Starch modification using starch branching enzymes from cassava manihot esculenta crantz

Abstract

Starch branching enzymes (SBE) is a member of the α-amylase family. SBE, an α-1,4-glucan-6-glucosyltransferase (EC 2.4.1.18), introduces branch points in the amylose and amylopectin molecules by hydrolysis of α-1,4-glucan chain from the non-reducing end and catalyzes the formation of an α-1,6 cross linkage between the reducing end of the cleaved chain and a glucose residue on the same oligosaccharide chain. In our laboratory, a few cassava starch metabolizing enzymes have been isolated and their genes were cloned. One of the enzymes was SBE cloned from cassava sbe genes. In this research work, two recombinant SBEs, SBERI and SBERII, were expressed and purified. The purified recombinant SBEs were used to modify cassava starch, amylose and amylopectin. SBERI and SBERII were incubated with cassava starch, amylose and amylopectin. The products were identified by HPAEC-PAD technique on a carbopac PA-1 and PA-100 columns. Optimum condition for SBERI activity on cassava starch was 37oC, 15 units of activity, 10% (w/v) of cassava starch and 12 hours incubation time while SBERII showed optimum activity at the same condition but at 3 hours incubation time. When substrate was changed to amylose or amylopectin, SBERI showed activity towards amylose but not amylopectin and the optimum conditions was 37oC, 10 units activity, 2% (w/v) of amylose and 12 hours incubation time. SBERII was active to only amylopectin and the optimum conditions for SBERII activity was 37oC, 10 units activity, 2% (w/v) of amylopectin and 3 hours incubation time. At the determined optimum conditions, analysis of side chain distribution on PA-1 showed that the starch modified by SBERI and SBERII contained higher branches than unmodified substrates and SBERII also yielded medium rings cycloamyloses when analyzed with PA-100 column on HPAEC-PAD. Characterization of the physical properties of the modified product of cassava starch, amylose and amylopectin showed low retrogradation, high thermo reversibility and low freeze-thaw stability.