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Purification of a serine protease of Vibrio parahaemolyticus and its characterization
Masami Ishihara1, Ayako Kawanishi, Hirofumi Watanabe
1Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Abstract:
A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease. N-terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.
Insights
A novel calcium-dependent serine protease, VPP1, was purified from Vibrio parahaemolyticus. This protease is similar to and widely distributed in related Vibrio species.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Vibrio parahaemolyticus is a significant marine bacterium causing seafood-borne illnesses.
- Proteases play crucial roles in bacterial pathogenesis and physiology.
- Characterization of novel enzymes from pathogenic bacteria is essential for understanding their mechanisms.
Purpose of the Study:
- To purify and characterize a novel protease, VPP1, from a clinical isolate of Vibrio parahaemolyticus.
- To determine the enzymatic properties and potential similarities of VPP1 with other bacterial proteases.
- To investigate the distribution of VPP1 or related proteases in other Vibrio species.
Main Methods:
- Purification of VPP1 using ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration, and Fractogel EMD TMAE 650 ion-exchange chromatography.
- Enzyme inhibition assays using EDTA, EGTA, and serine protease inhibitors.
- N-terminal amino acid sequencing and antibody-based cross-reactivity assays.
Main Results:
- A 50 kDa protease, VPP1, was successfully purified.
- VPP1 was identified as a calcium-dependent serine protease.
- VPP1 exhibited significant similarity to a protease from Vibrio metschnikovii, with cross-reactivity observed.
- VPP1 or related proteases were found to be prevalent in both Vibrio parahaemolyticus and Vibrio alginolyticus.
Conclusions:
- VPP1 is a novel calcium-dependent serine protease from Vibrio parahaemolyticus.
- VPP1 shares characteristics with proteases from other Vibrio species, suggesting conserved enzymatic functions.
- The widespread distribution of VPP1-like proteases indicates their potential importance in the ecology and pathogenicity of Vibrio species.
