Purification and characterization of starch synthase from cassava Manihot esculenta Crantz tubers

Abstract

Cassava starch is one of the major raw materials used in many industries. Starch has two major components, amylose and amylopectin, which were synthesized by starch synthase and other enzymes. Starch synthase exists in two forms, granule-bound and soluble starch synthase. They are enzymes that synthesize alpha(1, 4)-glucosidic linkage in amylose and amylopectin. In this study, soluble starch synthase was extracted from cassava tubers and purified by precipitation at 20-60% saturated ammonium sulfate, followed by Phenyl Sepharose, Sephadex G-200 and Q-Sepharose column chromatographies. The chromatographic proriles showed a single peak of starch synthase activity with molecular weight of 53.4 kDa on Sephadex G-200. The enzyme preparation obtained was purified up to 220 folds with 2.8% recovery. The purified cassava soluble starch synthase showed optimum pH and temperature at 8.5 and 28 ํC, respectively. The Q-Sepharose preparation of starch synthase showed 2 bands on non- denaturing polyacrylamide gel which appeared on second dimension electrophoresis as 3 bands with molecular weight of 79, 76 and 53.8 kDa. The pI of these proteins were 6.91, 6.41, respectively. These data suggested the possible existence of 3 isoforms of cassava starch synthase. The enzyme utilized rabbit liver glycogen as primer better than oyster glycogen, amylopectin, amylose, starches, and short chain glucose oligomers, respectively. The Km's for ADP-glucose and rabbit liver glycogen were 0.10 mM and 1.31 mg/ml respectively. It can be inhibited by thiol modifying reagents, indicating the involvement of SH-group on cassava starch synthase activity.