Purification and Characterization of β-galactosidase from Enterobacter sp. 3TP2A

In this study, a mesophilic Enterobacter sp. 3TP2A isolated from petroleum station in Batman in the southeast of Turkey was identified and found to produce a high amount of mesophilic β-galactosidase. The lactose was found to increase the β-galactosidase production to a great extent, meaning that this enzyme is inducible. An intracellular β- galactosidase from Enterobacter sp. 3TP2A was purified and characterized. The enzyme was purified by ammonium sulphate precipitation 70%, dialysis, ultrafiltration and finally using Sephadex G-75 chromatography method. The enzyme was purified to 17.3-fold after Gel permeation chromatography with a yield of approximately 11%. The purified enzyme was found to be stable in pH 8.0 and temperature of 35 oC. The Molecular weight of the purified enzyme was found to be about 60 kDa by both SDS-PAGE and native-PAGE. The effects of various metal ions at different concentrations of CaCl2, MgCl2, ZnCl2, CuCl2, and EDTA (1, 2, 5, 10 and 20 mM) were tested. EDTA and Cu2+ had an inhibitory effect on the β-galactosidase purified from Enterobacter sp. 3TP2A. EDTA inhibited the enzyme activity (upto 76%) and Cu2+ had strong inhibitory effect on β- galactosidase even at low concentrations (96.9%). However, Mg2+ caused activation of the purified enzyme. Ca2+ did not effect enzyme activity to a great extent, causing deactivation of the enzyme at 20 mM (only 16%), while Zn2+ at 1, 2 and 5 mM inhibited enzyme activity (32, 27, 8%, respectively). Increase in the concentration of Mg2+ causing activation upto 47% and also inhibition by EDTA show that the enzyme is metal-dependent or a metalloenzyme. Also determining the effect of different concentration of inhibitors on purified enzyme; PCMB (0.2, 0.4,1, 2 mM) , Iodo, DTT, β-mer, N-ethyl, (1, 2, 4, 8 mM). The enzyme was completely inhibited by N-ethyl (100%), but not affected by DTT. The enzyme was slightly affected by β-mer enhancing β-galactosidase activity at 8mM with 14% . The Iodo had a slight effect on β-galactosidase activity (upto 13%). PCMB inhibited the enzymatic activity to a great extent upto approx. 87%. The Lineweaver-Burk plot was linear, suggesting a simple Michealis-Menten kinetics. The Vmax was found as 0.701 (μmol/ min mg) and Km was found as 0.104 mM. It was also found that the time for lactose hydrolysis continues up to 10 h with the reaction catalyzed by purified β-galactosidase. The aim of this study was to purify and characterize the mesophilic β-galactosidase from Enterobacter sp. 3TP2A and then to test for use in biotechnology such as lactose hydrolysis. The results obtained indicated that this species may well be a good candidate.