Expression, purification, and characterization of a membrane-bound D-amino acid dehydrogenase from Proteus mirabilis JN458

Biotechnol Lett. 2017 Oct;39(10):1559-1566. doi: 10.1007/s10529-017-2388-0. Epub 2017 Jul 4.

Abstract

Objectives: To characterize a novel membrane-bound D -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).

Results: The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with D -alanine the most preferred substrate. The K m and V max values for D -alanine were 9 mM and 20 μmol min-1 mg-1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.

Conclusions: D-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.

Keywords: Characterization; D -amino acid; D -amino acid dehydrogenase; Proteus mirabilis.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Cell Membrane / enzymology
  • Cloning, Molecular / methods*
  • D-Amino-Acid Oxidase / genetics*
  • D-Amino-Acid Oxidase / isolation & purification*
  • Hydrogen Peroxide / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • Proteus mirabilis / enzymology*
  • Proteus mirabilis / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Sequence Analysis, Protein
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Hydrogen Peroxide
  • D-Amino-Acid Oxidase