Cloning and expression of an oligopeptidase, PepO, with novel specificity from Lactobacillus rhamnosus HN001 (DR20).
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.
Christensson
Camilla
C
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Bratt
Henrik
H
Collins
Lesley J
LJ
Coolbear
Tim
T
Holland
Ross
R
Lubbers
Mark W
MW
O'Toole
Paul W
PW
Reid
Julian R
JR
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
D001426:Bacterial Proteins; D002364:Caseins; D010447:Peptide Hydrolases; C028759:oligopeptidase; D008666:Metalloendopeptidases; C080385:oligopeptidase PepO