Antibacterial serine protease from Wrightia tinctoria: Purification and characterization
Sakthivel Muthu1, Venkatesh Babu Gopal1, Selvakumar Soundararajan1
1Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu, India.
Plant Physiology and Biochemistry : PPB
|January 15, 2017
Summary
A serine protease was isolated from Wrightia tinctoria leaves, demonstrating significant antibacterial properties against various bacteria. This enzyme effectively degrades the bacterial peptidoglycan layer, offering potential therapeutic applications.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Wrightia tinctoria is a medicinal plant with various bioactive compounds.
- Serine proteases are enzymes with diverse biological functions, including antimicrobial activity.
- Understanding plant-derived enzymes can lead to novel therapeutic agents.
Purpose of the Study:
- To purify and characterize a serine protease from Wrightia tinctoria leaves.
- To evaluate the antibacterial activity of the purified serine protease.
- To investigate the mechanism of antibacterial action.
Main Methods:
- Sequential flow through purification methods including screening, optimization, ammonium sulfate precipitation, gel filtration, and ion exchange chromatography.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D gel electrophoresis for molecular mass determination.
- Determination of optimal pH and temperature stability, and effect of metal ions.
- Antibacterial activity assay using zone of inhibition method against Gram-positive and Gram-negative bacteria.
- Transmission electron microscopy (TEM) to visualize bacterial cell wall degradation.
Main Results:
- A serine protease was successfully purified with a yield of 11.58% and a purification fold of 9.56.
- The purified serine protease exhibited a molecular mass of 38.5 kDa.
- Optimal activity was observed at pH 8.0 and 45°C, with enhanced activity in the presence of Mg2+ and Mn2+.
- Significant antibacterial activity was noted against Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.
- TEM analysis confirmed that the serine protease degrades the peptidoglycan layer of bacteria.
Conclusions:
- Wrightia tinctoria leaves contain a potent serine protease with broad-spectrum antibacterial activity.
- The enzyme's ability to degrade peptidoglycan suggests a novel mechanism for combating bacterial infections.
- This purified serine protease holds promise as a potential therapeutic agent for bacterial diseases.
