Isolation And Characterization Of Starch, Starch Biosynthetic Genes And Protease Inhibitors From Marama Bean (Tylosema Esculentum)

ABSTRACT

Marama bean (Tylosema esculentum) is a highly nutritious plant and is currently regarded as a prospective crop for the future in arid zone agri-ecologies of the world. Starch is a major storage component in higher plants and in marama bean it’s mostly found stored in the tuber. Starch is used in both food and non-food industries. Starch biosynthesis involves groups of committed enzymes. Aims of the present study were to determine the physicochemical and pasting properties of native marama bean starch isolate and characterize marama starch biosynthesis genes and detect serine protease inhibitor activities in green and mature marama seeds. The total starch content of marama bean tubers was determined by amyloglucosidase/α-amylase enzymatic digestion and amylose content by Concanavalin A precipitation. A complementary Deoxyribonucleic acid (cDNA) library was constructed from marama tuber for the screening and isolation of Soluble Starch Synthase I (SSSI) and a Polymersae Chain Reaction (PCR) based strategy was used to isolate Adenosine diphosphate-glucose pyrophosphorylase (AGPase) and Starch Branching Enzymes (SBEs) using degenerative primers designed at the conserved motif of corresponding cloned plant starch synthesizing genes. Detection of serine protease inhibitor activities in green and mature marama seeds was established using the reverse zymogram technique and fluorogenic substrate N-alpha-benzoyl-l-arginine 7-amido4-methylcoumarin hydrochloride and cDNA clone encoding a serine protease gene from marama was isolated using newly developed degenerate PCR primers. Native marama starch content was 87.38 mg starch/gram fresh weight and the total amylose content was 35 %. Phosphate at the C-6 position determined as Glucose-6-Phosphate was 0.788 nmol G6P/mg. The starch granules were round to elliptical with smooth surfaces and their sizes ranged from 8 -20 µm. The pasting properties of pasting temperature, host paste, peak, final viscosity, breakdown and set back showed higher values for marama starch in contrast to commercial potato starch. A cDNA clone encoding a SSSI from T. esculentum was isolated and identified by cDNA screening. The cDNA clone is 684 bp in length and encodes 228 amino acid residues. Sequencing of cloned cDNA showed 100% identity with potato SSSI.