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Published on: August 29, 2025
Purification and characterization of secretory serine protease from necrotrophic oomycete, Pythium myriotylum
Ravindranathan Aswati Nair1, Chellappan Geethu
1School of Biotechnology, National Institute of Technology Calicut (NITC), NITC Campus P.O, Calicut, 673 601, Kerala, India, aswati@nitc.ac.in.
Abstract:
Progressive increase in extracellular proteolytic activity with respect to growth was detected in cultures of necrotrophic oomycete Pythium myriotylum Dreschler with maximum activity detected at stationary phase of growth. The secretory protease from P. myriotylum designated, spPm1 was purified to homogeneity giving a single band of 47 kDa molecular mass on non-reducing SDS-PAGE and exhibiting caseinolytic activity in the zymogram. Under reducing conditions, an additional band of 27.0 kDa molecular size observed was subjected to matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Resulting peptide identified as an autolytic product and generated under reducing conditions on SDS-PAGE, showed homology to domains of oomycete effector molecules. spPm1 retained proteolytic activity over broad pH (5.0-12.0) and temperature (10-80 °C) ranges with optimal pH and temperature at 8.0 and 60 °C respectively. spPm1 was identified as a serine protease following experiments with inhibitors specific to various protease groups. spPm1 displayed high stability to surfactants, organic solvents, oxidizing agents and to metal-ion chelator, EDTA. Kinetic parameters, Km and Vmax were determined as 0.04 mM and 7.52 U min(-1) mg(-1) respectively. Preferential hydrolysis of synthetic fluorogenic substrate, SAAPPPA (N-succinyl-L-alanyl-alanyl-proline-phenylalanine-p-nitroanilide) confirmed spPm1 as belonging to subtilisin serine protease family. The excellent stability of spPm1 protease characterized from P. myriotylum is discussed with respect to its potential industrial applications.
Insights
A novel serine protease, spPm1, was purified from Pythium myriotylum, showing broad stability and potential for industrial applications. This extracellular protease exhibits high activity across various conditions.
Area of Science:
- Biochemistry
- Molecular Biology
- Oomycete Research
Background:
- Extracellular proteolytic activity increases with growth in Pythium myriotylum.
- This activity peaks during the stationary phase.
Purpose of the Study:
- To purify and characterize a secretory protease from P. myriotylum.
- To investigate its biochemical properties and potential industrial applications.
Main Methods:
- Protease purification using SDS-PAGE.
- Enzyme activity assays (caseinolytic, zymogram).
- MALDI-TOF-MS for peptide identification.
- Enzyme inhibition assays.
- Kinetic parameter determination.
- Substrate specificity analysis.
Main Results:
- A 47 kDa serine protease, spPm1, was purified.
- spPm1 showed homology to oomycete effector domains.
- Optimal activity at pH 8.0 and 60°C.
- High stability across broad pH, temperature, and chemical conditions.
- Belongs to the subtilisin serine protease family.
Conclusions:
- spPm1 is a robust serine protease with potential industrial utility.
- Its stability suggests applications in various biotechnological processes.
- Further research into its effector functions in oomycetes is warranted.
