A Comprehensive Method for Casein-based Assay of Soil Protease Activity

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Proteolytic enzymes play an important role in soil N cycling and are assayed by measuring protease activity. The casein substrate-based method of Ladd and Butler is commonly used to assay soil protease activities, but usage is constrained by several issues: methodological details in the original protocol are incomplete; controls for non-enzymatic activity are missing; and modifications of the assay have led to inconsistencies in how activities are measured. To clarify soil protease assays and improve their accuracy, we describe a comprehensive protocol in which field-moist soil is incubated with casein for 2 h at 50°C, and protease activity is determined by colorimetric measurement of the tyrosine produced after correction for soil- and substrate-only controls. The importance of such correction was investigated by a comparative study involving a single soil-substrate control as well as the dual-control approach. Soil-specific optimization of assay conditions was achieved by kinetic characterization of the maximum rate of activity (Vmax) and the substrate concentration required to obtain half of Vmax (Km). Soil protease activities were higher with a single control due to differential extraction of humic-phenolic substances, and for this reason, the dual-control approach is recommended. The procedure detailed herein stands to enhance the utility of casein-based assays of soil protease activities and improve the value of these assays for comparative applications.

Original languageEnglish (US)
Pages (from-to)507-520
Number of pages14
JournalCommunications in Soil Science and Plant Analysis
Issue number4
StatePublished - 2022


  • Extracellular enzymes
  • casein hydrolysis
  • kinetic characterization
  • nitrogen mineralization
  • protein degradation
  • tyrosine

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Soil Science


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