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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Environmental Science

Background:

• Laccase is an enzyme crucial for various biological processes and environmental applications.
• Accurate and sensitive methods for laccase activity assay are essential for research and practical applications.
• Existing methods may have limitations in sensitivity, stability, or applicability.

Purpose of the Study:

• To develop and validate a novel fluorescence-based method for quantifying laccase activity.
• To investigate the kinetic parameters and optimal conditions for the assay.
• To assess the method's applicability and reliability in real-world samples, such as soil.

Main Methods:

• Utilized the enzymatic transformation of Amplex Red to fluorescent resorufin, catalyzed by laccase in the presence of oxygen.
• Determined kinetic parameters (Vmax, Km) using the Lineweaver-Burk equation.
• Validated the method's linearity, detection limit, repeatability, recovery, and interference from common substances.

Main Results:

• Established a sensitive assay with a detection limit of 1.76 U/L and excellent linearity (r=0.9992) over a wide range (5.62-702 U/L).
• Demonstrated high repeatability (RSD=1.9%) and recovery (93.7%-100.0%), with minimal interference from various ions and compounds.
• Successfully applied the method to quantify laccase activity in 38 soil samples, revealing a significant correlation with total nitrogen content.

Conclusions:

• The developed Amplex Red-based fluorescence assay is a stable, reproducible, and selective method for laccase activity determination.
• The assay exhibits strong anti-interference capabilities and good selectivity, making it suitable for practical applications.
• This method holds significant promise for ecological investigations and soil environment research due to its effectiveness in soil sample analysis.