IDENTIFICATION OF A STARCH-BRANCHING ENZYME AND COEXISTING STARCH BIOSYNTHETIC ENZYMES FROM PARTIALLY PURIFIED MUNG BEAN (VIGNA RADIATA L.) FRACTIONS

Abstract

Three starch-branching enzyme (BE) bands (I, II and III) were detected by zymogram in the soluble extract of developing mung bean (Vigna radiata L. cv KPS1), and radioactive method was used to trace BE during purification by liquid chromatography. Protein profiles of bands I and II were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by proteomic analysis with liquid chromatography–mass spectroscopy/mass spectrometry and bioinformatic searching. The 96 kDa BE possessing specific amino acid sequences of the A-type family of BEs was identified from band II. Mung bean 92 kDa sucrose synthase and the previously identified 105 kDa starch phosphorylase (SP) and its 55 kDa fragments were also found in both bands I and II. Western blotting with SP antibodies showed that SP was present in all of the purified BE fractions. In summary, with the aid of proteomic analysis, we have identified a major mung bean BE and coexisting starch biosynthetic enzymes from partially purified fractions. PRACTICAL APPLICATIONS Mung bean starch possesses a unique structure of 45% dry weight of branched amylose content and is expected to be synthesized by unusual biological machinery. Among the enzymes in the starch biosynthesis pathway of higher plants, branching enzyme (BE) is considered responsible for producing branched structure from amylose and amylopectin chains. We utilized zymogram and radioactive methods to trace BE during purification by liquid chromatography. Protein profiles of active bands were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by proteomic analysis with liquid chromatography–mass spectroscopy/mass spectrometry and bioinformatic searching. A mung bean 96 kDa A-type BE and coexisting starch biosynthetic enzymes, starch phosphorylase and sucrose synthase, able to synthesize starch in vitro, were identified from partially purified fractions. The internal sequences found in mung bean BE enabled the design of degenerate primers for the cloning and expression of BE by molecular techniques to further investigate its physiological role and biotechnology applications in the food industry.