Streptomyces griseus protease

The yeast Saccharomyces cerevisiae wild-type BY4741 (MATa his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) stored at −80 °C was revivified on YPD agar plate and then cultivated in YPD medium (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) at 30 °C with shaking at 200 rpm until OD₆₀₀ = 1 (∼1.4 × 10⁷ cells/ml). Unless otherwise stated, cells were harvested by centrifugation (3000 rpm × 3 min), washed with PBS or acetate buffer (18 mM, pH 5.2) and re-suspended to obtain around 5 OD unit at 600 nm. Yeast cells were immobilized by mechanical trapping into polydimethylsiloxane (PDMS) stamps (Formosa et al., 2015a (link)). Firstly, a bare AFM tip was used to visualise and image a single cell before to change it with a functionalized dendritip. For protease treatment, YPD-cultivated yeast cells were treated with 2 mg/ml of Streptomyces griseus protease (P6911, Sigma-Aldrich) at 37 °C for 6 h at pH 7.5, then collected by centrifugation and processed as above. Eight cells from 3 independent experiments were analysed for each treatment and a total of 8192 force curves were analysed.

Mouse skin samples were weighed and homogenized in freshly made, pH optimized lysis buffer comprised of 0.15 M Tris base, 0.15 M NaCl, 0.01 M CaCl₂, 5 mM deferoxamine, and 20 U/ml Streptomyces griseus protease (Sigma) in a ratio of 15 µl buffer per 1 mg of tissue. Each sample was digested at 55 °C for 16 hours on a shaker at 1400 rpm. To stop digestion, the protease was denatured on a hot plate at 95 °C for 10–15 minutes. The supernatant was collected from each tissue sample. HA quantification in skin and serum samples was then performed using an ELISA-like sandwich protein binding assay as per the commercial manufacturer's instructions (Corgenix). Briefly, the assay uses microwells coated with highly specific HA binding proetin (HABP) to capture HA from the test samples. 50 μl of the samples or standards were pipetted into the HABP coated microwells and incubated for 1 hr, and an enzyme conjugated version of HABP was used to detect HA. The absorbance of each sample was measured at 450 nm using a plate reader and optical density was blanked against reagent reference. HA concentration in the samples was measured by comparing absorbance againt standard curve. The HA values were normalized to total protein determined from the undigested homogenate using Coomassie plus assay.

The capsular EPS of K. pneumoniae PCM2713 was extracted from the freeze-dried bacterial mass using 10% trichloroacetic acid (TCA) according to the method described by Gorska-Frączek et al.^{41 (link)}. The freeze-dried preparation of crude EPS (20 mg) was dissolved in 1 ml of buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2) and treated with DNase (210 μg; Sigma) and RNase (210 μg; Sigma) at 37 °C for 6 h. This step was followed by an overnight hydrolysis with Streptomyces griseus protease (447 μg, 37 °C; Sigma). Finally, the preparation was dialyzed against water at 4 °C for 24 h. To test for depolymerization activity, 0.1 ml of EPS (1 mg/ml) was mixed with 0.1 ml of TTPA (0.5 mg/ml). The reaction mixture (pH 5.5) was incubated for 2 h at 25 °C on a rotary shaker. The reducing sugar (RS) concentration was determined with glucose as a standard, according to the Nelson-Somogoyi method⁴² . As a positive control, we used the RS obtained following acidic hydrolysis of EPS (1 mg/ml) with 6 M HCl at 115 °C in an autoclave for 1 h^{43 (link)}. The negative control was run with EPS but no TTPA.