Biochemical Insights into the functionality of a novel thermostable β-amylase from Diocleareflexa

Abstract

β-amylases are exohydrolases that catalyze the hydrolysis of α-1,4 glycosidic linkages in polysaccharides to produce maltose. Despite various applications of plant β-amylases, thermolability of the enzyme has been a major challenge that continuously prompts the search for thermostable β-amylases. In this study, β-amylase was isolated from an underutilized legume, marble vine (Dioclea reflexa) and purified to apparent homogeneity, using a combination of 30–80% ammonium sulfate precipitation, ion exchange and gel filtration chromatography. It was characterized for its physicochemical properties. The enzyme gives a purification fold and specific activity of 12.25 and 9.43 U/mg respectively, it has a molecular weight of 56.23 kDa using 12% SDS-PAGE. The kinetic parameters Km and Kcat of the enzyme for p-nitrophenyl-maltopentaoside were 0.22 mM and 1340 s−1. The enzyme had an optimum pH of 6.0 while the optimum temperature was 60 °C. while the thermal inactivation rate constants (kd) value of the enzyme was 0.002, thermal inactivation (ΔG) was 99.1 kJ/mol, ΔH and ΔS were 27.7 kJ/mol and −305.80 J/mol.K respectively at 333 k respectively. The thermal inactivation of the enzyme followed first-order kinetics at 60 °C. The enzyme was relatively stable for 140 min retaining 70% of its initial activity at 60 °C and 67% of the activity at pH 6.0. The activity of purified D. reflexa β-amylase was enhanced in the presence of Zn2+, Ca2+ and Mg2+ and strongly inhibited by Cu2+ and Hg2+. Remarkably, the enzyme exhibited thermostable properties which increase its prospects as a veritable potential enzyme for various industrial and biotechnological applications.