Cloning and characterization of a chickpea α-galactosidase befitting industrial bioprocess applications

Chickpea (Cicer arietinum) contains at least four alpha-galactosidase genes (LOC101512744, LOC101513035, LOC101503593, and LOC101502309). The alpha-galactosidase gene (LOC101513035) was isolated from chickpea, cloned as eGFP fusion in pET 15b-eGFP, and overexpressed in E. coli BL21(DE3). The enzyme alpha-galactosidase was purified 32.9 fold using Ni-NTA affinity column with a recovery per cent of 53.7. The enzyme was qualitatively analysed for expression on denaturing and non-denaturing PAGE. The enzyme’s optimal pH and temperature was 5.4 and 46 °C, respectively. The Michaelis-Menten constant (Km) and Vmax for the synthetic substrate p-nitrophenyl-ɑ-D-galactopyranoside (p-NPG) was 0.3535 mM and 107.6 µmol.min− 1 respectively. Furthermore, 1-deoxygalactonojirimycin (DGJ) a galactose mimic inhibited alpha-galactosidase competitively with a linear curve fit Km of 6.8 mM and non-linear curve fit Km 4.4 mM. Higher temperature optima, acidic pH, lower Km, and higher Vmax collectively open suitable avenues for its applications in diversified process industries.