0.45 μm pvdf filter

Soybean residues (Nihon Beans, Tokyo, Japan), called okara, were obtained commercially. These residues [14% (w/w)] were diluted in 50 mM MES (pH 5.5) and sterilized. The enzymes described below were purchased from Amano Enzyme (Nagoya, Japan). The protease mixture contained 2 mg/mL ProteAX, Peptidase R, PROTIN SD-AV10, Protease M “Amano” SD, PROTIN SD-NY10, THERMOASE R PC10F, and Protease A “Amano” SD in 50 mM MES (pH 5.5). The cellulase mixture contained 2 mg/mL Hemicellulase “Amano” 90, Cellulase A “Amano” 3, Mannanase BGM “Amano” 10, Cellulase T “Amano” 4, and Pectinase G “Amano” in 50 mM MES (pH 5.5). The protease mixture and cellulase mixture were filtrated through a 0.45 μm PVDF filter (Merck Millipore, MA, USA) and poured into sterilized soybean residues. The mixture of soybean residues containing these enzymes was incubated at 55 °C for 72 h with shaking at 250 rpm (Bio-Shaker BR-300LF, Japan). For denaturing the enzymes, the mixture was incubated at 80 °C for 30 min. Then, the mixture was filtrated with ADVANTEC131 (3 μm particle retention capacity; Toyo Roshi Kaisha, Tokyo, Japan) after the filtration with ADVANTEC2 (5 μm particle retention capacity; Toyo Roshi Kaisha). After that, the flow-through was filtrated through a 0.45 μm PVDF filter (Merck Millipore) and used for the substrate of ammonia production.

The following protocol is adapted from Harwood and Cutting,38 . Overnight cultures of the relevant B. subtilis strain were grown at 30 °C in MMB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, 5 mM MgCl₂ and 0.1 mM MnCl₂). These cultures were used to inoculate fresh MMB medium to an OD₆₀₀ of 0.02. The cultures were grown at 37 °C until they reached mid-log phase (OD₆₀₀∼0.5), at which point mitomycin C (Melford) was added to a final concentration of 0.5 μg ml⁻¹. Cell cultures were grown at 37 °C with shaking for a further 2 h at which point lysis occurred. Lysis occurred in all strains, including ΔyonO and ΔSPβ, due to mitomycin C-dependant induction of non-SPβ lytic enzymes encoded elsewhere in the B. subtilis genome.
Cell lysates were centrifuged (20,000g, 2 min) and supernatant filtered through a 0.45 μm PVDF filter (Merck-Millipore). Strain CU1065 (SPβ sensitive-SPβ^S) was grown until 0.5 OD₆₀₀. Three hundred microlitres of CU1065 culture were incubated at room temperature with 100 μl of lysate containing SPβ particles for 2 min. MMB overlay agar (MMB media supplemented with 0.5% agar) was added and cells were plated onto MMB bottom agar (MMB media with 2% agar). Plates were incubated overnight at 37 °C.

Chromatographic analyses (HPLC-DAD) carried out at the Laboratory of Environmental Toxicology, Fiocruz, with a Shimadzu Nexera XR^® chromatograph and a Shimadzu UV-VIS detector with diode array SPDM20A (CBM20A controller, DGU20A degasser, LC20AD binary pump, CTO20A oven, and SILA20A auto-injector). Chromatograms were analyzed with a Shimadzu LabSolutions Software 5.92v (Shimadzu, Japan).
Parameters were tested to improve analytical conditions. Columns were silica-based C18 Ascentis (250 mm × 4.6 i.d.; 5 μm particle size) and a modified silica phenyl Ascentis (250 mm × 4.6 mm i.d.; 5 μm particle size), both from Supelco (Merck, Darmstadt, Germany). Mobile phase tests tried different pH values, combining ultrapure water (pH 3.0, with anhydrous acetic acid), acetonitrile, methanol, and potassium dihydrogen phosphate buffer and ammonium acetate buffer. All buffer solutions were filtered through a 0.45 μm PVDF filter (Merck-Millipore, Darmstadt, Germany) before use.
In addition, we examined mobile phase flow, isocratic or gradient elution mode, and total time analysis, in this validation. Keeping oven temperature at 50 °C, analyses were performed in triplicate (20 μL sample injection), and 3D-anth detected at 480 nm.