Recombinant expression, purification and characterization of a novel DyP-type peroxidase in Escherichia coli.
- Author:
Liqun WANG
1
;
Alan K CHANG
;
Wenjie YUAN
;
Fengwu BAI
Author Information
1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Catalysis;
Coloring Agents;
metabolism;
Escherichia coli;
genetics;
metabolism;
Molecular Sequence Data;
Peroxidases;
biosynthesis;
genetics;
isolation & purification;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Zymomonas;
enzymology
- From:
Chinese Journal of Biotechnology
2013;29(6):772-784
- CountryChina
- Language:Chinese
-
Abstract:
Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
