Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: Purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli

Moon Sun Jang, Young Mi Lee, Cheorl Ho Kim, Jai Heon Lee, Dong Woo Kang, Seok Jo Kim, Young Choon Lee*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

Abstract

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 6O°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

Original languageEnglish (US)
Pages (from-to)7955-7960
Number of pages6
JournalApplied and Environmental Microbiology
Volume71
Issue number12
DOIs
StatePublished - Dec 2005

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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