Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG
1Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec Canada H9X 3V9.
Insights
This study isolated and purified an intracellular proteinase from Lactobacillus casei. The enzyme, with a molecular weight of 55 kDa, showed optimal activity at pH 6.5 and 37°C, preferring beta-casein hydrolysis.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Lactobacillus casei is a probiotic bacterium with potential industrial applications.
- Intracellular proteinases play crucial roles in cellular processes and can be valuable biotechnological tools.
Purpose of the Study:
- To isolate and characterize the intracellular proteinase from Lactobacillus casei ssp. casei LLG.
- To determine the enzyme's biochemical properties and substrate specificity.
Main Methods:
- Isolation and purification using Fast Protein Liquid Chromatography (FPLC) with ion-exchange and gel filtration.
- Determination of molecular weight, isoelectric point, optimal pH, and temperature.
- Enzyme activity assays with various inhibitors and metal ions, and casein hydrolysis analysis.
Main Results:
- A single monomeric proteinase (55 kDa, pI ~4.9) was purified.
- Optimal activity observed at pH 6.5 and 37°C.
- Enzyme inactivated by EDTA, activated by Ca++, Mn++, Co++; showed higher activity on beta-casein.
Conclusions:
- The characterized intracellular proteinase from Lactobacillus casei possesses unique biochemical properties.
- Its specificity towards beta-casein suggests potential applications in dairy processing or other biotechnological fields.
Abstract:
The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.
