Automated chromatographic laccase-mediator-system activity assay
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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.
KeywordsHPAEC-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.
- 1.Anders N, Abb M, Sorkau E, Kubinec R, Lorenz W. Analysis and occurrence of polycyclic aromatic hydrocarbons on household dust. Fresenius Environ Bull. 2012;21(2):372–9.Google Scholar
- 11.Han M-J, Choi H-T, Song H-G. Purification and characterization of laccase from white rot fungus Trametes versicolor. J Microbiol. 2005;43(6):555–60.Google Scholar
- 17.Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl Environ Microbiol. 1995;61:1876–80.Google Scholar
- 23.Stoob K, Singer HP, Goetz CW, Ruff M, Mueller SR. Fully automated on-line solid phase extraction coupled directly to liquid chromatography-tandem mass spectrometry quantification of sulfonamide antibiotics, neutral and acidic pesticides at low concentrations in surface waters. J Chrom A. 2005;1097:138–47.CrossRefGoogle Scholar
- 24.Minussi RC, Miranda MA, Silva JA, Ferreira CV, Aoyama H, Marangoni S, et al. Purification, characterization and application of laccase from Trametes versicolor for colour and phenolic removal of olive mill wastewater in the presence of 1-hydroxybezotriazole. Afr J Biotechnol. 2007;6(10):1248–54.Google Scholar