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
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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.
Aminopeptidases, Milk proteins, Lactic acid bacteria