β-Aminopeptidases have the unique capability to hydrolyze 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 characterized， five from Gram-negative bacteria and one from a fungus， Here we report on the purification and characterization of an additional four β-aminopeptidases， one from a Gram-positive bacterium， (BapA)， one from a yeast， (BapA)， and two from Gram-negative bacteria isolated from... More
β-Aminopeptidases have the unique capability to hydrolyze 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 characterized， five from Gram-negative bacteria and one from a fungus， Here we report on the purification and characterization of an additional four β-aminopeptidases， one from a Gram-positive bacterium， (BapA)， one from a yeast， (BapA)， and two from Gram-negative bacteria isolated from activated sludge identified as spp. (BapA and BapA). The genes encoding β-aminopeptidases were cloned， expressed in ， 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 immobilized-metal affinity chromatography (IMAC) purification， even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β-homo-Leu (βhLeu) with high activities. These enzymes displayed higher activity with H-βhGly--nitroanilide (H-βhGly-NA) than previously characterized 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 also showed that the β-aminopeptidases may play a role in some cellular functions. β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized 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 β-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.
