土壤水解酶活性测定方法的研究进展

Research advances on the measurement methods for soil hydrolytic enzymes activities

  • 摘要: 土壤水解酶直接参与有机物质的矿质化过程, 对维持生态系统碳和养分的循环过程起重要作用。但不同研究者和实验室测定过程的差异, 常给土壤酶测定方法的选定带来困难。针对这一问题, 本文围绕土壤酶活性测定过程中样本贮存方式、酶底物选择以及培养条件等, 对近二十多年来的研究成果进行了评述。土壤酶来源广泛, 存在形式复杂, 直接提取土壤酶还有很大的难度。目前土壤酶活性的测定主要是通过一定量底物在酶催化反应过程中的生成物或剩余物量来实现。基于对硝基酚衍生物为底物的分光比色法, 因其成本低廉而被广泛使用。针对国际上未能建立统一的测定方法而导致研究数据之间难以进行比较的现状, 作者认为现阶段土壤水解酶测定过程中应注意以下几点: 1)尽量采用新鲜土样或短时间低温冷藏的土样; 2)酶底物种类的选择应与国际接轨, 采用饱和底物浓度使其反应速度接近最大值; 3)缓冲液的pH值可与土壤pH值相近; 4)4 h内培养的土壤酶, 可忽略微生物抑制剂的使用。建议我国在近期内: 1)应丰富土壤水解酶种类的研究; 2)注重灵敏度高、需样量小以及培养时间短的荧光分析技术的应用; 3)在强化典型土壤酶动力学特征研究基础上, 尽快规范土壤酶活性的测定方法。

     

    Abstract: Soil hydrolytic enzymes play an important role for maintaining carbon and nutrient cycle of ecology system as they are directly involved in the soil organic matter mineralization. The methodological differences among researchers and laboratories influenced the determining on proper measurement methods. The review summarized the research results on measuring method of soil enzymes activities involved soil storage methods, selection of substrate and incubation conditions in recent circa 20 years. To extract and purify the enzyme from soil have been left unsuccessful for the complexity of broad origin and existing states, so soil enzyme activity assays were commonly based on the quantitative evaluation of the product released or of the substrate consumed. Colorimetric methods based on artificial p-nitrophenol substrate had been used widely for the low cost. Based on the absence of the uniform international standard, and hard contrast among investigator results, the corresponding measures should be token to run measurement of soil hydrolytic enzymes activities recently. We recommended some manipulation: 1) conduct assay with fresh soil whenever possible, or soil stored shortly at 4 ℃ or -20 ℃; 2) opt substrate based on international standards and use saturated substrate concentrations to ensure Vmax being measured; 3) run the assay at a pH of buffer similar to that of soil; 4) omit inhibitors of microbial proliferation within 4 h incubation. Some research orientation currently were also suggested: 1) to enrich the research categories of soil hydrolytic enzymes; 2) develop the measurement methods of fluorimetric determination with high sensitivity, small quantity of soil sample and short incubation time; 3) establish primary standard methods by studying the catalytic kinetic of hydrolytic enzymes in typical soil types.

     

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