β-peptides are resistant to proteolysis and are metabolically stable. They have the capability to mimic α-peptidic secondary structures. Naturally occurring β-peptides composed solely of β-amino acids are not known to exist. However, six naturally occurring β-aminopeptidases that degrade β-peptides have previously been isolated from Gram negative microorganisms and a fungus exhibiting both β-peptide degrading and synthetic properties. These β-aminopeptidases possess varied substrate specificities. Based on BLAST analysis against characterised β-aminopeptidase genes, four uncharacterised genes were selected. These genes were identified in a Gram positive bacterium, Mycobacterium smegmatis MC2 155, a Gram negative bacterium, Sphingobium yanoikuyae 342-B2, a bacterium belonging to the hyperthermophilic archaea (Pyrococcus horikoshii OT3), and a fungus (Yarrowia lipolytica).
These genes were commercially synthesised, ligated to pET28a (+) and expressed in Escherichia coli BL21 (pLysS). SDS-PAGE and Western blot analysis indicated that the enzymes consist of two polypeptide subunits (α and β). In some E. coli strains the proteins appeared uncleaved, while in others they appear to be cleaved into the two subunits. Qualitative whole cell enzyme activity assays with chromogenic β-alanine-paranitroanilide as the substrate were conducted at room temperature. The active β-aminopeptidases hydrolysed the substrate and released paranitroanilide (pNA), which is yellow in colour. The released pNA was measured spectrophotometrically at 405 nm. The β-aminopeptidases from M. smegmatis, P. horikoshii, Y. lipolytica and S. yanoikuyae 342-B2 were observed to hydrolyse the substrate with the release of pNA at varying rates.
This study has resulted in the isolation of four uncharacterised enzymes with putative β-peptidase activity. Further studies on these enzymes will contribute to a better understanding of β-amino acid biotransformation and β-peptidases in the natural environment and more importantly may lead to the production of β-peptide-based materials.