Purification and characterization of isoforms of starch branching enzyme from tuber of cassava Manihot esculenta CRANTZ

Abstract

Cassava starch is one of the major raw material used in many industries. Starch is composed of the polysaccharides, amylose and amylopectin, which were synthesized by a group of enzymes including starch branching enzyme (SBE). Starch branching enzyme involved amylopectin synthesis by catalyzing formation of alpha-1, 6-glucosyl-alpha-1,4-glucan. There are two classes of, class A preferentially branches amylopectin whereas class B preferentially branch amylose. In this study, starch branching enzyme was monitored in cassava tubers from two cultivars, Rayong 1 and KU 50. SBE activity rapidly increased around 9 months old. Purification and isoform studies were performed with tubers from 9 months old KU 50. The crude enzyme was purified by precipitation with 10% (w/v) polyethylene glycol, followed by DEAE-Toyopearl column which could separate SBE into 3 isoforms. Hitrap Q-sepharose column was further used to purify each isoform which resulted in purities of 125, 77 and 250. All isoform showed molecular weight of 57 kDa on Sephacryls HR S-200. Isoform 1 separated by SDS- PAGE, into 2 bands with molecular weight 108 and 57 kDa whereas isoform 2 and 3 showed single band with molecular weight of 57 and 60 kDa. The pI of the 3 isoform was 4.9, 4.9 and 5.0, respectively. Optimum pH{7f2019}s were 8.0, 6.5, 7.5 and optimum temperatures were 37, 37 and 30 ํC for isoform 1, 2 and 3, respectively. All isoforms were stable up to 40 ํC. The K[subscript m]'s for amylose were 1.12, 1.37 and 2.17 mg/ml. Isoform 1 and 2 were more active than isoform 3 towards amylose as substrate, whereas isoform 3 was more active than isoforms 1 and 2 with amylopectin as substrate. All isoforms were absolutely inhibited by divalent metal ion.