P nitrophenyl β d galactopyranoside

Enzyme kinetic parameters (k_cat, K_m and k_cat/K_m) were determined for purified enzymes by measuring the initial rates (v₀) of hydrolysis of at least 10 different concentrations of substrates, including p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-fucopyranoside and p-nitrophenyl-β-D-galactopyranoside (Sigma, St. Louis, MO, USA) prepared in 100 mM sodium citrate – sodium phosphate buffer pH 6.0. Experiments were performed at 30°C. The hydrolysis of these substrates was detected following the formation of p-nitrophenolate by absorbance at 420 nm after addition of 250 mM sodium carbonate – sodium bicarbonate buffer pH 11 to the reaction samples. Kinetic parameters K_m and k_cat were determined by fitting the v₀ and [S] data to the Michaelis-Menten equation using the Enzfitter software (Elsevier-Biosoft, Cambridge, UK).
Differential scanning fluorimetry (DSF) experiments of both wild-type and mutant Sfβgly were performed using SYPRO® Orange solution (500-fold dilution) (Sigma, St. Louis, MO, USA). Melting studies were performed in optical tubes using a 7500 Real-Time System (Applied Biosystems, Foster City, CA, USA). The temperature gradient ranged from 25°C to 95°C with a slope of 0.5% per step. The melting data were fitted according to recent literature [22] (link), resulting in a theoretical T_m. Fitting processes were performed using EnzFitter software.

The activity of GAL was determined according to the method described by Chojnowska et al. [28 (link)]. 40 μL citrate-phosphate buffer (0.1 M, pH 4.3) and 30 μL 1.6 mM substrate solution (p-nitrophenyl-β-d-galactopyranoside, Sigma-Aldrich; Steinheim, Germany) were added to 10 μL supernatant fluid. The reaction mixture was incubated on a shaker for 60 min at 37 °C. The reaction was stopped by adding 200 μL borate buffer (0.2 M, pH 9.8). The absorbance of the released p-NP was measured at 405 nm wavelength.

Gallic acid, rutin, toluidine blue, DNS reagent, cephalexin, and ampicillin were purchased from Yuanye Biotechnology Co. (Shanghai, China). Acarbose, α-amylase, α-glucosidase, p-nitrophenyl-β-D-galactopyranoside (PNPG), 2,3,5-triphenyl tetrazolium chloride (TTC), 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Folin–Ciocalteu reagent, dimethyl sulfoxide (DMSO), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS), and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide, hydrochloric acid, glacial acetic acid, ferric chloride, anhydrous sodium carbonate, potassium persulfate, sodium acetate, aluminum nitrate, sodium nitrite, and other regents were obtained from Bodi Chemical Co. (Tianjin, China). HPLC-grade formic acid, acetonitrile, and methanol were bought from CNW Technologies GmbH (Duesseldorf, Germany).