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Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea with broad substrate specificity against aromatic alcohols

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Abstract

Objectives

The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2).

Results

The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol.

Conclusions

Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

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Acknowledgements

This work was financially supported by JSPS KAKENHI (Grant No. 15H04526).

Supporting information

Supplementary Fig. 1—Multiple alignments of known AAO sequences.

Supplementary Fig. 2—SDS-PAGE analysis of deglycosylated and purified rCcAAO.

Supplementary Fig. 3—The pH dependence of rCcAAO.

Supplementary Fig. 4—Three-dimensional structure model of substrate access channel of PeAAO and CcAAO.

Supplementary Fig. 5—Amino acid sequence logo using known AAO and putative AAO sequences corresponding to positions 411–421 of mature CcAAO generated with WebLogo3.

Author information

Author notes

  1. Kiwamu Umezawa

    Present address: Department of Applied Biological Chemistry, Kindai University, Nara, 631-8505, Japan

Authors and Affiliations

  1. Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan

    Yoshiaki Tamaru, Kiwamu Umezawa & Makoto Yoshida

Authors
  1. Yoshiaki Tamaru
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  2. Kiwamu Umezawa
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  3. Makoto Yoshida
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Corresponding author

Correspondence to Makoto Yoshida.

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Tamaru, Y., Umezawa, K. & Yoshida, M. Characterization of an aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea with broad substrate specificity against aromatic alcohols. Biotechnol Lett 40, 1077–1086 (2018). https://doi.org/10.1007/s10529-018-2534-3

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  • DOI: https://doi.org/10.1007/s10529-018-2534-3

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