Purification and characterization of β-Galactosidase from Thermophilic bacillus zhangzhounesis 2ÇM4

β-Galactosidase (E.C. 3.2.1.23) is a glycoside hydrolase enzyme that hydrolyzes galactopyranosides such as lactose and produces galactooligosaccharides by catalyzing the trans-galactosylation reaction. Recent studies have focused on thermostable b-galactosidases obtained from thermopHiles. ThermopHilic β-galactosidases are used in many biotechnological fields such as in medicine to treat milk lactose tolerance, in food industry to prevent lactose crystallization in dairy products and to reduce water pollution caused by whey. The aim of this study is to produce, purify and characterize β-galactosidase from thermophilic Bacillus zhangzhounesis 2ÇM4 isolated from Çermik (Diyarbakır) thermal spring. For this, both intracellular and extracellular enzyme production were examined depending on time (0-96 hours). The optimum temperature (25-80 oC) and pH (4.0-11.0) values of the enzyme were determined. Optimum time, temperature and pH values of extracellular β-galactosidase of B. zhangzhounesis 2ÇM4 were found at 33h (335,5 U/mg), 60 oC and pH 6.0, respectively. For partial purification of the enzyme, it was purified 10,2 times with 5,8% yield using precipitation and chromatography methods. For the characterization of the partially purified enzyme, substrate specificity using different concentrations of substrate (oNPG and lactose), effect of different concentrations of some divalent metals and chemicals, the effect of pH and thermal stability as well as the molecular weight of the enzyme were determined by SDS-PAGE. It was determined that 1 mM Mg+2 (154%) and 5 mM Ca+2 (126%) increased activity; Mn+2 and Zn+2 decreased enzyme activity with increasing concentration, while Cd+2, Co+2, Ni+2 and Cu+2 inhibited enzyme activity. In addition, it was determined that IAA completely inhibited the enzyme activity, N-EtM, Phen, PCMB and EDTA caused inhibition in enzyme activity, and β-Mer increased the activity. The Km and Vmax values of the enzyme for oNPG were 0,112 mM and 0,712 μmol/min, respectively, while the Km and Vmax values for lactose were 14,20 mM and 1,596 μmol/min, respectively. As a result, this study shows that the obtained enzyme can be used for biotechnological applications with its thermostable feature in a short time and cost-effective production.