Effects of Major Ingredients in Cattle Milk on Enzyme Kinetics of Recombinant β-galactosidase (BGalP) Expressed in Pichia pastoris

Background and objective: β-galactosidase enzymes hydrolyze lactose into glucose and galactose for production of lactose free dairy products. However, different ions and fat content in milk may act as the inhibitor or activator for β-galactosidase enzymes. A cold-active β-galactosidase enzyme (BGalP), originally from Planococcus sp. L4, was previously expressed in Pichia pastoris to perform lactose hydrolysis in the refrigerated milk. In this study, the effects of milks major ingredients were evaluated on the enzymatic kinetics to confirm its capacity for hydrolyzing milk lactose. Material and methods: The activity was determined in different concentrations of NaCl, KCl, MgCl2, and CaCl2 as well as in the milk with low, medium or high-fat content. In these experiments ortho-Nitrophenyl β-galactoside was used as the substrate. Additionally, glucose was measured as the product after incubation of milk with BGalP enzyme for 24 h at room temperature. Results and conclusion: This study demonstrated that ions and fat content did not adversely affect the enzyme activity in the concentration corresponding to the milk contents. Ca (27.5-32.5 mM), Cl (25.3-30.9 mM), Na (15.2-39.1 mM) and Mg (3.75-5.83 mM) had no inhibitory effects, but KCl decreased the enzyme activity. Since Cl existed in MgCl2, and CaCl2 exerted no inhibitory effects, it can be concluded that inhibitory effects of KCl resulted from potassium rather than chloride. The results indicate that BGalP enzyme was not inhibited by milks major ingredients and has the potential to be used for the production of lactose-free dairy products.