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2,6-Dimethoxyphenol-Based Assay for Quantitation of Polysaccharide Monooxygenase in Multienzyme Cocktails

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Abstract

A rapid method for quantitation of lytic polysaccharide monooxygenase (LPMO) in multienzyme cocktails produced by mutant strains of filamentous fungi is developed. In this method, 2,6-dimethoxyphenol and hydrogen peroxide are used as cosubstrates in a nonspecific LPMO-catalyzed reaction leading to the formation of a colored product. With this method, we determine the LPMO content in the multienzyme cocktails produced by Penicillium verruculosum recombinant strains that homologously express LPMO. The results agree closely with the findings obtained using a method based on the fine chromatographic fractionation of the cocktails followed by the qualitative and quantitative evaluation of their content by electrophoresis and mass-spectrometry and protein quantitation assays in fractions collected from chromatographic separation.

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Funding

The study was supported within the research program “Molecular Design, Structure–Function Analysis and Regulation of Enzyme Systems, Cellular Constructs, and Bionanomaterials: Fundamentals and Application in Technology, Medicine, and Environmental Protection” (state registration no. АААА-А16-116052010081-5).

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Authors and Affiliations

  1. Divisions of Chemical Enzymology, Department of Chemistry, Moscow State University, Moscow, Russia

    V. D. Telitsin, A. V. Gusakov & A. P. Sinitsyn

  2. Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, Moscow, Russia

    M. V. Semenova, D. O. Osipov & A. P. Sinitsyn

Authors
  1. V. D. Telitsin
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  2. M. V. Semenova
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  3. D. O. Osipov
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  4. A. V. Gusakov
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  5. A. P. Sinitsyn
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Corresponding author

Correspondence to M. V. Semenova.

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The authors declare that they have no conflict of interest.

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Translated by A. Kukharuk

Abbreviations: LPMO, lytic polysaccharide monooxygenase; EC, enzyme cocktail; 2,6-DMP, 2,6-dimethoxyphenol; CBH, cellobiohydrolase.

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Telitsin, V.D., Semenova, M.V., Osipov, D.O. et al. 2,6-Dimethoxyphenol-Based Assay for Quantitation of Polysaccharide Monooxygenase in Multienzyme Cocktails. Moscow Univ. Chem. Bull. 75, 96–100 (2020). https://doi.org/10.3103/S0027131420020157

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  • DOI: https://doi.org/10.3103/S0027131420020157

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