Characterization of recombinant 4-α glucanotransferase from cassava Manihot esculenta Crantz and Arabidopsis thaliana

Abstract

Cassava is one of the most important starch sources in tropical region. The synthesis and degradation pathway of starch in the tubers are considerable commercial important. Currently, there is no clear picture of the metabolic processes involved in cassava starch metabolism. One of the enzymes believed to be involved in the starch catabolic pathway is the disproportionating enzyme (4-α-glucanotransferase) (DPE1) (E.C. 2.4.1.25). DPE1 transfers a maltosyl unit of (1,4)-alpha-D-glucan to an acceptor sugar that can produce longer chain oligosaccharides. Previous report on DPE1 in leaves of Arabidopsis thaliana indicated that DPE1 can specifically use maltotriose as substrate to produce longer oligosaccharides and glucose. This work reports the cloning and expression of DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz caltivar KU50 (MeDPE1) in Escherichia coli. Purified recombinant AtDPE1 and MeDPE1 were biochemically characterized on their molecular weight, optimum pHs, optimum temperatures, the stability of the enzymes and the enzyme kinetic parameters. Recombinant MeDPE1 and AtDPE1 were able to produce longer chain oligosaccharides from maltotriose by transferring maltosyl unit and showed different properties to amylomaltase (E.coli) and DPE2 from Arabidopsis thaliana (AtDPE2) in the same glycoside hydrolase family 77 (GH77). Enzyme in GH77 family can transfer glucan to fluoroglucose derivatives to produce longer chains, MeDPE1 was also able to produce fluorooligosaccharide derivatives. The fluoromaltose derivatives produced from AtDPE2 reaction mixture were purified and characterized by ¹⁹F-NMR, ¹H-NMR and high resolution mass spectrometry. Oligosaccharide array was used to find the acceptor specificity of DPE1 which showed that AtDPE1 can use maltooligosaccharide with alpha-4-glucan as acceptor. Large ring cyclodextrins were produced from amylose and degree of polymerization analysed by HPAEC-PAD and MALDI-TOF-MS techniques. Mixture of cycloamyloses can form complexes with polyaniline which resulted in change of UV-Vis absorption.