Identification and characterization of β-aminopeptidases - insight into enzymes without a known natural substrate

β-Aminopeptidases have the unique capability to hydrolyse N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date six β-aminopeptidases have been discovered and functionally characterised, five from Gram negative bacteria and one from a fungus, Aspergillus . Here we report on the purification and characterisation of an additional four β-aminopeptidases from a Gram-positive bacterium, Mycolicibacterium smegmatis (Ms BapA), a yeast, Yarrowia lipolytica (Ylip BapA), and two β-aminopeptidases (BcA5 BapA and BcC1 BapA) from Gram-negative bacteria isolated from activated sludge identified as Burkholderia sp. The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated as the active enzyme was recovered after IMAC purification even though only the α-subunit was His 6 -tagged. The enzymes were shown to hydrolyse chromogenic substrates with the N-terminal L-configuration βhGly and β 3 hLeu with high activities. These enzymes displayed higher activity with H-βhGly- p NA than previously characterised enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions. Importance β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilise synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterised with preferences for different β-amino acid substrates and have demonstrated the capability to not only catalyse the degradation of synthetic β-peptides, but also catalyse the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with varying β-amino acid substrate preferences and kinetics. These enzymes have potential to be utilised in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important because β-amino acids and peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.