Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146)

An extracellular thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146) has been purified to homogeneity by chromatographic step, using Phenyl Sepharose CL-4B column. The specific activity of the enzyme was increased approximately 389-fold, from 1.03 U/mg protein to 400 U/mg protein. The molecular mass of the purified enzyme as determined by SDS-PAGE and gel filtration was 79.9 and 165.9 kDa, respectively, suggesting dimeric nature. The purified α-galactosidase is a non-glycosylated protein with a pI of 4.9. The pH and temperature optima for the purified enzyme are 6.5–7.0 and 65 °C, respectively. The α-galactosidase is stable over a broad pH range (3–9) and its half-life of inactivation (t1/2) at 70 °C is 30 min. The partial N-terminal sequence of α-galactosidase showed remarkable homology (80% similarity) with earlier reported α-galactosidase from B. stearothermophilus NUB 3621. The secondary structure of the enzyme determined by circular dichroism (CD) spectroscopy exhibited α/β class of protein and showed temperature induced conformational forms below and above the transition temperature. The purified enzyme showed biphasic Arrhenius plot with break point at 55 °C for pNPG and 50 °C for melibiose, raffinose and stachyose. The enzyme hydrolyzes α-1-3, α-1-4, and α-1-6 galactosidic linkages and not the β-galactosidic linkages. Synthetic substrates pNPG and oNPG had lower Km and higher Kcat as compare to natural substrates, melibiose, raffinose, and stachyose.