Purification and characterization of cell-envelopeproteinase from Lactobacillus casei DI-1
Using a Ca2+-free method, the cell-envelope proteinase (CEP) of Lactobacillus casei DI-1 isolated from duck small intestine was released from cells and purified by ammonium sulfate precipitation, and by diethylaminoethyl (DEAE)-Sephadex A-25 and Sephadex G-100 gel chromatography. The purified CEP had a monomer structure with a molecular mass of about 35 kDa. Optimal activity occurred at pH 7.0 and 37°C. The purified CEP was a metallopeptidase, which was activated by Co2+, Ba2+, Mg2+ and Fe3+, and inhibited by Ca2+, Zn2+, K+, Ni2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It was a serine proteinase which was inhibited by phenylmethylsulfonyl fluoride (PMSF). Its kinetic constant (Km) is 0.29 mM and the first 10 amino acids of the CEP’s N-terminal sequences were Asp-Asn-Asp-Phe-Glu-Ile-Phe-Glu-Ser-Ser. The hydrolysates of α-, β- and κ-casein produced by CEP showed different angiotensin-I-converting enzyme (ACE) inhibitory activity; the hydrolysates of β-casein displayed the greatest ACE inhibitory activity.
Key words: Cell-envelope proteinase, purification, characterization.