The initial steps of complex organic matter degradation in soil comprise several oxidative processes catalysed by phenol oxidases and peroxidases. Commonly, their activity is determined by colorimetric approaches based on a range of substrates, making comparison between studies difficult. To improve this shortcoming, we conducted a calibration procedure for assessing phenol oxidase and peroxidase activities in soils by a sensitive fluorometric assay. This assay is based on the colourless and non-fluorescent Amplex Red® reagent as a substrate, which forms a fluorescent product, resorufin, upon oxidation. Commercially available resorufin was used as a standard for calibration. We applied Michaelis–Menten kinetics to determine functional traits of phenol oxidases and peroxidases in a case study on Haplic Chernozem from different land-use and climate treatments of a field experiment. The kinetic parameters, limit of detection and limit of quantification demonstrated a high sensitivity of the fluorometric assay to differences caused by these treatments. For the total oxidative enzymes, the catalytic efficiency (K_cat, calculated as V_max/K_m) was 30% higher in the cropland compared with the pasture plot. However, compared with soil samples collected from ambient climatic conditions, K_cat of total oxidoreductases was 60% lower in cropland exposed to elevated air temperature and drought. The phenol oxidase activity was substantially higher than peroxidase activity in cropland land use. However, higher K_cat of peroxidase than phenol oxidase resulted in 60% faster substrate turnover under both climatic conditions. In our case study, the Amplex Red-based assay enabled rapid and high-throughput analyses of phenol oxidase and peroxidase activity in soil. The sensitivity of this assay for soil enzymology applications needs further testing for a larger range of soils and environmental conditions.

中文翻译：

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一种改进的基于 Amplex Red 的酚氧化酶和过氧化物酶活性荧光测定法：Haplic Chernozem 的案例研究

土壤中复杂有机质降解的初始步骤包括由酚氧化酶和过氧化物酶催化的几个氧化过程。通常，它们的活性是通过基于一系列底物的比色方法确定的，这使得研究之间的比较变得困难。为了改善这一缺点，我们进行了校准程序，通过灵敏的荧光测定法评估土壤中的酚氧化酶和过氧化物酶活性。该测定基于无色且无荧光的 Amplex Red® 试剂作为底物，在氧化后形成荧光产物试卤灵。市售的试卤灵被用作校准标准。我们应用 Michaelis-Menten 动力学来确定酚氧化酶和过氧化物酶的功能性状，以对来自田间试验的不同土地利用和气候处理的 Haplic Chernozem 进行案例研究。动力学参数、检测限和定量限表明荧光测定法对这些处理引起的差异具有高灵敏度。对于总氧化酶，催化效率（与牧场地块相比，农田的K _cat（计算为V _max /K _{m ）高 30%。}然而，与从环境气候条件下采集的土壤样品相比，暴露于高温和干旱的农田中，总氧化还原酶的K _{cat降低了 60%。}酚氧化酶活性显着高于农田利用中的过氧化物酶活性。然而，更高的K_猫在两种气候条件下，过氧化物酶比苯酚氧化酶的底物周转速度快 60%。在我们的案例研究中，基于 Amplex Red 的分析能够快速、高通量地分析土壤中的酚氧化酶和过氧化物酶活性。该测定法对土壤酶学应用的敏感性需要对更大范围的土壤和环境条件进行进一步测试。