Extraction and Partial Characterization of Proteolytic Activities from the Cell Surface of Lactobacillus helveticus Zuc2

Percentage of the total proteolytic activity extracted from Lactobacillus helveticus Zuc2 cells by phosphate buffer (P_i) at increasing ionic strength. The activity is expressed as OD₃₄₀.

Sodium dodecyl sulfate-PAGE separation of various Lactobacillus helveticus Zuc2 cell envelope proteinase (CEP) extracts. Lane 1: purified CEP. Lanes 2 to 6: various extracts: white precipitate from dialysis (2); 5 M LiCl (3); 0.5 M phosphate buffer (P_i) (4); 0.5 M P_i + 1mM PMSF (5); 0.5 M P_i + 20mM EDTA (6). Lane 7: SDS molecular mass marker. A = 180-kDa proteinase; B = S-layer monomer.

Digestion of α_s1-CN (lanes 1 to 4), β-CN (lanes 5 to 8), and κ-CN (lanes 9 to 12) by Lactobacillus helveticus Zuc2 cell envelope proteinase at different incubation times: 0min, lanes 1, 5, 9; 10min, lanes 2, 6, 10; 60min, lanes 3, 7, 11; 120min, lanes 4, 8, 12. α_s1I = α_s1-CN (f24–199); GMP = glycomacropeptide.

Reversed-phase HPLC patterns of α_s1-CN (f1–23) hydrolysis products after 10min (A) and 30min (B) of incubation at 37°C with purified Lactobacillus helveticus Zuc2 cell envelope proteinase.

Reversed-phase HPLC patterns of GMP hydrolysis products after 30min of incubation at 37°C with purified Lactobacillus helveticus Zuc2 cell envelope proteinase. GMP = undigested glycomacropeptide (not glycosylated fraction); glycosylated GMP = undigested glycomacropeptide (glycosylated fractions).

Reversed-phase HPLC profile of α_s1-CN (f1–23) hydrolysis products after 30min of incubation with Lactobacillus helveticus Zuc2 cells treated by 3 M LiCl.