Research Article

Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives

Number: 35 June 12, 2026
  • Saadet Alpdagtas *

Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives

Abstract

Harnessing fungal lignocellulose-degrading enzymes offers sustainable pathways for converting biomass into value-added chemicals. Glyoxal oxidases (GLOX), copper metalloenzymes involved in lignin degradation, can catalyze the oxidation of several furan derivatives into 2,5-furandicarboxylic acid (FDCA), a bioplastic precursor. However, natural GLOX enzymes display limited catalytic activity, necessitating optimization via protein engineering. This study applied directed evolution to the Trametes versicolor glyoxal oxidase gene (tvglox), generating mutants via error-prone PCR and evaluating activity using a high-throughput, colorimetric, agar-based HRP-ABTS assay. In addition, both episomal and genome-integrated expression systems in Pichia pastoris were assessed. While no activity was detected from episomal expression variants, 2.5% of the genome-integrated mutants, along with wild-type controls, exhibited activity on methylglyoxal. Notably, three mutants displayed enhanced activity towards 5-hydroxy-2-furaldehyde carboxylic acid (FFCA), suggesting improved biocatalytic potential for FDCA synthesis. This study represents the first comparison of episomal and genome-integrated GLOX expression in P. pastoris, and highlights a rapid, cost-effective screening platform for furan-active GLOX variants.

Keywords

Supporting Institution

Scientific and Technological Research Council of Turkey (TÜBİTAK) through the 2219 International Research Fellowship Programme

Thanks

This study was carried out by SA under the supervision of Prof. Vlada Urlacher and Dr. Katja Koschorreck at Heinrich Heine University Düsseldorf (HHU). SA gratefully acknowledges the invaluable guidance and support provided by the supervisors throughout the study.

References

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Details

Primary Language

English

Subjects

Industrial Molecular Engineering of Nucleic Acids and Proteins

Journal Section

Research Article

Authors

Saadet Alpdagtas * This is me
0000-0002-8322-2658
Türkiye

Publication Date

June 12, 2026

Submission Date

June 11, 2025

Acceptance Date

April 7, 2026

Published in Issue

Year 2026 Number: 35

APA
Alpdagtas, S. (2026). Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives. Biotech Studies, 35, 1969768-1969768. https://doi.org/10.38042/biotechstudies.1969768
AMA
1.Alpdagtas S. Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives. Biotech Studies. 2026;(35):1969768-1969768. doi:10.38042/biotechstudies.1969768
Chicago
Alpdagtas, Saadet. 2026. “Directed Evolution of Glyoxal Oxidase and High-Throughput Activity Screening for Biocatalytic Valorization of Furan Derivatives”. Biotech Studies, nos. 35: 1969768-68. https://doi.org/10.38042/biotechstudies.1969768.
EndNote
Alpdagtas S (June 1, 2026) Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives. Biotech Studies 35 1969768–1969768.
IEEE
[1]S. Alpdagtas, “Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives”, Biotech Studies, no. 35, pp. 1969768–1969768, June 2026, doi: 10.38042/biotechstudies.1969768.
ISNAD
Alpdagtas, Saadet. “Directed Evolution of Glyoxal Oxidase and High-Throughput Activity Screening for Biocatalytic Valorization of Furan Derivatives”. Biotech Studies. 35 (June 1, 2026): 1969768-1969768. https://doi.org/10.38042/biotechstudies.1969768.
JAMA
1.Alpdagtas S. Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives. Biotech Studies. 2026;:1969768–1969768.
MLA
Alpdagtas, Saadet. “Directed Evolution of Glyoxal Oxidase and High-Throughput Activity Screening for Biocatalytic Valorization of Furan Derivatives”. Biotech Studies, no. 35, June 2026, pp. 1969768-, doi:10.38042/biotechstudies.1969768.
Vancouver
1.Saadet Alpdagtas. Directed evolution of glyoxal oxidase and high-throughput activity screening for biocatalytic valorization of furan derivatives. Biotech Studies. 2026 Jun. 1;(35):1969768-. doi:10.38042/biotechstudies.1969768


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