Substrate specificity and structural investigation into PepO and PepW : two peptidases from Lactobacillus rhamnosus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

The proteolytic systems of lactic acid bacteria have important roles in the maturation and flavour development of cheese. Lactic acid bacteria pepetidases contribute to the taste of cheese through the production of low-molecular weight peptides and free amino acids. Although some lactic acid bacteria peptidases have been structurally and enzymatically characterised for their substrate specificity, there are some that are yet to be completely biochemically characterised. The aim of the present study was to investigate the substrate specificity and three-dimensional structure of two peptidases that could potentially be used as a tool to modify and control cheese bitterness and possibly other flavour attributes from Lactobacillus rhamnosus, PepO and PepW. The pepW gene was successfully cloned from L. rhamnosus into an E. coli expression system. Recombinant PepW was purified to homogeneity and was shown to exist as a hexamer of 50 kDa subunits. Recombinant PepO was expressed from a previously established L. lactis expression system and purified to homogeneity. PepO was shown to exist as a 70 kDa monomer, and function as a metallopeptidase. Pepo and PepW were shown to selectively hydrolyse chymosin-derived bovine β- and κ-casein peptides, and casein peptides extracted from Cheddar cheese. One conclusive PepO cleavage site that had not been previously characterised was identified. This was the β-casein peptide bond between Leu₆-Asn₇. Several possible PepO and PepW cleavage sites in αs₁-, β- and κ- casein were identified, suggesting that PepO has a broad endopeptidase activity, whilst PepW has a specific exopeptidase activity. Pepo and PepW crystals were successfully grown for structure determination by x-ray crystallography. Native data sets were collected for both PepO and PepW, and derivative data were collected for PepO. Structure determination was attempted using Multiple Isomorphous Replacement and Molecular Replacement techniques. Results from the substrate specificity and structural investigation of the L. rhamnosus peptidases, PepO and PepW, are presented in this thesis.