Aminopeptidase from Sphingomonas capsulata

Abstract

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min-1 for glutamate. At pH 7.5 (pH optimum) and 25 °C, the kinetic parameters for alanine para-nitroanilide were found to be kcat = 7600 min-1 and Km = 14 mM. For alanine 6-naphthylamide, they were kcat = 860 min-1 and K m = 6.7 mM. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.