Buffers are commonly employed in soil enzyme assays to maintain a constant pH during the assay incubation, but soils are already buffered and buffer can alter apparent V_max and K_m. To test for potential artifacts of buffer on soil enzyme activities, we selected 32 soils to furnish a broad range of physiochemical characteristics and assayed soil β-glucosidase (BG) and phosphomonoesterase (PME) activities at varied substrate concentrations either in water or in modified universal buffer (MUB). The pH of assays was different (up to 1.6 units) from the measured soil pH (1:2, m/v in water), but MUB did not maintain pH better than water. Compared to water, MUB generally suppressed activities (by∼31%), apparent V_max (by∼32%) and K_m (by∼52%) of PME, but yielded similar activities (by∼4% difference) and apparent V_max (by∼9% difference) for BG. Soils with higher pH tended to have larger degree of suppressed PME actvities in MUB compared to assays in water. Based on the best practice of using a substrate concentration that is 5×K_m for substrate saturation of the enzyme, the median substrate requirement to assay PME in these 32 soils was ≈ 50 mM g^-1 in water and 25 mM g^-1 in MUB. Regardless of matrix type, the commonly employed PME substrate concentration of 10 mM g^-1 (e.g., Tabatabai, 1994) is insufficient for accurate activity assays. In contrast, for BG assays the commonly used pNP-linked substrate concentration of 10 mM g^-1 appears appropriate for most soils with a median substrate requirement of ∼4 mM g^-1 in water and ∼6 mM g^-1 in MUB. Our results support previous claims that buffers are unnecessary for assaying soil enzyme activities and can alter apparent kinetic parameters (K_m, V_max). Potential soil- and enzyme-specific substrate requirements should be determined a priori to ensure accurate measurements of enzyme activities in soils.

中文翻译：

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土壤酶活性的显色测定需要缓冲液吗？在基于对硝基苯基的磷酸单酯酶和 β-葡萄糖苷酶测定中，改良通用缓冲液的缺点多于优点


缓冲液通常用于土壤酶测定，以在测定孵育期间保持恒定的 pH 值，但土壤已经缓冲，缓冲液会改变表观 V_max 和 K_m。为了测试缓冲对土壤酶活性的潜在伪影，我们选择了 32 种土壤来提供广泛的理化特性，并测定了不同基质浓度下的土壤 β-葡萄糖苷酶 （BG） 和磷酸单酯酶 （PME） 活性在水中或改性通用缓冲液 （MUB）。测定的 pH 值与测得的土壤 pH 值（1：2，水中 m/v）不同（高达 1.6 个单位），但 MUB 的 pH 值并不优于水。与水相比，MUB 通常抑制 PME 的活性 （∼31%）、表观 V_max （∼32%） 和 K_m （∼52%），但对 BG 产生相似的活性 （差异∼4%） 和表观 V_max （差异∼9%）。与水中的测定相比，pH 值较高的土壤往往在 MUB 中具有更大程度的 PME 活性抑制。基于使用 5×K_m 的底物浓度进行酶底物饱和度的最佳实践，在这 32 种土壤中测定 PME 的底物要求的中位数为 ≈ 50 mM ^g-1 的水溶液和 25 mM ^g-1 的 MUB 溶液。无论基质类型如何，通常采用的 10 mM ^g-1 的 PME 底物浓度（例如，Tabatabai，1994）都不足以进行准确的活性测定。相比之下，对于 BG 测定，常用的 pNP 连接底物浓度 10 mM ^g-1 似乎适用于大多数土壤，水中底物需求中位数为 ∼4 mM ^g-1，MUB 中中位基质需求量为 ∼6 mM ^g-1。 我们的结果支持了以前的说法，即缓冲液对于测定土壤酶活性是不必要的，并且可以改变表观动力学参数 （K_m， V_max）。应先验确定潜在的土壤特异性基质要求和酶特异性基质，以确保准确测量土壤中的酶活性。