Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: Effect of assay conditions and soil type
Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: Effect of assay conditions and soil type
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DOI：
10.1016/j.soilbio.2013.08.022
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发表时间：
2013-12-01
影响因子：
9.7
通讯作者：
German, Donovan P.
中科院分区：
文献类型：
--
作者：
Bach, Christopher E.;Warnock, Daniel D.;German, Donovan P.
微生物酚氧化酶和过氧化物酶介导土壤中的生物地球化学过程，包括微生物获取碳和氮、木质素降解、碳矿化和封存以及溶解有机碳输出。由于反应的非特异性、自由基性质以及酶、测定底物和土壤基质之间复杂的相互作用，测量土壤中的氧化酶活性比测定水解酶活性更成问题。我们比较了常用于测定土壤中酚氧化酶和过氧化物酶的三种底物：邻苯三酚（PYGL，1,2,3-三羟基苯）、L-DOPA（L-3,4-二羟基苯丙氨酸）和 ABTS（2,2'-连氮基-双（3-乙基苯并噻唑啉-6-磺酸）。 测量了三种土壤中 pH 梯度从 3.0 到 10.0 的底物氧化情况，以确定每种底物的最佳 pH 值。此外，我们还比较了使用三种基质的 17 种土壤的活性。一般来说，底物活性遵循PYGL>L-DOPA>ABTS的趋势，并且与底物氧化还原电位成反比。 PYGL 和 ABTS 在 pH > 5 时不是合适的底物，并且 ABTS 经常氧化 在测定中添加过氧化物后下降。与土壤类型和测定 pH 值相关的底物之间的绝对和相对氧化速率差异很大。我们还测试了高压灭菌或燃烧的土壤是否可以用作非生物因素（例如土壤矿物学）对氧化活性影响的阴性对照。然而，高压灭菌和燃烧都没有产生可靠的阴性对照，因为底物氧化仍然发生；在 在某些情况下，这些处理提高了底物的氧化速率。对于大规模研究，我们建议研究人员使用所有三种底物来评估土壤氧化电位。对于重点研究，我们建议在选择单一选项之前评估底物，并且我们建议在土壤 pH 值和参考 pH 值（例如 pH 5.0）下进行测定，以确定测定 pH 值对氧化酶活性的影响。这些建议应有助于提高各个研究中氧化酶潜在活性的可比性。 (C) 2013 Elsevier Ltd. 保留所有权利。
Microbial phenol oxidases and peroxidases mediate biogeochemical processes in soils, including microbial acquisition of carbon and nitrogen, lignin degradation, carbon mineralization and sequestration, and dissolved organic carbon export. Measuring oxidative enzyme activities in soils is more problematic than assaying hydrolytic enzyme activities because of the non-specific, free radical nature of the reactions and complex interactions between enzymes, assay substrates, and the soil matrix. We compared three substrates commonly used to assay phenol oxidase and peroxidase in soil: pyrogallol (PYGL, 1,2,3-trihydroxybenzene), L-DOPA (L-3,4-dihydroxyphenylalanine), and ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). We measured substrate oxidation in three soils across a pH gradient from 3.0 to 10.0 to determine the pH optimum for each substrate. In addition, we compared activities across 17 soils using the three substrates. In general, activities on the substrates followed the trend PYGL > L-DOPA > ABTS and were inversely related to substrate redox potential. PYGL and ABTS were not suitable substrates at pH > 5, and ABTS oxidation often declined with addition of peroxide to the assay. Absolute and relative oxidation rates varied widely among substrates in relation to soil type and assay pH. We also tested whether autoclaved or combusted soils could be used as negative controls for the influence of abiotic factors (e.g., soil mineralogy) on oxidative activity. However, neither autoclaving nor combustion produced reliable negative controls because substrate oxidation still occurred; in some cases, these treatments enhanced substrate oxidation rates. For broad scale studies, we recommend that investigators use all three substrates to assess soil oxidation potentials. For focused studies, we recommend evaluating substrates before choosing a single option, and we recommend assays at both the soil pH and a reference pH (e.g., pH 5.0) to determine the effect of assay pH on oxidase activity. These recommendations should contribute to greater comparability of oxidase potential activities across studies. (C) 2013 Elsevier Ltd. All rights reserved.