Isolation and characterization of isofroms of soluble starch synthase from Manihot esculenta CRANTZ cultivar Kasetsart 50 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)- glycosidic linkage in amylase 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 and Q-Sepharose column chronatographies. The purification step could isolate three isoform of soluble starch synthase that are SSS1, SSS2 and SSS3. These enzymes were purified up to 426, 513 and 142 fold, respectively. When analyzed the enzymes by SDS-polyacrylamide gel electrophoriesis found that SSS1 showed two protein bands with molecular weight 92.4 and 81.7 kDa, SSS2 showed three protein bands with molecular weight 112, 90.1 and 79.8 kDa and SSS3 showed two protein bands with molecular weight 90.1 and 79.8 kDa. All isoforms of soluble starch synthase showed optimum pH and temperature at 9.5, 7.9, 9.5 and 37, 35, 37 [degree celsius], respectively. SSS1, SSS2 and SSS3 used branched polysaccharides and starch with high amylopectin contents as primer better than linear polysaccharides. The apparent K [subscript m] values of SSS1, SSS2 and SSS3 for ADP-glucose were 0.09, 0.06 and 0.08 mM, respectively, whereas the K[subscript m] values of enzymes for rabbit liver glycogen were 1.46, 0.26 and 0.13 mg/ml, respectively. Moreover, all three isoforms could be inhibited by thiol modifying reagents, indicating the involvement of SH-group on cassava starch synthase activity.