Julian R. Reid

Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine αs1-casein

SummaryThe cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with αs1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the aminp acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of αs1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the αs1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No cleat consensus sequence of amino acid residues surrounding the cleavage sites could be identified.

Cloning and Expression of an Oligopeptidase, PepO, with Novel Specificity from Lactobacillus rhamnosus HN001 (DR20)

ABSTRACT Oligopeptidases of starter and nonstarter lactic acid bacteria contribute to the proteolytic events important in maturation and flavor development processes in cheese. This paper describes the molecular cloning, expression, and specificity of the oligopeptidase PepO from the probiotic nonstarter strain Lactobacillus rhamnosus HN001 (DR20). The pepO gene encodes a protein of 70.9 kDa, whose primary sequence includes the HEXXH motif present in certain classes of metallo-oligopeptidases. The pepO gene was cloned in L. rhamnosus HN001 and overexpressed in pTRKH2 from its own promoter, which was mapped by primer extension. It was further cloned in both pNZ8020 and pNZ8037 and overexpressed in Lactococcus lactis subsp. cremoris NZ9000 from the nisA promoter. The purified PepO enzyme demonstrated unique cleavage specificity for αs1-casein fragment 1–23, hydrolyzing the bonds Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9, and Gln-9-Gly-10. The impact of this enzyme in cheese can now be assessed.