Wang Liqun, Chang Alan K, Yuan Wenjie, Bai Fengwu
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China.
Sheng Wu Gong Cheng Xue Bao. 2013 Jun;29(6):772-84.
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.
染料脱色过氧化物酶（DyP型过氧化物酶）是一类含血红素的过氧化物酶，能够使多种有机染料脱色，其中大多数是外源性物质。为了从运动发酵单胞菌ZM4（ATCC 31821）中鉴定和表征一种新的DyP型过氧化物酶（ZmDyP），通过PCR从运动发酵单胞菌的基因组DNA中扩增ZmDyP，并将其克隆到大肠杆菌表达载体pET-21b(+)中。ZmDyP的氨基酸序列与DyP型过氧化物酶的其他成员进行比对，结果显示存在活性位点保守残基D149、R239、T254、F256以及典型的GXXDG基序，表明ZmDyP是DyP型过氧化物酶家族的一个新成员。含有ZmDyP基因的pET-21b(+)通过IPTG诱导在大肠杆菌中表达。表达的酶通过镍螯合层析进行纯化。对纯化后的酶进行SDS-PAGE分析，结果显示分子量为36 kDa，而活性染色显示分子量为108 kDa，这表明该酶可能是三聚体。此外，如Soret带典型的血红素吸收峰所示，ZmDyP是一种含血红素的酶。而且，ZmDyP以2, 2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)为底物时表现出较高的催化效率。这些结果丰富了DyP型过氧化物酶的种类，为进一步研究奠定了基础。
