Peptidases from two strains ofPseudomonas fluorescens: partial purification, properties and action on milk

Abstract

SummaryPseudomonas fluorescensstrains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysedN-α-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33000 and a pH optimum of 8·0. The enzyme was stimulated by Ca2+and Mg2+and inhibited by Co2+, Mn2+, Hg2+, Zn2+and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44000 and a pH optimum of 8·0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases fromPseudomonasstrains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.