A study of aminopeptidases from lactic streptococci : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Two arninopeptidase enzymes from the proteolytic system of Streptococcus lactis 4760 have been studied. An X-Prolyl dipeptidyl arninopeptidase has been purified and characterised. The enzyme has a native molecular weight of approximately 150 kDa determined by gel filtration, and a subunit molecular weight of 83 000, determined by denaturing polyacrylarnide gel electrophoresis, showing the native enzyme to be a dimer. It is inhibited by phenyl methyl sulphonyl fluoride and is active over a pH range of 6 - 9. A range of X-Prolyl-amido methyl coumarin (X-Pro-AMC) derivatives with different aminoacyl residues in the X position have been used to define the steady state kinetic parameters. The Km and kcat values obtained with all of the X-Pro-AMC substrates tested were similar, with the exception of Glu-Pro-AMC, which gave a somewhat higher Km value. The action of the enzyme in degrading small peptides has been studied. It was found to be capable of removing X-Proline residues from peptides, except where two proline residues are situated in consecutive positions. A Lysyl-arninopeptidase has been partially purified and its characteristics studied. This enzyme has been shown to have a native molecular weight of approximately 78 000. It hydrolyses lysyl-, arginyl-, and leucyl-arnido methyl coumarin derivatives, but has little or no activity with other arninoacyl-AMC substrates. It also catalyses the removal of lysine and arginine residues from the amino-terminus of short peptides. The partially purified arninopeptidase preparation also has endopeptidase activity which is probably due to contamination by a separate enzyme. The individual and combined effects of these two enzymes on -casein-derived oligopeptides (produced by proteolytic action of the S.lactis proteinase) have been studied. These results indicate that these enzymes may be important in degradation of some casein-derived peptides during cheese ripening, while other peptides are resistant to hydrolysis