Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution

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Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution

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Publication Article, peer reviewed scientific
Title Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution
Author Mateljak, Ivan ; Monza, Emanuele ; Fatima Lucas, Maria ; Guallar, Victor ; Aleksejeva, Olga ; Ludwig, Roland ; Leech, Donal ; Shleev, Sergey ; Alcalde, Miguel
Date 2019
English abstract
Fungal high-redox-potential laccases (HRPLs) are multi-copper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (E-T1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased E-T1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic highredox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the E-T1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the E-T1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices.
DOI https://doi.org/10.1021/acscatal.9b00531 (link to publisher's fulltext.)
Link https://doi.org/10.1021/acscatal.9b00531 .Icon
Publisher American Chemical Society
Host/Issue Acs Catalysis;5
Volume 9
ISSN 2155-5435
Language eng (iso)
Subject laccase
redox potential
redox mediator
stability
directed evolution
computer-aided design
Sciences
Research Subject Categories::NATURAL SCIENCES
Handle http://hdl.handle.net/2043/29518 Permalink to this page
Link to publication in DiVA Find this research publication in DiVA (n/a for student publ.)
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