Optimization and Characterization of β-Galactosidase from Bacillus aryabhattai – A Marine Bacterium

Abstract

β-Galactosidase (EC 3.2.1.23) is an industrially important enzyme used in the dairy and food industries, particularly for lactose hydrolysis. The aim of the present study was to isolate and identify a marine bacterial β galactosidase producer, optimize enzyme production, and purify and characterize the enzyme. Marine seawater and sediment samples were subjected to microbiological isolation; β-galactosidase-producing isolates were screened by an X-Gal chromogenic assay and quantitatively evaluated using the ONPG assay. Among 24 morphologically distinct isolates, MBC1 and MBC3 were β-galactosidase-positive, and MBC1 showed the highest intracellular activity (29 U/mL). MBC1 was identified as Bacillus aryabhattai by VITEK 2 biochemical profiling, 16S rRNA gene analysis, and phylogenetic analysis (GenBank accession MG831954). One-factor-at-a time optimization identified glucose as the preferred carbon source (398 U/mL), 1.5% lactose as the optimum lactose concentration (216 U/mL), yeast extract as the preferred nitrogen source (425 U/mL), MgCl₂ at 1 mM (299 U/mL), pH 7.5 (399 U/mL), 37 °C (487 U/mL), 36 h incubation (319 U/mL), 5% inoculum (269 U/mL), 180 rpm agitation (219 U/mL), and 3% NaCl (148 U/mL). Corn steep liquor produced 248 U/mL among the alternative substrates tested. The crude extract showed 573 IU/mL activity and 220.38 IU/mg specific activity; after ammonium sulphate precipitation and dialysis, specific activity increased to 778.75 IU/mg with a 3.53-fold purification. Following DEAE-cellulose and Sephadex G-200 chromatography, the final preparation showed 1326 IU/mL activity, 491.10 IU/mg specific activity, 2.23-fold purification, and 60% overall yield. SDS-PAGE indicated an apparent molecular mass of approximately 45 kDa. These findings support the potential of marine derived B. aryabhattai MBC1 as a source of β-galactosidase for further food and dairy biotechnology studies.