Publication Details

Category Text Publication
Reference Category Journals
DOI 10.1111/ejss.13225
Licence creative commons licence
Title (Primary) An improved amplex red-based fluorometric assay of phenol oxidases and peroxidases activity: A case study on Haplic Chernozem
Author Khosrozadeh, S.; Dorodnikov, M.; Reitz, T.; Blagodatskaya, E.
Source Titel European Journal of Soil Science
Year 2022
Department BOOEK
Volume 73
Issue 2
Page From e13225
Language englisch
Topic T5 Future Landscapes
Keywords Amplex Red; Fluorometric assay; Michaelis-Menten kinetics; Peroxidase; Phenol oxidase activity
Abstract The initial steps of complex organic matter degradation in soil comprise several oxidative processes catalyzed by phenol oxidases and peroxidases. Commonly, their activity is determined by colorimetric approaches based on a range of substrates, making comparison between studies difficult. To improve this shortcoming, we conducted a calibration procedure for assessing phenol oxidase and peroxidase activities in soils by a sensitive fluorometric assay. This assay is based on the colorless and non-fluorescent Amplex Red as a substrate, which forms a fluorescent product, resorufin, upon oxidation. Commercially available resorufin was used as a standard for calibration. We applied Michaelis-Menten kinetics to determine functional traits of phenol oxidases and peroxidases in a case study on Haplic Chernozem from different land-use and climate treatments of a field experiment. The kinetic parameters, limit of detection and limit of quantification demonstrated a high sensitivity of the fluorometric assay to differences caused by these treatments. For the total oxidative enzymes, the catalytic efficiency (Kcat, calculated as Vmax/Km) was 30% higher in the cropland compared to the pasture plot. However, compared to soil samples collected from ambient climatic conditions, Kcat of total oxidoreductases was 60% lower in cropland exposed to elevated air temperature and drought. The phenol oxidase activity was substantially higher than peroxidase activity in cropland land-use. However, higher Kcat of peroxidase than phenol oxidase resulted in 60% faster substrate turnover under both climatic conditions. In our case study, the Amplex Red-based assay enabled rapid and high-throughput analyses of phenol oxidase and peroxidase activity in soil. The sensitivity of this assay for soil enzymology applications needs further testing for a larger range of soils and environmental conditions.
Persistent UFZ Identifier
Khosrozadeh, S., Dorodnikov, M., Reitz, T., Blagodatskaya, E. (2022):
An improved amplex red-based fluorometric assay of phenol oxidases and peroxidases activity: A case study on Haplic Chernozem
Eur. J. Soil Sci. 73 (2), e13225 10.1111/ejss.13225