Characterization of a Purified β–Amylase from Black Marble Vine (Dioclea reflexa) Seeds

Abstract

A thermostable β-amylase was purified from the seeds of black Marble vine (Dioclea reflexa). The purification of this enzyme involved a combination of 30-80% ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was determined by 10% sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE). Effects of pH, temperature and some metallic ions on the enzyme activity, substrate specificity as well as the kinetic parameters of the purified enzyme were determined. The enzyme was purified 12.25 fold with 20.60% yield, giving a final specific activity of 9.38 µmol/min/mg. The molecular weight was 56.23 kDa. The enzyme was found to be thermostable with optimum pH and temperature of 6.0 and 60 oC respectively. The enzyme activity was activated by Zn2+, Na+, and Mg2+. The enzyme activity was slightly inhibited by Ca2+ but strongly inhibited by Cu2+, Hg2+, SDS, ethylenediaminetetraacetic and Urea at both 10 mM and 20 mM. The apparent Km and Vmax of the enzyme were 0.42 mg/mL and 1.23 μmol/min/mL respectively when soluble starch was used as substrate. The distinctive physicochemical properties of this enzyme in coexistence with its abundance in D. reflexa suggest it will be a good enzyme for use in starch utilizing industries especially food and fermentation industries.