一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

Recombinant expression, purification and characterization of a novel DyP-type peroxidase in Escherichia coli

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白。为了研究运动发酵单胞菌Zymomonas mobilis ZM4(ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中。通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员。经IPTG诱导大肠杆菌中pET21b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化。SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体。光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白。对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率。这些研究结果丰富了DyP-type过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础。

Dye-decolorizing peroxidase （DyP-type peroxidase） represents a group of heme-containing peroxidases able todecolour 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-21 b（＋）. 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.