Characterization of the cell wall-bound proteinase of Lactobacillus casei HN14

Abstract

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca2+-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolizes β-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.