Cloning of ß-galaktosidase gene of thermophilic Bacillus licheniformis KG9 to Escherichia coli and its characterization

In this study Bacillus licheniformis KG9, which was isolated from Taşlıdere hot water springs in Batman/Turkey, was used for gene cloning and purification due to its stable ß-galactosidase, abundant production of the enzyme and since it displayed similarity at 99.9% to B. licheniformis DSM 13, whose all genomes were known when 16S rRNA sequence analysis of this bacterium was compared. Chromosomal DNA of B. licheniformis KG9 was isolated. ß-Galactosidase gene of thermophilic bacteria was scanned at gene library. Four different putative ß-galactosidase genes (ß-gal I, ß-gal II,ß-gal III,ß-gal IV) isolated with the method based on PCR were amplified. The PCR fragments were inserted into pUC18?lacZ vector, transformed into Escherichia coli, and by selecting the expressed colonies they were sequenced. According to the sequence analysis results: PCR based isolation of putative ß-galactosidase genes from B. licheniformis KG9 showed the presence of four ß-galactosidase genes, which when translated, displayed similarity to genes from other Gram positive bacteria: ß-gal I showed 42.9% identity to a ß-gal from Bacillus cereus; ß-gal II showed 68.3% identity to a ß-gal from Bacillus circulans; ß-gal III displayed 68.8% identity to a ß-gal from Bacillus subtilis; and ß-gal IV showed 76.7% identity to a ß-gal from B. subtilis.? -galactosidase, isolated from both B. licheniformis KG9 and recombinant E. coli was purified by DEAE-cellulose, Sephadex G-75 and p-aminobenzyl-1-thio-ß-D-galactopyranoside (PABTG-agarose) chromatography methods. Purification fold was determined to be 9.4, and yield as 85%. Molecular weight of the purified enzyme was found to be about 90 kDa by non-denatured PAGE.In the studies of characterization of purified enzyme; Artificial (ONPG) and natural (lactose) substrates for substrate specifity and CoCl2, MgCl2, NiCl2, CaCl2, MnCl2, HgCl2, AgCl2, FeCl2 and EDTA of different concentrations (1, 2.5, 5, 10, 25 mM) for determining the effect of the metals and EDTA were used and tested. Enzyme activity was increased in presence of Ca+2 (147%), Mn+2 (106%) and Mg+2 (135%) while activity of the enzyme was inhibited in presence of Ag+2, Fe+2 and Hg+2. Km and Vmax values of the enzyme depending on ONPG concentration were calculated as 2.0792 mM and 2.037 ?mol/min respectively according to the Lineweaver-Burk plot. Km and Vmax values of the enzyme depending on lactose concentration were calculated as 9.068 mM and 1.0476 ?mol/min respectively according to the Lineweaver-Burk plot.