Purification and characterisation of an intracellular arninopeptidase from Brevibacterium linens ATCC 9174

Abstract

An intracellular aminopeptidase from Brevibacterium linens ATCC 9174 was purified 4300-fold to homogeneity using ammonium sulphate fractionation, anion exchange chromatography, hydrophobic interaction chromatography and anion exchange chromatography. The pH and temperature optima were 8.5 and 35 °C, respectively. The purified aminopeptidase was stable over the range pH 8 to 10, and was thermally stable up to 20 °C at pH 8.5. The molecular mass of the enzyme was found to be 59 kDa by SDS-PAGE and 69 kDa by gel filtration, indicating that the native enzyme exists as a monomer. The aminopeptidase was strongly inhibited by the thiol blocking agent, p-hydroxymercuribenzoate, and by Co2+ and Zn2+: activity was unaffected by metal chelators, reducing agents or phenylmethylsulphonyl fluoride. Km and kcat values for L-Ala-p-NA were 3.3 mmol/L and 4.3 s-1, respectively, while the corresponding values for L-Gly-p-NA were 0.2 mmol/L and 7.6 s-1, respectively. The aminopeptidase hydrolysed dipeptides with an alanine residue at the N-terminal but tripeptides were not hydrolysed. The sequence of the first 19 N-terminal amino acids was NH 2-Pro-Phe-Asp-Gly-Pro-Asp-Thr-Ala-Ala-Ile-Ile-Asp-Arg-Leu-?-Asn-Ala-? -Thr.