Purification And Characterization Of CEP From Lactobacillus

Sour milk fermented by Lactobacillus was found to have the ability of anti-hypertension because of the Angiotensin I-Converting Enzyme (ACE). The object of this article was to separation and purification the cell-envelop-protease (CEP) of Lactobacillus, and the physicochemical characterization of CEP and the capability of hydrolyze various casein to ACE inhibitory peptides were also studied in this research.Two lactobacilli producing high proteinase were screened from gastrointestinal tract of duck, and the stains were identified as Lactobacillus casei subsp. rhamnosus, Lactobacillus casei by 16S rRNA. Consideration the higher activity of CEP, named Lactococcus casei DI-1, which was selected for the further study.The CEP from Lactobacillus casei DI-1was released from the cells by Ca²⁺-free method. pH, temperature and EDTA-Na2 concentrations were studied through single factor experiment and response surface methodology. The optimum conditons of temperature, pH and EDTA-Na2 concentration were 39.75℃, pH6.98 and 50.03mM, respectively.The CEP from Lactobacillus casei DI-1 was purified by ammonium sulfate precipitation, ultrafiltration Demineralization, chromatographed on DEAE-Sephadex A-25 ion exchange column and Sephadex G-100 gel column. The result shows that the specific activities of enzyme was 19.01 mg/mL, the recovered activity was about 24.85% and the enzyme was purified about 74.04-fold.The purified CEP was a monomer structure and had a molecular mass of about 35 kDa, michaelis constant Km was 0.29mM, and optimal activity occurred at pH 7.0 and 37℃. It is a metallopeptidase, activated by Mn²⁺, Mg²⁺, Ba²⁺, Co²⁺ and Fe3+, inhibited by K+, Ca²⁺, Zn²⁺, Ni²⁺ and EDTA. The CEP can also be was inhibited by PMSF which shows it is a serine proteinase. According to the N-terminal sequencing of Lactobacillus casei, the 10 amino acids sequence of N-terminal was determined as Asp-Asn-Asp-Phe-Glu-Ile-Phe-Glu-Ser-Ser- for the purified enzyme.The effect of process conditions on the ACE inhibitory activity of casein concentrates which were separated by DEAE Sepharose Fast-Flow ion exchange column hydrolysed with crude proteinases and purified enzyme preparation from Lactobacillus casei was also investigated. ACE inhibitory activity producted by purified enzyme occupyed more than 50% ACE inhibitory activity of crude proteinases.This further proved the CEP is the key enzyme of ACE inhibition of hydrolysis of milk protein peptide and the synergistic enzyme in other protease hydrolysis process.