Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Abstract

Summary.A leucine aminopeptidase was purified to homogeneity from Streptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column.The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent o n C a 2+ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids or D-amino acid residues.

Purification and Properties of an Extracellular Aminopeptidase from Streptomyces Lividans 1326

“…Kcat for hydrolysis of leu-pNA is 2-3 s-l, and K J K m is 7.7 x lo3. These values are similar to those obtained by Vitale et al (1986) for the S. rimosus LAP.…”

“…Nevertheless, S. lividans LAP was inhibited by o-phenanthroline, Na,-EDTA, NTA and EGTA, indicating that it is a metalloproteinase. Furthermore, the enzyme was partially inhibited by bestatin, a known inhibitor of aminopeptidases (Vitale et al, 1986). The combined data indicate that S. lividans LAP is a metallo-enzyme, produced during stationary phase, that has a rather narrow range of substrate specificities.…”

Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus , inhibits autogenous aminopeptidases

“…Some other aminopeptidase inhibitors containing a 3-amino-2-hydroxy-carboxylic acid also inhibit bacterial aminopeptidases: Wilkes and Prescott (1985) have shown that bestatin and amastatin are slow-and tight-binding competitive inhibitors of leucine aminopeptidase from A. proteolytica. Leucine aminopeptidase from S. rimosus is also inhibited by bestatin and amastatin (Vitale et al 1986), and leucine aminopeptidase from S. lividans is inhibited by bestatin (Aphale and Strohl 1993). All these aminopeptidases belong to the PepA-type metallo-aminopeptidases that appear to be quite common in Streptomyces species (Gonzales and Robert-Baudouy 1996).…”

“…Other aminopeptidases used were isolated in our laboratories: extracellular leucine aminopeptidase from S. rimosus (Vitale et al 1986), intracellular arginine aminopeptidase from S. rimosus (Vitale et al 1996), intracellular proline aminopeptidase from S. rimosus (Lj. Vitale, T. Tripić, I. Škrtić, unpublished work), dipeptidyl aminopeptidase III from human erythrocytes (Abramić et al 1988), and aminopeptidase N from egg white (Škrtić and Vitale 1994).…”

“…We have previously isolated a leucine aminopeptidase from the waste broth of oxytetracycline production by Streptomyces rimosus (Vitale et al 1986). Since this enzyme was inhibited by two low-molecular-weight inhibitors produced by streptomycetes (bestatin and amastatin), we investigated whether or not the culture medium of S. rimosus contains a low-molecular-weight aminopeptidase inhibitor.…”