Evaluation of protease activity in culture supernatant of ΔPa PRO1 , ΔPa PRO2 , and wild-type by SDS-PAGE with gelatin as a substrate.

• Main Authors: Natsuki Omae (10318504), Yuka Sameshima-Yamashita (6292238), Kazunori Ushimaru (3689119), Hideaki Koike (410323), Hiroko Kitamoto (6076307), Tomotake Morita (526538)
• Format: Still Image
• Language: unknown
• Published: 2021
• Subjects: Biophysics; Biochemistry; Microbiology; Cell Biology; Genetics; Molecular Biology; Developmental Biology; Science Policy; Infectious Diseases; Biological Sciences not elsewhere classified; Chemical Sciences not elsewhere classified; vacuolar protease function; yeast Pseudozyma antarctica; GB; plastic-degrading enzyme; 48 h incubation; orthologous genes encoding proteases; 3- L jar fermenter; XG 8; XG 8 Δ Pa; strain; XG 8 control; B orthologous genes; PaE; degradation fragments; Δ Pa; Antarc*; Antarctica
• Online Access: https://doi.org/10.1371/journal.pone.0247462.s012

Culture supernatants of ΔPa PRO1 , ΔPa PRO2 , and wild-type GB-4(0) were centrifuged at 20, 000 × g for 5 min, and subjected to SDS-PAGE containing 0.1% gelatin without heat denaturation. After electrophoresis, to remove SDS [ 35 ], the polyacrylamide gel was incubated with 2.5% tritonX-100 at 25°C for 1h, washed with distilled water, and then incubated with buffer fluids, i.e, 0.1M sodium acetate (pH5.2), 0.1M Tris-HCl (pH7.2), or 0.1M Tris-HCl (pH9.8). The gel was stained with Symply Blue Safe Stain (Invitrogen, CA). Protease activity was visualized as white band caused by gelatin degradation. (TIF)