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Open AccessCommunication
Int. J. Mol. Sci. 2017, 18(8), 1793; doi:10.3390/ijms18081793

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

1
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
2
National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
*
Author to whom correspondence should be addressed.
Received: 14 July 2017 / Revised: 9 August 2017 / Accepted: 11 August 2017 / Published: 18 August 2017
(This article belongs to the Special Issue The Lignin Challenge: Exploring Innovative Applications)
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Abstract

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin–Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates. View Full-Text
Keywords: laccase; lignosulfonate; high-throughput screening; phenolic content; enzyme directed evolution laccase; lignosulfonate; high-throughput screening; phenolic content; enzyme directed evolution
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Rodríguez-Escribano, D.; de Salas, F.; Pardo, I.; Camarero, S. High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization. Int. J. Mol. Sci. 2017, 18, 1793.

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