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Extracellular proteinase from Enterococcus faecalis subsp. liquefaciens. II. Partial purification and some
G D García de Fernando1, P E Hernández, J Burgos
1Departamento de Nutrición y Bromatología III (Higiene y Tecnología de los Alimentos), Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Folia Microbiologica
|January 1, 1991
Summary
Researchers purified an extracellular proteinase from Enterococcus faecalis subsp. liquefaciens, finding it stable and active against various proteins. This enzyme shows potential for biotechnological applications due to its robust characteristics.
Area of Science:
- Microbiology
- Enzymology
- Protein Chemistry
Background:
- Enterococcus faecalis subsp. liquefaciens produces extracellular proteinases.
- Characterization of these enzymes is crucial for understanding their roles and potential applications.
Purpose of the Study:
- To purify and characterize an extracellular proteinase from Enterococcus faecalis subsp. liquefaciens.
- To assess the enzyme's stability and substrate specificity.
Main Methods:
- Purification using gel filtration (Sephadex G-50) and affinity chromatography (gramicidin J).
- Further purification via chromatofocusing.
- Enzyme characterization including molar mass determination (Sephadex G-50, SDS-PAGE), isoelectric point, optimal pH and temperature, and substrate hydrolysis analysis.
Main Results:
- Achieved 780-fold purification with 15% yield, potentially reaching 14,800-fold with 11.4% yield.
- Molar mass estimated at ~30 kDa (gel filtration) and ~26 kDa (SDS-PAGE).
- Isoelectric point found to be 4.6; optimal activity at pH 7.5 and 45°C.
- Hydrolyzed various proteins including BSA, casein, and pork muscle proteins.
- Demonstrated high stability across a wide temperature range (-25 to 37°C) and pH (5.5–8.0 at 12°C).
Conclusions:
- The purified extracellular proteinase from Enterococcus faecalis subsp. liquefaciens is a robust enzyme.
- Its broad substrate specificity and high stability suggest potential biotechnological applications, such as in food processing or detergent formulations.