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Automated chromatographic laccase-mediator-system activity assay
Nico Anders1, Maximilian Schelden2, Simon Roth3
1Aachener Verfahrenstechnik-Enzyme Process Technology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany. nico.anders@avt.rwth-aachen.de.
Analytical and Bioanalytical Chemistry
|June 3, 2017
Summary
A new automated method using HPAEC-PAD quantifies laccase-mediator system (LMS) activity by measuring adlerol consumption. This innovation enables efficient screening of LMS for lignin modification, overcoming limitations of traditional assays.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Enzymology
Background:
- Laccase-mediator systems (LMS) are crucial for lignin modification.
- Understanding the interactions between laccases, mediators, and substrates is essential for optimizing LMS efficiency.
- Existing assays often lack the specificity or automation required for comprehensive analysis.
Purpose of the Study:
- To develop and optimize an on-line measurement for studying LMS interactions.
- To quantify adlerol consumption and LMS activity using a novel automated method.
- To enable high-throughput screening of LMS for lignin modification applications.
Main Methods:
- Development of an on-line measurement system combining High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).
- Optimization of chromatographic run time and automated sample collection.
- Application of the method to measure Trametes versicolor laccase-induced oxidation of adlerol using 1-hydroxybenzotriazole (HBT) as a mediator.
Main Results:
- The developed HPAEC-PAD method successfully quantified adlerol consumption and LMS activity.
- Automated monitoring of LMS reaction progress over 48 hours minimized manual errors.
- LMS activity was determined to be 0.47 U/mg protein for a specific laccase/mediator ratio.
Conclusions:
- The automated HPAEC-PAD assay provides a robust tool for quantifying LMS activity on lignin-related substrates.
- This method facilitates rapid screening of enzyme-mediator-substrate combinations for efficient lignin modification.
- The assay overcomes limitations of colorimetric methods by directly measuring substrate conversion.
