection 2023.

Authors

Karan Wangpaiboon¹, Thanapon Charoenwongpaiboon², Methus Klaewkla¹, Robert A Field³, Pawinee Panpetch^{1

4}

Affiliations

¹ Center of Excellence in Structural and Computational Biology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
² Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand.
³ Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
⁴ Center of Excellence in Molecular Crop, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.

Abstract

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 (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL 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 Ca²⁺ ions. Pullulan is the most favourable substrate for rMePUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

Keywords: cassava (Manihot esculenta Crantz); debranching enzyme; pullulan; pullulanase; starch; starch degradation; synergistic debranching.

Grants and funding

This research was supported by Thailand Science research and Innovation Fund Chulalongkorn University [BCG66230003 to PP]. KW received a Second Century Fund (C2F) Postdoctoral Fellowship of Chulalongkorn University. This work [Grant No. RGNS 64-014] is also supported by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI) and Chulalongkorn University. This research project is supported by grants for development of new faculty Staff, Ratchadaphiseksomphot Fund, Chulalongkorn University”.