Specific aminopeptidases of indigenous Lactobacillus brevis and Lactobacillus plantarum

Abstract

Lactic acid bacteria play an important role in milk coagulation and cheese ripening. To select strains showing interesting industrial features, two indigenous lactobacilli (Lactobacillus brevis and Lactobacillus plantarum) were studied for aminopeptidase activity. Cell and cells free extract were tested for leucyl aminopeptidase activity on the chromogenic leucyl-p-nitroanilide substrate. Intracellular and membrane enzymes were solubilized with glycine /lyzozyme treatment then purified by ammonium sulphate precipitation followed by Sephadex G100 and diethylaminoethyl (DEAE) ions exchange chromatography's separation. The molecular weight of denatured proteins was estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Effects of several parameters, pH, temperature, some ions and inhibitors on purified enzyme activity were studied. Cellular aminopeptidase activity was higher for CHTD 27 strain than BH14 strain. No aminopeptidase activity was noted in the cell free extract. The results of chromatography sephadex G100 combined to those of electrophoresis allowed suggesting a dimer structure for the native enzyme. The Lb CHTD27 purified enzyme showed maximal activity at pH 6.6 and at 40°C. This enzyme was partially inhibited by ethylenediamine acetic acid (EDTA) and Cu 2+ ions but increased by Na 2+ and Co 2+ ions. The aminopeptidase extracted from Lb BH14 was inhibited by EDTA and phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride (PMSF), its maximal activity was observed at pH 7.5 and 40°C. In addition to other characteristics as proteolysis and autolysis, in this paper we showed that both studied strains were also able to degrade peptide with specific peptidases which are important characters in cheese manufacturing.

Antibacterial and technological properties of Lactococcus lactis ssp. lactis KJ660075 strain selected for its inhibitory power against Staphylococcus aureus for cheese quality improving

“…The extracellular proteolytic activity of culture supernatant was examined using well diffusion, in milk-agar medium (1 and 10 %) plates. After incubation at 30, 25 and 12°C for 4 days, proteolytic activity was detected as a clear zone around the colonies or wells (Belkhir et al 2012).…”