Analytical and Bioanalytical Chemistry

, Volume 409, Issue 20, pp 4801–4809 | Cite as

Automated chromatographic laccase-mediator-system activity assay

  • Nico Anders
  • Maximilian Schelden
  • Simon Roth
  • Antje C. Spiess
Research Paper


To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and—based on adlerol conversion—for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays.


HPAEC-PAD On-line measurement Laccase-mediator system Adlerol Ion chromatography 



This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Biomass,” which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities. Additionally, part of this work was done as part of the Bioeconomy Science Center which is financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13).

The authors thank Jakob Mottweiler (RWTH Aachen, Organic Chemistry, group of Prof. Bolm) for providing both adlerol and adlerone.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_423_MOESM1_ESM.pdf (501 kb)
ESM 1 (PDF 501 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Aachener Verfahrenstechnik-Enzyme Process TechnologyRWTH Aachen UniversityAachenGermany
  2. 2.Aachener Verfahrenstechnik-Biochemical EngineeringRWTH Aachen UniversityAachenGermany
  3. 3.Institute of Biochemical EngineeringTU BraunschweigBraunschweigGermany

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