Purification, characterization and thermal inactivation kinetics of β-galactosidase from Lactobacillus leichmannii 313

Abstract

β-galactosidase from Lactobacillus leichmannii 313 (LL313) was purified (4.5-fold, 11% purification yield), and characterised, giving optimal enzyme activity at pH 5.5 and 55 °C. Thermal inactivation of crude and purified enzyme showed first order inactivation kinetics. Deactivation energy (Ed) of 390.58 ± 34.94 kJ/mol (crude enzyme) and 404.17 ± 46.19 kJ/mol (purified enzyme), based on the Arrhenius equation were not significantly different. Thermal stability, determined by decimal reduction time (D value), z value, and half-life (t1/2) of purified enzyme were significantly lower than those of crude enzyme. This, together with thermodynamic parameters (ΔH#, ΔG# and ΔS#) suggested that the purification procedure affected the thermal stability of the enzyme. The purified enzyme gave Vmax and Km values of 9.15 ± 0.23 mol g−1.min−1 and 2.97 ± 0.32 mM respectively, with ο-nitrophenol-β-d-galactopyranoside as substrate. The purified enzyme was activated by Na+ ions (>1 mM); remained unaffected by K+; and was inhibited by Ca2+ and Mn2+ (1–100 mM). Inhibition by EDTA (1 mM) and activation by 2-mercaptoethanol (1 mM) demonstrated respectively that the enzyme is a metalloenzyme and required cysteine in the active site. The enzyme exhibited hydrolytic and transgalactosylation activities with lactose as substrate, demonstrating its potential for use in the food industry.