Towards the directed evolution of an L-aspartate oxidase from Pseudomonas putida

Amino acid oxidases (AAOs) are enantioselective flavoenzymes that catalyse the oxidation of amino acids into imino acids, which spontaneously hydrolyse in water to form keto acids. AAOs have several potential applications, most notably as biocatalysts in the production of enantiomerically pure amino acids and keto acids, or in enzymatic biosensors. Therefore, there is a demand for a range of AAOs with specific activity against various substrates, encouraging the characterisation of less well understood oxidases.
Seven putative oxidases were cloned into the pET-YSBLIC-3C expression vector, expressed in E. coli-DE3 expression strains and assayed for activity against all proteinogenic amino acids. Of these seven targets, the L-amino acid oxidase from Pseudomonas putida (PpLAAO) was found to be highly soluble, had detectable activity against L-aspartate and L-asparagine and had not been investigated in depth previously.
The purified PpLAAO protein showed high substrate specificity against L-aspartate and lower activity with substrate inhibition against L-asparagine. Very low activity was also detected against L-glutamate. The purified protein had optimal activity around pH 7.5 and at temperatures between 4°C and 30°C.
To investigate the role of residues in the active site area of the PpLAAO protein thirteen active site residues, determined by comparison with the structure of the L-aspartate oxidase from E. coli (L-AspO), were mutated to alanine. Eleven of these mutants were purified and assayed against L-aspartate, L-asparagine and L-glutamate. Results were largely consistent with knowledge of L-AspO.
Ingenza Ltd. has an interest in potential applications of L-tyrosine and L-alanine oxidases. Because of this iterative combinatorial active site saturation testing, using the active structure of L-AspO was performed alongside a small scale epPCR mutagenesis in an attempt to introduce activity against L-alanine and L-tyrosine. L-homoserine was also targeted as part of a substrate walking approach towards L-alanine; however no novel activity was detected in any transformants.