Characterization of starch debranching enzyme from tuber of cassava Manihot esculenta crantz cv. KU50

Abstract

Starch debranching enzyme (DBE), one of the emzymes involved starch biosynthesis in plant, hydrolyzed the a-1,6 linkages of polyglucans was classified into two types; lsoamylase and pullulanase. DBE from nine moths old cassava tubers cv.KU50 was purified by 60% saturated ammonium sulfate precipitation followed by chromatographies on DEAE-Sepharose and Sephacryl S-200. lsoamylase and pullulanase were 14.6 and 20 folds purified. on sephacryl S-200, molecular weight of lsoamylase and pullulanase were 98 and 175 kDa respectively. on SDS-PAGE, lsoamylase showed 2 major protein bands containing 41 and 34 kDa, and pullulanase showed 3 major protein bands of 54, 46 and 41 kDa. Both lsoamylase and pullulanase had optimum pH at 6.0 while the optimum temperatures for their activities were 70 and 50oC, respectively. pullulanase showed high specificity towards pullulan and law specificity with amylopectin. lsoamylase showed high specificity with amylopectin but could not hydrolyza pullulan. the Km for amylopectin with lsoamylase was 21.14 mg/ml while the Km for pullulan of pullulanase was 39.49 mg/ml. the sulfhydryl reagents such as DTT, GSH and B-mercaptoethanol showed positive effect on CBE, indicating that –SH group involved in DBE activity. lsoamylase was inhibited by Cu2+ and activated by Co2+ while pullulanase was inhibited by Cu2+ and Ni2+ and activated by Co2+, Mn2+.