8) containing 5 mM final concentration of lactose or nitrophenyl substrate. The kinetic parameters (K m and k cat) for the recombinant enzyme were investigated by assaying the enzymatic activity in 0.1 M phosphate buffered saline (PBS, 0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1 M NaCl, pH 6.8) at 78°C with two substrates, ONPG and lactose. All kinetic studies were performed three times, and kinetic data were

fitted C646 price to hyperbola by using the Michaelis-Menton equation. Kinetic analyses by curve fitting were performed with the SigmaPlot software (Systat Software, Chicago, IL, USA). Furthermore, Lineweaver-Burk plots (1/V vs. 1/[S]) were used to investigate the inhibition type of galactose and glucose on the enzymatic activity. The inhibition constants (K i values) of galactose and glucose

to Gal308 were obtained by fitting to Cornish-Bowden plot using various concentrations of galactose (0 – 20 mM) and glucose (0 – 400 mM) with various concentrations of ONPG (0.05 – 1 mM) as a substrate [32]. Effects of galactose and glucose on the enzyme activity The effects of galactose and glucose on the activity of Gal308 were determined at the concentrations of galactose from 25 to 400 g/L and glucose from 50 to 400 g/L using ONPG as substrate [13]. The relative activity was defined URMC-099 nmr as the relative value to the maximum activity without galactose or glucose. Hydrolysis of lactose in milk Milk containing 5% (w/v) lactose was added with equal amount of enzyme (20 U for 1 g of lactose) including recombinant Gal308 or a commercial product of β-galacosidase (Maxilact, DSM China, Shanghai, China), and the solutions were incubated for 30 min, 45 min, and 60 min with shaking (150 rpm) at 65°C, respectively. Then, mixed the aliquots of the digest with the same volume of 10% trichloroacetic

Thymidine kinase acid solution and centrifuged, and adjusted pH of the supernatant to 7.0 with NaOH immediately. Finally, a commercial enzymatic test kit (Sunbio, Beijing, China) was used to test the concentration of glucose liberated by the enzyme, and glucose concentration was determined based on A 530 measurements of the dye produced by oxidation of a chromogen (4-aminopyrine). Nucleotide sequence accession number The nucleotide sequence data reported here have been submitted to the nucleotide sequence databases (GenBank) under accession number (click here JQ009372). Acknowledgements This work was supported by the grant of National Natural Science Foundation of China (31170117, 31270156), National marine research funds for public welfare projects of China (201205020), Major Science & Technology Projects of Guangdong Province, China (2011A080403006), the Fundamental Research Fund for the Central Universities of Sun Yat-sen University (No. 11lgpy23), Science and Technology Plan Project in Guangdong Province (2012B010300021, 2009B020313005), and Natural Science Foundation of Guangdong Province (S2012010010464, 9451022401003873). References 1.