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Applied and Environmental Microbiology, January 2002, p. 254-262, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.254-262.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Camilla Christensson,^1,2, Henrik Bratt,^1,2 Lesley J. Collins,² Tim Coolbear,² Ross Holland,² Mark W. Lubbers,² Paul W. O’Toole,¹ and Julian R. Reid^2*

Institute of Molecular BioSciences, Massey University,¹ New Zealand Dairy Research Institute, Palmerston North, New Zealand²

Received 8 May 2001/ Accepted 12 October 2001

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 _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.

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* Corresponding author. Mailing address: Microbiology, Nutrition and Enzyme Science Section, New Zealand Dairy Research Institute, Private Bag 11029, Palmerston North, New Zealand. Phone: 64 6 350 4649. Fax: 64 6 350 4616. E-mail: julian.reid{at}nzdri.org.nz.

Present address: Department of Applied Microbiology, Lund University, SE-221 00 Lund, Sweden.

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Applied and Environmental Microbiology, January 2002, p. 254-262, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.254-262.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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