Venusil xbp c18

The activity of BdsA was determined by measuring the amount of HBPSi using high-performance liquid chromatography (HPLC) according to a method described previously (Ohshiro et al., 1997 (link), 1999 (link)). The enzyme reaction system for measuring the activity of BdsA contained 100 mM potassium phosphate buffer (pH 7.0), 0.25 mM DBT sulfone, 6 mM NADH, 10 μM FMN, 20 nM Fre (flavin reductase from E. coli O157), and 2 μM BdsA, in a total volume of 1 mL. The reaction was performed with rotary shaking (2000 rpm) at 35°C for 20 min, and stopped by the addition of 100 μL of 12 M HCl. The mixture was extracted with ethyl acetate, centrifuged at 15,000 g for 5 min, and then the supernatant was injected into an HPLC system using 20 mM KH₂PO₄ (pH 2.5) and methanol as the mobile phase, at a ratio of 2:3.
The reaction mixture was loaded onto a C18 reversed-phase column (Venusil XBP-C18, Agela Technologies) attached to an HPLC system (LC-10AT pump, SPD-10A UV/VIS detector). An increase in absorbance at 280 nm, which indicates the amount of HBPSi, was observed. The enzymatic assays for all mutants were performed in the same manner as that for the wild-type BdsA protein.

The co-precipitates were analyzed on a Q-exactive mass spectrometer equipped with a Dionex Ultimate 3000 Nano (Thermo Fisher Scientific). Peptides were trapped on a Acclaim PePmap 100 (75 μm × 2 cm, nanoviper, C₁₈, 3 μm, Thermo Fisher Scientific) and separated on a Venusil XBP C₁₈ (2.1 × 150 mm, 5 μm, Agela Technologies) using a gradient formed between solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient started at 5% solvent B and the concentration of solvent B was increased to 80% within 60 min. Database search was performed using the MASCOT software against the E. coli K12 database.

After refrigerating (4°C, 12 hr) and removing oil and fat, 5 g of the beef broth sample supernatant was centrifuged for 10 min at 4°C and 6,010 g relative centrifugal force (RCF). After being diluted 20 times, the samples were filtered through 0.22‐µm nylon filter membrane; then, 4 5′‐nucleotides in the samples were analyzed by Thermo U3000 UPLC (Thermo Fisher Scientific Inc., USA), and the data were collected, processed, and analyzed by Chromeleon software. The conditions of 5′‐nucleotide detection were as follows: column (Venusil XBP C₁₈(4.6 mm × 250 mm, 5 μm) purchased from Agela Technologies (Tianjin, China); mobile phase was Buffer salt I (0.05 mol/L KH₂PO₄) and methanol (5:95, V/V), equal gradient elution, with a flow rate of 1ml/min; the column temperature was 25°C, the injection volume was 20 μl, and the UV detection wavelength was 254 nm. The mixed 5′‐nucleotide external standard (5′‐IMP, 5′‐GMP, 5′‐AMP, and 5′‐CMP) was prepared with ultrapure water and diluted into 7 gradients. According to the concentration range of four 5′‐nucleotides in the samples, the concentration of mixed external standard was 66.700, 40.000, 33.300, 20.000, 10.000, and 2.000 µg/ml, respectively. Each sample was analyzed three times.