Identification And Biochemical Characterization Of A Novel Dye-decolorizing Peroxidase

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases that can decolour various organic dyes, most of which are xenobiotics. These enzymes can also enhance the resistance of bacteria to oxidative stress through participating in its regulation.A putative heme-containing peroxidase gene was found in the genomic DNA of Zymomonas mobilis ZM4(ATCC31821). It codes for a enzyme (designated as ZmDyP) that contains the conserved active residues D149, R239, T254, F256as well as the typical GXXDG motif found in the active sites of other members of the DyP-type peroxidases, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. So far, there has been no report on the characterization of ZmDyP.To characterize ZmDyP. the entire coding region of its gene was amplified from the genomic DNA of Z. mobilis ZM4by PCR and cloned into the Escherichia coli expression vector pET-21b(+). The resulting plasmid (pET-21b-ZmDyP) was transformed into E. coli BL21(DE3)/pLysS and the enzyme was expressed by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a single band with an apparent molecular weight of36kDa. However, enzyme activity staining gave a molecular weight of108kDa, suggesting that the enzyme could be a trimer. ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band by spectrophotometric analysis. Moreover. ZmDyP shows a high catalytic efficiency with2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. Surprisingly, the activity of ZmDyP was enhanced by high concentration of isoamyl alcohol without H2O2, a property that has not been demonstrated for DyP-type or other heme-containing peroxidases, and thus this may open a new aspect of research into heme-containing peroxidases.Finally, the relevance of ZmDyP in term of its physiological function ZmDyP was explored in E. coli. and the result showed that ZmDyP could offer some advantage in growth, probably through enhancing the resistance of the cells to oxidative stress. The result of this study therefore enriches the knowledge pool of DyP-type peroxidases and also lays a foundation for further study and potential application of ZmDyP.