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

Leese, Charlotte, Fotheringham, Ian, Escalettes, Franck, Speight, Robert and Grogan, Gideon (2013) Cloning, expression, characterisation and mutational analysis of L-aspartate oxidase from Pseudomonas putida. Journal of Molecular Catalysis B: Enzymatic, 85-86 17-22. doi:10.1016/j.molcatb.2012.07.008


Author Leese, Charlotte
Fotheringham, Ian
Escalettes, Franck
Speight, Robert
Grogan, Gideon
Title Cloning, expression, characterisation and mutational analysis of L-aspartate oxidase from Pseudomonas putida
Formatted title
Cloning, expression, characterisation and mutational analysis of L-aspartate oxidase from Pseudomonas putida
Journal name Journal of Molecular Catalysis B: Enzymatic   Check publisher's open access policy
ISSN 1381-1177
1873-3158
Publication date 2013-01-01
Year available 2012
Sub-type Article (original research)
DOI 10.1016/j.molcatb.2012.07.008
Volume 85-86
Start page 17
End page 22
Total pages 6
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Collection year 2014
Language eng
Formatted abstract
l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates 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 KM for l-aspartic acid of 2.26 mM and a kcat = 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.
Keyword L-aspartate
LASPO
Amino acid oxidase
FAD
Biocatalysis
Deracemisation
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ
Additional Notes Available online: 25 July 2012.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
Australian Institute for Bioengineering and Nanotechnology Publications
 
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Created: Thu, 12 Sep 2013, 19:35:11 EST by Cathy Fouhy on behalf of Aust Institute for Bioengineering & Nanotechnology