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Applied and Environmental Microbiology, December 2005, p. 7955-7960, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7955-7960.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

Moon-Sun Jang,1 Young-Mi Lee,1 Cheorl-Ho Kim,2 Jai-Heon Lee,1 Dong-Woo Kang,1 Seok-Jo Kim,1 and Young-Choon Lee1*

College of Natural Resources and Life Science, Dong-A University, Busan 604-714,1 Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyung-Pook 780-350, South Korea2

Received 26 March 2005/ Accepted 26 August 2005

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 60°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.

* Corresponding author. Mailing address: Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea. Phone: 82-51-200-7591. Fax: 82-51-200-6536. E-mail: yclee{at}daunet.donga.ac.kr.

Applied and Environmental Microbiology, December 2005, p. 7955-7960, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7955-7960.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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