Analytical and Bioanalytical Chemistry

, Volume 409, Issue 20, pp 4801–4809

Automated chromatographic laccase-mediator-system activity assay

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

Abstract

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.

Keywords

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

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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