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Microplate fluorimetric assay of soil leucine aminopeptidase activity: alkalization is not needed before fluorescence reading

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Abstract

The fluorescence responses of 7-amino-4-methylcoumarin (AMC) and L-leucine-7-amido-4-methylcoumarin (L-leucine-AMC) (the fluorescent dye and substrate used in the soil leucine aminopeptidase assay, respectively) to alkalization by NaOH and tris(hydroxymethyl)aminomethane (THAM) buffer were analyzed. The leucine aminopeptidase activities were also measured without and with the alkalization procedure using five soils with contrasting properties. The pH optimum of leucine aminopeptidase activity was 8 ± 1 for the five studied soils. In this pH range, the alkalization procedure cannot increase the fluorescence of AMC to improve the assay sensitivity. Excess alkalization (final medium pH of 12 or higher) led to decreasing fluorescence of AMC with time and non-enzymatic hydrolysis of L-leucine-AMC, which caused irreproducible values of the calculated enzyme activities. Moreover, a medium pH of ~ 11 cannot terminate the enzymatic reaction, and thus it seems impractical to stop the enzyme reaction by alkalization without causing the unstable behavior of AMC and L-leucine-AMC. We recommend performing the soil leucine aminopeptidase assay at the optimal pH without alkalization and reading the fluorescence immediately after incubation. This approach may help to simplify and standardize the leucine aminopeptidase assay.

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Funding

This research was supported by the National Natural Science Foundation of China (41701288 and 41630751), Youth Innovation Promotion Association, Chinese Academy of Sciences (2017424), Science & Technology Department of Sichuan Province (2018JY0163), and China Scholarship Council (201708515106).

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Authors and Affiliations

  1. College of Ecology and Environment, Chengdu University of Technology, Chengdu, 610059, China

    Jipeng Wang & Shunyu Huang

  2. Institute of Geography and Geoecology, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany

    Jipeng Wang & Wolfgang Wilcke

  3. Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, #9, Block 4, Renminnanlu Road, Chengdu, China

    Qingqing He, Haijian Bing, Xifeng Zhang, Xin Tian, Jun Zhou & Yanhong Wu

  4. University of Chinese Academy of Sciences, Beijing, 100049, China

    Qingqing He & Xin Tian

  5. Institute of Inorganic Chemistry, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany

    Xiao Chen

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  1. Jipeng Wang
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  2. Shunyu Huang
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  3. Qingqing He
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  4. Haijian Bing
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  5. Xiao Chen
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  7. Xin Tian
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  8. Jun Zhou
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  9. Wolfgang Wilcke
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  10. Yanhong Wu
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Correspondence to Haijian Bing or Yanhong Wu.

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Wang, J., Huang, S., He, Q. et al. Microplate fluorimetric assay of soil leucine aminopeptidase activity: alkalization is not needed before fluorescence reading. Biol Fertil Soils 56, 281–285 (2020). https://doi.org/10.1007/s00374-019-01419-x

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  • DOI: https://doi.org/10.1007/s00374-019-01419-x

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