Isolation and characterization of starch debranching enzyme from tuber of cassava manihot esculenta crantz

Abstract

Debranching enzyme, one of enzymes involved in starch biosynthesis in plants, catalyzes the hydrolysis of alpha-1,6-glucosidic linkages specifically in alpha-glucan. The enzyme can show either pullulanase or isoamylase activity or both. Debranching enzyme was detectable in parenchyma of cassava tuber but not in cortex. Starch debranching enzyme was purified from parenchyma of cassava tuber by 20-50% saturated ammonium sulfate precipitation followed by chromatographies on DEAE-Sepharose and Sephadex G-150, which resulted in purity of 18 folds. The chromatographic profile showed a peak of pullulanase activity with molecular weight of 103 kDa on Sephadex G-150 and 35 kDa on SDS-PAGE. The debranching enzyme had a pI of 4.75, a pH optimum of 6.0, optimum temperature 37 ํC, and stable at 4-40 ํC. The enzyme showed high specificity for pullulan as a substrate, but lower activity with soluble starch and amylopectin. The K[subscript m] for pullulanase was 0.88 mg/ml. It can be activated by DTT but inhibited by NEM and IAA, indicating the involvement of SH-group on pullulanase activity. The enzyme was activated by Mn[superscript 2+] and Co[superscript 2+] but inhibited by Ni[superscript 2+], Hg[superscript 2+]and Cu[superscript 2+].