| Summary: | The activity of extra- (A) and intracellular (B) acid proteases was determined in the same manner as published studies [ 14 , 36 ] with minor modifications. All strains were cultivated at 30°C for 5 days as with the flask cultivation in the Materials and methods section. The 1 mL of cultures after the cultivation were collected by a centrifuge at 14,000 g for 2 min and the supernatants were used as extracellular enzyme samples. The cell pellets were washed by 1 mL of 150 mM NaCl, resuspended in 1 mL of pure water. The resuspended cells (1 mL) were disrupted using the bead beater homogenizer (μT-12, TAITEC, Saitama, Japan) and zirconia beads (20 beads with 2 mm diameter and 3 beads with 3 mm diameter in 2 mL tube). After the disruption (3200 rpm for 20 sec and cooling on ice for 1 min, the cycle was repeated for 15 times), the disrupted cells were centrifuged at 22,000 g for 5 min at 4°C, then the supernatants were used as intracellular enzyme samples. The acid-denatured hemoglobin from bovine blood (Product No. H2625, Sigma Aldrich, St. Louis, MO, USA) was used as a substrate. The denaturation was performed by the incubation of hemoglobin in HCl (pH 1.8) at 35°C for 1 h, followed by pH adjustment at 3.2 by NaOH (Final concentration of hemoglobin was 20 g/L). The enzyme samples (400 μL) was mixed with 400 μL of acid-denatured hemoglobin (20 g/L, pH 3.2) and 400 μL of glycine-HCl buffer (100 mmol/L, pH 3.2) at 37°C. A portion (390 uL) of the mixture was taken at 0, 30, and 60 min, then, mixed with 700 μL of ice-cold trichloroacetic acid (TCA, 50 g/L) to stop the reaction. The TCA containing samples were incubated for 20 min at room temperature to progress the denaturation. Obtained samples were centrifuged at 22 , 000 g for 5 min at 4°C to remove denatured hemoglobin and cell-derived proteins, and these supernatants (500 μL) were mixed with 500 μL of 1 M NaOH. Finally, tyrosine-containing peptide in soluble fraction was determined using Folin & Ciocalteu’s phenol reagent (Product No. F9252, Sigma Aldrich, St. Louis, MO, USA, diluted by the same volume of pure water before the use). The NaOH added samples (1000 μL) was mixed with diluted Folin & Ciocalteu’s reagent (200 μL), incubated for 30 min at room temperature, and measured their light absorbance at 750 nm. The concentration of tyrosine-containing peptide was calculated from the calibration curve using tyrosine. One unit of activity (U) was defined as the amount of enzyme that released 1 μg of tyrosine-containing peptides per minute at 37°C. (TIF)
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