lyophilization of NK
The final fermentation broth was collected and centrifuged (GL-25M, Luxiangyi, Shanghai, China) immediately at 18,300 × g for 10 min. Solid (NH4)2SO4 was then gradually added until the supernatant was 20% saturated, after which it was stored at 4°C overnight. Next, the broth was centrifuged at 18,300 × g for 10 min and the precipitate was discarded. Additional (NH4)2SO4 was subsequently added to make the supernatant 60% saturated, after which the mixture was stored at 4°C overnight again. Following centrifugation at 18,300 × g for 30 min, the supernatant was removed and the precipitate was dissolved in 10 mmol L-1 PBS (pH 7.4) as a crude enzyme. The crude enzyme was further purified by Ni–NTA affinity chromatography according to the manufacturer’s (Novagen, San Diego, CA, USA) protocols. After purification, 10 µl of eluted fractions of different dilution times were used to assay their fibrinolytic activities. Residual solution was desalted by dialysis and lyophilized using a GLZY-0.5B vacuum freeze drier (Pudong Freeze Drying Equipment, Shanghai, China) to obtain NK Lyophilized powder.

SDS-PAGE and Western blotting analysis

SDS-PAGE protein analysis was performed in a Mini-Protean Tetra system (BioRad, Hercules, CA, USA) by loading 30 μL of 1: 1 (v: v) boiled supernatant and dye buffer onto a 4–20% precast Mini protean TGX gel (BioRad) and then running the samples in 1 × Tris-glycine-SDS running buffer (BioRad, Shanghai, China) at 100 V for 15 min followed by 200 V for 25 min. A protein standard (10-250 kDa, Precision Plus Protein Kaleidoscope, Bio-Rad) was used as a ladder.
NK was identified by Western blotting analysis as described by Towbin, with slight modification [18]. Briefly, 10 μL aliquots of different supernatants were separated by 15% SDS–PAGE. After electrophoresis, the protein bands were transferred from the gel to an Amersham nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA) using a Mini Trans Blot electrophoretic transfer cell (BioRad). NK on the membrane was reacted with a mouse monoclonal antibody (Yisheng Biological Technology, Shanghai, China) against His-tag (1:1000 v/v), then incubated with anti-mouse Ig G alkaline phosphatase (Sigma) conjugate (1:2000 v/v). After washing, the NC membrane was stained using Super ECL Detection Reagent (Yisheng Biology Technology, Shanghai, China) according to the manufacturer’s instructions, then covered by an X-ray film and exposed for 1 min. A Tanon v.3500 Gel Imaging System (Tanon Co., Shanghai, China) was used for the analysis of proteins.