摘要
The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.
木糖醇脱氢酶(XDH)是能够分解戊糖的酵母和真菌中参与木糖利用的关键酶。除了可以生产木酮糖以外,XDH还可以用于木糖醇浓度检测的生物传感器的开发,具有重要的研究意义。在本研究中,构建了来源于嗜热真菌埃默森篮状菌Talaromyces emersonii的木糖醇脱氢酶(TeXDH)的大肠杆菌表达质粒pF58⁃TeXDH。TeXDH在16℃下以可溶性形式在大肠杆菌BL21(DE3)中表达。通过使用Ni⁃NTA亲和柱进行纯化,获得了纯度较高的重组TeXDH。分子排阻层析和SDS⁃PAGE电泳分析表明,纯化的重组TeXDH以天然三聚体的形式存在,分子量约为116 kD,由3个相同的亚基组成,每个亚基的分子量约39 kD。TeXDH严格依赖NAD^(+)作为辅酶,而不依赖NADP^(+)。TeXDH的最适温度和pH值分别为40℃和10.0。经过EDTA处理,TeXDH的酶活性降低到起始酶活的43.26%。二价金属离子Mg^(2+)或Ca^(2+)能够恢复TeXDH的酶活性,分别达到起始酶活的10332%和110.69%,是TeXDH酶的最佳二价金属离子辅因子。而二价金属离子Mn^(2+)、Ni^(2+)、Cu^(2+)、Zn^(2+)、Co^(2+)和Cd^(2+)能够显著抑制TeXDH的活性。ICP⁃MS和分子对接分析表明,1 mol/L的TeXDH结合了2 mol/L的Zn^(2+)离子和1 mol/L的Mg^(2+)离子。另外,TeXDH对木糖醇具有较好的底物特异性,为未来木糖醇生物传感器的发展奠定了基础。
出处
《中国生物化学与分子生物学报》
北大核心
2025年第7期1007-1018,共12页
Chinese Journal of Biochemistry and Molecular Biology
基金
湖南省教育厅基金优秀青年项目(No.22B0482)
湖南科技大学博士启动基金(No.E51992 and E51993)资助。
