A comparative study of an aminopeptidase from lactic acid bacteria: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Aminopeptidase enzymes from the proteolytic systems of S.salivarius subsp.thermophilus Lactococcus lactis subsp.cremoris and Lactococcus lactis subsp.lactis have been investigated. An aminopeptidase was purified to near homogeneity from a crude cell free extract of S.thermophilus 5109. The enzyme had a native molecular weight of approximately 96kDa determined by gel-filtration, and a subunit molecular weight of 98kDa, determined by denaturing polyacrylamide gel electrophoresis, showing the native enzyme to be a monomer. The aminopeptidase activity was optimal at pH 7.0 and 35°C. The enzyme was inactivated by p-chloromercuribenzoic acid, iodoacetic acid,the chelating agents EDTA and 1,10-phenanthroline and the divalent cations Cu2+, Zn2+ and Co2+. The aminopeptidase was not inhibited by the serine protease inhibitor PMSF and only minor inhibition occured with the inhibitor No:-p-tosyl-L-lysine chloromethyl ketone (TLCK). The aminopeptidase was capable of hydrolysing several amino-acyl amido methyl coumarin (AMC) and p-nitroanilide (pNA) derivatives, particularly those of lysine, arginine and leucine. The enzyme showed greatest activity with lysyl derivatives (and is therefore referred to in this thesis as a lys-aminopeptidase). The enzyme was able to degrade several oligopeptides by progressive cleavage of the peptide bond but did not hydrolyse peptides containing a proline or aspartic acid residue in the second position. The aminopeptidase activity was dependent on the size of the peptide in that generally only peptides with more than three amino acids were degraded. The aminopeptidase had no endopeptidase or dipeptidase activity. Five different amino-acyl p-nitroanilides derivatives and two amido methyl coumarin derivatives were used to determine the kinetic parameters of the aminopeptidase. The Km values obtained for all the substrates tested were similar, with the exception of ala-pNA, for which the Km value was significantly higher. On the basis of the distribution of activity between different cell-fractions the lys­ aminopeptidase appears to be localised intracellularly. An aminopeptidase was also partially purified from cell-free extracts from Lactococcus lactis subsp.cremoris AM2 and Lactococcus lactis subsp.lactis ML3. The aminopeptidase from L.cremoris AM2 was shown to have a molecular weight of 106kDa and was a monomer. It showed optimal activity at a pH of 7.0 and 450c. The aminopeptidase activity was inhibited by metal-chelators, SH group inhibitors and TLCK. The aminopeptidase hydrolysed lysyl-, arginyl- and leucyl-p-nitroanilide derivatives, but had little or no activity with other pNA substrates. The aminopeptidase from L.lactis ML3 had a molecular weight of 100-105kDa and was monomeric. The optimal activity for the aminopeptidase was at pH of 7.0 and 40°C. The enzyme was inactivated by metal-chelators, sulphydryl inhibitors and by TLCK. Like the aminopeptidases from the other two strains the ML3 aminopeptidase was very specific hydrolysing lysyl-, leucyl- and arginyl-pNA but with very little or no activity with other amino-acyl derivatives