*4.8. Determination of Substrate Specificity and Kinetic Parameters*

The substrate specificity of the recombinant Gal was estimated using 5 mM (final concentration) ONPG, 4-Nitrophenyl-β-d-galactopyranoside (PNPG), 4-Nitrophenyl-α-d-galactopyranoside, 2-Nitrophenyl-β-d-glucopyranoside, 4-Nitrophenyl-β-d-glucopyranoside, 4-Nitrophenyl-α-d-glucopyranoside or 4-Nitrophenyl-β-d-xylopyranoside in 50 mM sodium phosphate buffer (pH 8) under the optimum conditions. For the activity of 4-nitrophenol-derived substrates, the absorbance of the released 4-nitrophenol (PNP) was measured at 405 nm and quantified using a PNP standard curve. Kinetic parameters of the recombinant Gal were determined at 35 ◦C and the reaction rate with ONPG (0.1–5 mM) and lactose (0.2–40 mM) as substrates was determined. The released glucose in the lactose hydrolysis reaction was measured using a commercial glucose oxidase-peroxidase assay kit (Shanghai Rongsheng Biotech Co., Ltd., Shanghai, China). One unit (U) of enzyme activity was defined as 1 μmol of glucose released per minute. The values of the kinetic constants were calculated using the Lineweaver-Burk method.
