An extracellular thermostable α-galactosidase from Bacillus megaterium VHM1 was purified 94.26-fold by precipitation with ethanol, followed by sequential column chromatography with DEAE-Sephacel and G75 column. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). It was found to be a monomeric protein with a molecular weight of about 66 kDa. The purified enzyme showed optimum activity in p-nitrophenyl α-D-galactopyranoside (PNPG) at pH 7.0 and a temperature of 60°C. The enzyme was thermostable, showing complete activity even after heating at 55°C for 60 minutes. The substrate specificity of α-galactosidase on PNPG, ortho-Nitrophenyl-β-galactoside, and raffinose was investigated and Km was found to be 0.508, 0.529, and 5.0 mM and Vmax was 3.492, 4.287, and 14.20 μ mol/ml/minute, respectively. Among the metal ions and reagents tested, Hg2+, Cu2+, and Ag2+ strongly inhibited the α-galactosidase activity, and ethylenediaminetetraacetic acid showed no effect on enzymes. The present study supports the application of α-galactosidase from B. megaterium VHM1 as a potent enzyme in the food processing industry.
Key words: Bacillus megaterium VHM1, α-Galactosidase, Purification, Food industry
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