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 alpha(s1)-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.

FIG. 1.

Cloning strategies in pNZ8020 and pNZ8037. The nisA promoter and ribosome binding sites for nisA and pepO are in bold, primers used to amplify pepO are double underlined, and nucleotides changed by site-directed mutagenesis are in bold italics. (A) pNZ8020 was cut with BamHI and ligated to the PCR product of pepO with forward primer PepO10 cut with BglII. This generated a NisA-PepO fusion protein. (B) pNZ8020 was cut with BamHI and ligated to the PCR product of pepO with forward primer PepO11 cut with BglII. Primer PepO11 introduced a stop codon for nisA by site-directed mutagenesis. (C) pNZ8037 was cut with NcoI and ligated to the PCR product of pepO with forward primer PepO12 cut with BspLU11I. An A instead of a T was introduced by primer PepO12 by site-directed mutagenesis, resulting in a codon for serine rather than threonine as the second amino acid.

FIG. 2.

Nucleotide and protein sequences of pepO in L. rhamnosus HN001. The promoter, ribosome binding site (RBS), and zinc motif are shown in bold. The translated protein is shown, and the full open reading frame is indicated by italics. Arrows show the inverted repeats.

FIG. 3.

L. rhamnosus HN001 pepO transcript analysis. (A) Northern hybridization of total RNA with a pepO probe. (B) Primer extension analysis of pepO. Sequence reactions (A, G, C, and T) and primer extension (PE) were done with the same primer. The arrows indicate the primer extension products. (C) Interpretation of the primer extension results. The putative pepO −35 and −10 promoter hexamers (bold), transcription start site (underlined), and putative ribosome binding site and ATG start codon (italics) are shown.

FIG. 4.

SDS-PAGE showing the molecular mass and purity of PepO. (A) Cell extract of L. lactis subsp. cremoris NZ9000 with pLB003 induced with nisaplin (lane 1) and the final purified PepO (lane 2). (B) Cell extract of L. lactis subsp. cremoris NZ9000 with pLB003 obtained without induction (lane 1) and induced with nisaplin (lane 2). Lane 3, standard proteins.

FIG. 5.

Bonds hydrolyzed in α_s1-casein f(1–23) and bradykinin by PepO from L. rhamnosus HN001 compared with specificities of previously described oligopeptidases: LEP II (44), Lactococcus lactis subsp. lactis MG1363 PepO (37), L. lactis neutral thermolysin-like oligoendopeptidase (NOP) (1), LEP I (45), L. lactis alkaline endopeptidase (1), L. delbrueckii subsp. bulgaricus B14 endopeptidase (3), L. lactis subsp. cremoris SK11 endopeptidase (34), L. lactis neutral neprilysin-like endopeptidase (NEP) (23), L. paracasei Lc-01 oligopeptidase (41), and L. lactis subsp. cremoris NCDO763 PepF1 (29).