Peer-Reviewed Journal Details
Mandatory Fields
Mateljak, I;Monza, E;Lucas, MF;Guallar, V;Aleksejeva, O;Ludwig, R;Leech, D;Shleev, S;Alcalde, M
2019
May
ACS Catalysis
Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution
Published
Optional Fields
PH ACTIVITY PROFILE TYPE-1 COPPER SITE ELECTRON-TRANSFER COTA-LACCASE OXIDATION BLUE PERTURBATIONS EXPLORATION EXPRESSION CUPREDOXIN
9
4561
4572
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.
2155-5435
10.1021/acscatal.9b00531
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