Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120.
The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35 degrees C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 muM.min(-1), respectively. The apparent K(m) and k(cat)/K(m) values for Reactive Blue 5 were 3.6 muM and 1.2 x 10(7) M(-1) s(-1), respectively, while the apparent K(m) and k(cat)/K(m) values for H(2)O(2) were 5.8 muM and 6.6 x 10(6) M(-1) s(-1), respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to approximately 50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.
Ogola
Henry Joseph Oduor
HJ
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan. ysawa@life.shimane-u.ac.jp.
Kamiike
Takaaki
T
Hashimoto
Naoya
N
Ashida
Hiroyuki
H
Ishikawa
Takahiro
T
Shibata
Hitoshi
H
Sawa
Yoshihiro
Y
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D000880:Anthraquinones / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000032:analysis; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D000880:Anthraquinones; D001426:Bacterial Proteins; D003067:Coenzymes; D006418:Heme; D006861:Hydrogen Peroxide; D009195:Peroxidase