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定点突变对tfudyp催化活性的影响
首发时间：2025-03-10
摘要：染料脱色过氧化物酶（dyps）作为一种血红素（heme）依赖性酶，在木质素降解、染料脱色等领域具有广泛的应用前景。异源重组表达是发酵法生产dyps的有效方式，但是重组蛋白与heme结合效率和酶活性低是存在的主要问题。本研究以源于褐色嗜热裂孢菌 (thermobifida fusca) 的tfudyp为对象，通过分子对接和氨基酸序列比对分析，对heme配体8?范围内的11个位点进行了定点突变，得到11个变体，并在大肠杆菌e.coli bl21（de3）中进行重组表达，分析了胞内heme水平、蛋白表达和tfudyp活性之间的关系。纯化后的酶显示，部分变体的酶活性较原酶显著提高，其中a349r和n347y的活性分别提高了45%和29%。本研究为dyps的工程化改造提供了新的策略和理论支持。
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the effect of site-directed mutations on the catalytic activity of tfudyp
abstract：dye-decolorizing peroxidases (dyps), as heme-dependent enzymes, have broad application prospects in fields such as lignin degradation and dye decolorization. heterologous recombinant expression is an effective method for the fermentation production of dyps; however, low efficiency of heme binding and enzyme activity in recombinant proteins remain major issues. in this study, tfudyp from thermobifida fusca was selected as the target. through molecular docking and amino acid sequence alignment analysis, 11 sites within an 8 ? range of the heme ligand were subjected to site-directed mutations, resulting in 11 variants. these variants were recombinantly expressed in escherichia coli bl21 (de3), and the relationship between intracellular heme levels, protein expression, and tfudyp activity was analyzed. the purified enzymes showed that the activity of some variants was significantly enhanced compared to the wild-type enzyme, with a349r and n347y variants exhibiting activity increases of 45% and 29%, respectively. this study provides new strategies and theoretical support for the engineering modification of dyps.
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