Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

Leese, Charlotte, Fotheringham, Ian, Escalettes, Franck, Speight, Robert, & Grogan, Gideon (2013) Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida. Journal of Molecular Catalysis B: Enzymatic, 85-86, pp. 17-22.

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Abstract

L-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid sub-strates when combined with abiotic reductants. The gene nadB encoding the L-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K M for l-aspartic acid of 2.26 mM and a k cat = 10.6 s −1 , with lower activity also displayed towards L-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 • C, but rapid losses in activity were observed at 50 • C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both L-asparagine and L-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.

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5 citations in Scopus
3 citations in Web of Science®
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ID Code: 70402
Item Type: Journal Article
Refereed: Yes
DOI: 10.1016/j.molcatb.2012.07.008
ISSN: 1381-1177
Divisions: Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Elsevier
Deposited On: 22 Apr 2014 04:57
Last Modified: 22 Apr 2014 23:47

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