Mutacin D-123.1 was produced in TSBYE (Difco) containing 0.5% agarose (Difco). Batches of this medium (4 L) were stab inoculated with a culture of S. mutans 123.1 grown in TSBYE and incubated for 72 h at 37°C. After growth, the culture was scraped, aliquoted into centrifuge bottles and frozen overnight at -20°C. The bottles were then centrifuged at 4000 × g for 60 min and 8000 × g for 30 min at room temperature. The resulting supernatant was filtered through glass fibers and Whatman no. 1 filter paper to remove agarose fines then stored at 4°C. Purification of mutacins Purification
of the two mutacins was achieved by two hydrophobic chromatography steps as previously described [15, 39] by replacing TFA with HCl (10 mM) . Briefly, the active preparation was loaded on a Sep-Pak® Vac 35 cc (10 g) t-C18 Cartridge (Waters Corporation, Milford, Rabusertib order MA, USA). Cartridges were first ROCK inhibitor equilibrated with 500 mL of methanol followed by 500 mL of deionized distilled water. Antibacterial compounds were eluted with successive steps of 500 mL of water:methanol mixtures increasing the gradient of methanol by 10% from 0 to 100% in 10 mM HCl. This was carried out at a flow rate of 1 mL/min and UV detection at 214 nm. The final purification step was carried out by reverse phase chromatography (RP)-HPLC analysis
(Beckman Gold Model, Coulter Canada Inc., Mississauga, ON, Canada) DUB inhibitor using an analytical C18 column (Luna 5 μ C18(2), 250 × 4.6 mm, 4 × 3.0 mm, Phenomenex, Torrance, CA, USA). Elution was carried
out with solvent A (5% acetonitrile, 10 mM HCl) and solvent B (60% acetonitrile, 10 mM HCl) and recorded stiripentol at 214 nm. The following program of elution was developed: 0 to 3 min, constant 100% A; 3 to 15 min, a linear gradient from 100% A to 100% B; 15 to 20 min, constant 100% B; 20 to 23 min, a linear gradient from 100% B to 100% A. A flow rate of 1 mL/min was used. The column was maintained at 39°C with a column heater. Active fractions were manually collected, subsequently dried in a Speed-Vac® concentrator (Model SC110A, Savant Instrument Inc. Farmingdale, NY, USA) and then kept at -20°C until processing. Protein concentration in active fractions was determined using the BioRad DC protein assay (BioRad, Mississauga, ON, Canada). Activity assay of mutacins Mutacin activity was determined by the spot test using Micrococcus luteus ATCC 272 as sensitive strain where two-fold dilutions were prepared in acidified (pH 2) peptone water (0.5%) . Antibacterial activity spectra of purified mutacins was tested against a panel of bacterial strains using the critical dilution method combined with the spot test method as described previously . Briefly, overnight cultures of test strains in TSBYE were diluted in fresh broth before inoculating 5 mL of soft agar (0.