Isolation, purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine Pseudomonas mendocina

Objectives An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina. Results BTPI-301was purified to homogeneity by (NH 4 ) 2 SO 4 , precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a K i of 3.5 × 10 −10 M. It was stable and active at pH 4–12 and also at 4–90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin. Conclusion A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.