Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: characterization and specificity for beta-casein

Abstract

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.

FIG. 1

The action of whole cells (A1) and cell wall crude proteinase (A2) on α-casein (lane 2) after 4 h (lanes 3 and 6), 8 h (lanes 4 and 7), and 24 h (lanes 5 and 8), the action of whole cells (B1) and cell wall crude proteinase (B2) on β-casein (lane 10) after 4 h (lanes 11 and 14), 8 h (lanes 12 and 15), and 24 h (lanes 13 and 16), and the action of whole cells (C1) and cell wall crude proteinase (C2) on κ-casein (lane 18) after 4 h (lanes 19 and 22), 8 h (lanes 20 and 23), and 24 h (lanes 21 and 24) are shown. Molecular mass markers are 116, 97, 66, 45, and 29 kDa (from top to the bottom) (lanes 1, 9, and 17). The reaction mixtures were incubated at 40°C and pH 6.0. Electrophoretic conditions were 12.5% acrylamide gels in 0.025 M Tris HCl–0.19 M glycine buffer (pH 8.3).

FIG. 2

Action of the cell wall crude proteinase on β-casein at various pH values. Lanes: 1, Molecular weight markers; 2, β-casein; 3, pH 4; 4, pH 5; 5, pH 6; 6, pH 7; 7, pH 8; 8, pH 9. Reaction mixtures were incubated at 40°C for 8 h. Electrophoretic conditions were as in Fig. 1.

FIG. 3

Action of the cell wall crude proteinase on β-casein at various temperatures. Lanes: 1, molecular weight markers; 2, β-casein; 3, 10°C; 4, 20°C; 5, 30°C; 6, 40°C; 7, 50°C. Reaction mixtures were incubated at pH 6.0 for 8 h. Electrophoretic conditions were as in Fig. 1.

FIG. 4

Action of the cell wall proteinase on β-casein in the presence of various inhibitors (10 mM). Lanes: 1, β-casein; 2, proteolysis assay in the absence of inhibitor; 3, proteolysis assay in the absence of inhibitor but in the presence of 3 μl of isopropanol; 4, PMSF; 5, DFP; 6, EDTA; 7, 1,10-phenanthroline; 8, iodoacetamide; 9, N-ethylmaleimide; 10, molecular mass markers (116, 97, 66, 45, and 29 kDa from top to bottom). Reaction mixtures were incubated at 40°C and pH 6.0 for 8 h. Electrophoretic conditions were as in Fig. 1.

FIG. 5

Separation by reversed-phase HPLC of the peptides from the 1% TFA soluble fraction obtained after 8, 24, and 48 h of hydrolysis of β-casein by the cell wall proteinase of L. delbrueckii subsp. lactis ACA-DC 178.

FIG. 6

Localization of the cell wall proteinase-generated peptides 1 to 4 in the amino acid sequence of β-casein.