甲醛氧化途径关键酶重组蛋白的生化特性及固定化酶吸收甲醛效果研究

甲醛脱氢酶(formaldehyde dehydrogenase,ADH)与甲酸脱氢酶(formate dehydrogenase,FDH)是甲醛氧化途径的两个关键酶.恶臭假单胞菌(Pseudomonas putida)的PADH是一种不依赖谷胱甘肽可以把游离甲醛直接氧化为甲酸的脱氢酶,博伊丁假丝酵母菌(Candida boidinii)的FDH在有NAD+存在时可以把甲酸氧化为二氧化碳.以基因组DNA为模板用PCR方法,从P.putida中扩增出PADH基因的编码区(padh),从C.boidinii中扩增出FDH的编码区(fdh),然后亚克隆到pET-28a(+)中分别构建这两个基因的原核表达载体pET-28a-padh和pET-28a-fdh,转化大肠杆菌,利用IPTG诱导重组蛋白PADH和FDH的表达.通过优化条件使重组蛋白的表达量占菌体总蛋白的70%以上,通过亲和层析法纯化出可溶性PADH和FDH重组蛋白.对重组蛋白的生化特性分析结果表明:PADH在最适反应温度50℃的活性为1.95 U/mg;FDH在最适反应温度40℃的活性为0.376 U/mg.所表达的重组蛋白与之前报道过的相比,具有更好的热稳定性和更广的温度适应范围.将PADH、FDH两个重组蛋白及辅因子NAD+固定到聚丙烯酰胺载体基质上,对固定化酶甲醛吸收效果的初步分析结果显示固定化酶对空气中的甲醛有一定的吸收效果,说明这两种酶被固定后具有开发成治理甲醛污染环保产品的潜力.

Biochemical Characteristics of Recombinant Enzyme Proteins for Formaldehyde Oxidation and the Formaldehyde Absorption Effects of the Immobilized Enzymes

GSH-dependent formaldehyde dehydrogenase(ADH) and formate dehydrogenase(FDH) are two key enzymes related to formaldehyde oxidation.PADH from Pseudomonas putida is a formaldehyde dehydro-genase which can oxidize free formaldehyde to formate independent of GSH.FDH from Candida boidinii can oxidize formate to carbon dioxide in the presence of NAD+.Using the genomic DNA of P.putida and C.boidinii as the template,respectively,the coding regions of the PADH gene(padh) and FDH gene(fdh) were ampli-fied by PCR.The PCR products were then subcloned into the prokaryotic expression vectors pET28a,re-spectively,to construct pET28a(+)-padh and pET28a(+)-fdh expression vectors.The expression vectors were introduced into Escherichia coli.The PADH and FDH recombinant proteins were expressed under the in-duction of IPTG.By optimizing the expression conditions,the expressed proteins accounted for 70% of the total proteins in E.coli cells.The soluble recombinant proteins of PADH and FDH were purified to homogeneity by His-tag affinity chromatography.The analysis of the biochemical characteristics of the purified PADH and FDH recombinant proteins showed that the highest specific activity(1.95 U/mg) of PADH protein was obtained at the optimal temperature of 50 ℃.The highest specific activity(0.376 U/mg) of FDH protein was achieved at 40 ℃.The recombinant proteins had a higher thermo-tolerance and more extensive reaction temperature than those reported previously.The ADH,FDH proteins and NAD+ were immobilized in a polyacry-lamide gel.A preliminary investigation showed that the immobilized enzymes had considerable formaldehyde absorption capacity,suggesting that the immobilized PADH and FDH proteins have a potential to develop as the products for remediation of indoor formaldehyde contamination.