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

ABSTRACT

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 (V_max) and the substrate concentration required to obtain half of V_max (K_m). 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.

KEYWORDS:

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

Acknowledgments

We thank Dr. Michelle Wander for providing access to the microplate reader used in our work, Dr. Chongyang Li for his assistance with Michaelis–Menten modeling, and Lynn Connor, USDA Agricultural Research Service, for providing access to some of the laboratory equipment used in our work.

Disclosure statement

No potential conflict of interest was reported by the author(s).