2 ± 2.1 s-1 and 7.1 ± 0.8 mM for lactose, respectively. The k cat/K m value of the enzyme for ONPG (172.1 s-1 mM-1) was 4.6-fold higher than that for lactose (37.2 s-1 mM-1), which clearly demonstrated that the catalytic efficiency

of Gal308 for ONPG was much higher than that for lactose. Table 2 Relative activity of purified Gal308 with several nitrophenyl-derived chromogenic substrates and its natural substrate lactose Substrate Activity a (%) o-Nitrophenyl-β-D-galactopyranoside (ONPG) 100 p-Nitrophenyl-β-D-galactopyranoside (pNPG) <1 o-Nitrophenyl-β-D-fucopyranoside <1 p-Nitrophenyl-β-D-mannoside 3.5±0.3 o-Nitrophenyl-β-D-glucoside Protein Tyrosine Kinase inhibitor <1 p-Nitrophenyl-β-D-xyloside 5.7±0.2 p-Nitrophenyl-β-D-cellobioside <1 p-Nitrophenyl-β-D-lactoside 7.8±0.3 p-Nitrophenyl-α-D-galactoside <1 Lactose 25.7±1.8 a The values are relative to the 100% value observed with ONPG (185 U/mg). Hydrolysis of lactose in milk by Gal308. Effects of galactose and glucose on

the activity of Gal308 Lineweaver-Burk plots (1/V vs. 1/[S]) were used to investigate the effects of the inhibitors galactose and glucose on the activity of Gal308 using ONPG as substrate. The results demonstrated that both of galactose and glucose were competitive inhibitors of Gal308 because V max value of Gal308 was unchangeable SB525334 research buy and K m value of Gal308 was increased with concentration enhancement of the inhibitors (data not shown). Furthermore, the inhibition constant (K i) of galactose and glucose to Gal308 were also determined. The enzyme displayed a very high tolerance of galactose and glucose, with the inhibition constants K i,gal of 238 mM and K i,glu of 1725 mM. In addition, the effects of galactose and glucose on enzymatic activity were investigated at various concentrations of galactose and glucose (Figure 4). Comparing

to the inhibition of galactose to other β-galactosidases reported previously, the inhibition of galactose to Gal308 is G protein-coupled receptor kinase less pronounced, and the relative activity of Gal308 still reached to 32.5% at a concentration of 400 mM galactose. On the other hand, glucose had an almost negligible inhibitory effect with 89.6% of the initial activity remaining at a concentration of 400 mM glucose. Figure 4 Effects of galactose ( circle ) and glucose ( square ) as inhibitors on the activity of Gal308. The reactions were performed under standard conditions with ONPG as a substrate. The relative activity was defined as the relative value to the maximum activity without galactose or glucose. Data represent the means of three experiments and error bars represent standard deviation. To investigate the lactose hydrolysis activity of Gal308, an experiment on lactose hydrolysis in milk was performed. After 30 min of incubation at 65°C, 66.5% of milk lactose was hydrolyzed by Gal308 and 31.2% of milk lactose was hydrolyzed by the commercial enzyme. When the incubation time of Gal308 was extended to 45 min, 60 min, the hydrolysis rate of lactose in milk was increased to 82.8% and 93.6%, respectively.