title: 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.
authors.affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
authors.forename: Camilla
authors.initials: C
authors.lastname: Christensson
authors.forename: Henrik
authors.initials: H
authors.lastname: Bratt
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Collins
authors.forename: Tim
authors.initials: T
authors.lastname: Coolbear
authors.forename: Ross
authors.initials: R
authors.lastname: Holland
authors.forename: Mark W
authors.initials: MW
authors.lastname: Lubbers
authors.forename: Paul W
authors.initials: PW
authors.lastname: O'Toole
authors.forename: Julian R
authors.initials: JR
authors.lastname: Reid
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002364:Caseins / Q000378:metabolism; D002611:Cheese / Q000382:microbiology*; D003001:Cloning, Molecular; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019936:Probiotics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D010447:Peptide Hydrolases; C028759:oligopeptidase; D008666:Metalloendopeptidases; C080385:oligopeptidase PepO