染料脱色过氧化物酶(dye-decolorizing peroxidase,DyP)属于以血红素为辅基的新型过氧化物酶超家族。由于能够降解蒽醌类染料,DyP成为当今绿色经济的研究热点,而染料的有效降解与酶活性密切相关,因此寻求提高酶活性的方法是DyP后续生产应用的关键所在。克隆来自红球菌(Rhodococcus jostii)的染料脱色过氧化物酶基因RhDypB,实现在大肠杆菌Escherichia coli BL21(DE3)中的高效表达,并对提高酶活性的方法进行了研究。利用一步克隆技术构建重组菌株pET24b-DypB /BL21, 利用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导使蛋白表达,镍柱亲和层析纯化蛋白,比较了外源添加血红素合成前体5-氨基乙酰丙酸(5-aminolevulinic acid ,5-ALA)和共表达大肠杆菌血红素合成途径中的谷氨酰-tRNA还原酶基因(hemA)对酶活性的影响。SDS-PAGE显示在40 kDa有明显的目的条带(纯度达到90%),蛋白质量浓度可达100 mg/L。添加5-ALA以及共表达hemA分别使血红素浓度增加至8.9和2.4 μmol/ L,相应的酶活性提高了102%和93%。RhDypB的可溶性表达与血红素含量的提升,为以血红素作为辅因子的过氧化物酶的相关研究提供依据。
Dye-decolorizing peroxidase (DyP) with heme as a prosthetic group is regarded as a novel type of peroxidase subfamily. DyP has become a hot research topic of green economy because of its ability to degrade anthraquinone dyes. Since the effective degradation of dyes is closely related to enzyme activity, it is important to seek the ways of improving enzyme activity for the subsequent production and application of DyP. DypB gene from Rhodococcus jostii (RhDypB) was cloned to realize high-efficiency expression in Escherichia coli BL21(DE3), and to study methods for improving the enzyme activity. Recombinant strain BL21(DE3)/pET24b-DypB was constructed by one-step cloning technology. The recombinant protein was expressed by IPTG induction and purified by affinity chromatography using a nickel column. The effect on the activity of RhDypB by exogenous addition of heme synthesis precursor 5-aminolevulinic acid (5-ALA) and co-expression of the glutamyl-tRNA reductase gene (hemA) in the heme synthesis pathway in E. coli was compared. SDS-PAGE showed that there was an obvious target band at 40 kDa (purity-90%), and the concentration of protein was 100 mg/L. The addition of 5-ALA and co-expression of hemA increased the concentration of heme to 8.9 and 2.4 μmol/L, and the corresponding activity of enzyme was increased by 102% and 93%, respectively. The soluble expression of RhDypB and the increase in content of heme provide a basis for related research on peroxidase with heme as a cofactor.
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