A novel fluorimetric method for laccase activities measurement using Amplex Red as substrate

Talanta. 2017 Jan 1:162:143-150. doi: 10.1016/j.talanta.2016.10.006. Epub 2016 Oct 3.


In this paper, a novel fluorescence-based method for laccase assay was presented. The method was based on the transformation of Amplex Red into a highly fluorescent and colored resorufin catalyzed by laccase in the presence of O2. The catalysis and transformation mechanism were investigated in detail. The kinetic parameters of the Amplex Red catalysis by laccase were determined using the Lineweaver-Burk equation. Vmax and Km were estimated to be 15.63μmolmin-1 and 76.88μmolL-1, respectively. Under optimal conditions, a good linear correlation was found between fluorescence intensity and laccase activities within 5.62-702UL-1 (r=0.9992), with a detection limit of 1.76UL-1 (S/N=3). A series of repeatability measurements (351UL-1 laccase) gave reproducible results with a relative standard deviation (RSD) of 1.9% (n=11). The recoveries ranged from 93.7% to 100.0% after standard additions. Common existing species such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, Fe2+, l-lysine, glycine, glucose, phenol, humic acid, lignin peroxidase, manganese peroxidase alkaline phosphatase, cellulose, glucose oxidase, urease, catalase, invertase, and horseradish peroxidase did not significantly exhibit interference. The test solution (i.e., Amplex Red stock solution) could stabilize at least three months via storage in dark at 4±0.1°C. These results confirmed that the laccase-Amplex Red system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that this method can has great potential in practical applications for the assay of laccase activity. The proposed method was further successfully used to detect laccase activities in 38 soil samples. We noticed that the laccase activity significantly correlated with total nitrogen content (r=0.559; p<0.01) of soil, indicating laccase activity assay holds great promise as an index of soil analysis. These findings indicate that this presented method has great perspective in ecological investigation and fundamental research of soil environment.

Keywords: Amplex Red; Fluorescence; Laccase activity; Resorufin; Soil enzyme.

MeSH terms

  • Biocatalysis
  • Enzyme Assays / methods*
  • Fluorescence
  • Fluorometry / methods*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Laccase / metabolism*
  • Models, Chemical
  • Molecular Structure
  • Oxazines / chemistry
  • Oxazines / metabolism*
  • Reproducibility of Results*
  • Soil / chemistry
  • Substrate Specificity


  • Oxazines
  • Soil
  • Amplex Red
  • resorufin
  • Laccase