Studies on the β-galactosidase gene and enzyme of some bacilli strains

In our study, intracellular β-Galactosidase gene and enzyme of Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK isolated from Bingol Binkap hot spring water, as well as the enzyme gene of Anoxybacillus ayderensis FMB1 have been studied. In addition, PCR amplification of β-galactosidase gene was performed by using some specific primers of the gene. β-Galactosidase gene presence of all three bacteria were detected by using using different primers and PCR products obtained were ~2.4kb. The effect of temperature and pH on the enzyme was investigated at temperatures of 25-65˚C and at pH 4.0-11.0. The optimal temperature and pH values for Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK were 45˚C, pH 6.0 and 55˚C, pH 6.0, respectively. Thermal stability for enzymes of Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK was examined at 40-50˚C and 45-60˚C for 30-120 min, respectively. β-Galactosidase of Bacillus subtilis 4NK was found to posses thermal stability. The enzyme of Bacillus paralicheniformis was found to have thermal stability at lower temperature, partially at 55˚C activity but the activity was not protected at 60˚C. Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK were partially purified by ammonium sulfate precipitation and dialysis. As a result of this partial purification, the yield of enzyme was 85.2% and the purification fold was 2.8 for enzyme of Bacillus subtilis 4NK. The yield of Bacillus paralicheniformis 5NK enzyme was 76.8% and the purification fold was 2.0. Some chemicals, metals and chelators were tested on the enzyme activity. It was found that PCMB (4-chloro mercuric benzoic acid) caused very high inhibition on Bacillus subtilis 4NK β-galactosidase, while inhibition was very low by DTT, PMSF and NEM, but iodoacetamide did not seriously affect enzyme activity. For Bacillus paralicheniformis 5NK β-galactosidase, it was found that iodoacetamide and PCMB caused high inhibition, PMSF caused high inhibition at 10 mM, and partial inhibition was observed by NEM at 5 and 10 mM. DTT had no significant effect on the enzyme. Studies on Bacillus subtilis 4NK β-galactosidase revealed that CoCl2 and MnCl2 highly activated the enzyme at low concentrations (1, 2.5 mM). CuCl2 and CdCl2 caused inhibition at high concentrations (10 and 20 mM). Whereas EDTA was found to activate the enzyme at all concentrations. Studies on Bacillus paralicheniformis 5NK β- galactosidase revealed that CuCl2 and CdCl2 inhibited the enzyme at high ratios at several concentrations. However, EDTA had no significant effect on enzyme activation. Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK bacteria cultured on a NB medium with lactose and lactose-free media for 6-48 h were examined for bacterial growth and enzyme production. The strain 4NK showed an increase in bacterial growth after 24 hours in the medium with lactose. However, bacterial growth in the lactose medium for the strain 5NK did not change compared to lactose free medium. It was found that lactose increased enzyme production for both bacteria. Different oNPG concentrations were used to find the Km and Vmax values of the enzyme. Km and Vmax values for the strains 4NK and 5NK were 23.80 mM, 1.978 abs / min. and 5.61 mM, 1.869 abs / min, respectively.