Development of universal purification protocols for fibrinolytic enzyme-producing bacilli

An aqueous protocol was developed for nattokinase purification under scalable conditions using a combination of salting out, anion exchange chromatography and ultra-filtration. The enzyme was firstly washed off from the fermented soybeans. The whole proteins were precipitated by ammonium sulfate and purified by anion exchange chromatography. The purified enzyme was finally refined by size exclusion chromatography and ultra-filtration at laboratory and industry-compatible level, respectively. There was a 476.18-fold increase in enzymatic activity with a 48.3% yield at the laboratory level and 429.75-fold increase in enzymatic activity with a 42.7% yield at the industrial-compatible scale. The protocol was universally adaptive for purifying the fibrinolytic enzymes produced by bacillus tequilensis, bacillus amyloliquefaciens, and bacillus cereus, showing a purification efficiency of 329.76-fold with 42.7% yield, 221.78-fold with 32.5% yield, and 288.56-fold with 38.7% yield, respectively. All procedures are scalable, aqueous, simple, cost-effective, and suitable for fibrinolytic enzymes industrial production.