Characterization of a trypsin-like protease 1 produced by a probiotic Lactobacillus plantarum subsp. plantarum PTCC 1896 from skimmed milk based medium

The present study investigates the catalytic activity of a trypsin-like protease (TLP1) and its production management from probiotic bacterium Lactobacillus plantarum subsp. plantarum PTCC 1896. Different culture media were screened for the production of TLP1 at 37 degrees C. TLP1 was only produced in skimmed milk broth (SMB) medium with the maximal activity of 1.377 +/- 0.026 mU/mL following 14 h of fermentation. The optimum temperature and pH for hydrolyzing the trypsin specific substrate N-benzoyl-nt-arginine-p-nitroanilide (BAPNA) were found to be 39 degrees C and 7.5, respectively. Heat stability of the secreted TLP1 from the bacterium was more than 37 degrees C, within the pH ranges of 7.0-9.0. The Michaelis-Menten constant (K-m) of the filtered TLP1 for BAPNA was 0.8 mM and the maximum velocity was 1.53 pmol/min. The crude TLP1 was purified from SDS-PAGE using the electroelution method. The purified TLP1 showed a single protein band of approximately 25 kDa on SDSPAGE and 93.3% similar amino acid composition to standard trypsin. Generally, we showed that optimized fermentation conditions can provide sufficient amounts of active and safe TLP1 from the bacterium. The produced TLP1 has great potential to be used for the production of bioactive peptides from natural sources for diabetes prevention.