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High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization
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. O...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578181/ https://www.ncbi.nlm.nih.gov/pubmed/28820431 http://dx.doi.org/10.3390/ijms18081793 |
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author | Rodríguez-Escribano, David de Salas, Felipe Pardo, Isabel Camarero, Susana |
author_facet | Rodríguez-Escribano, David de Salas, Felipe Pardo, Isabel Camarero, Susana |
author_sort | Rodríguez-Escribano, David |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5578181 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-55781812017-09-05 High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization Rodríguez-Escribano, David de Salas, Felipe Pardo, Isabel Camarero, Susana Int J Mol Sci Communication 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. MDPI 2017-08-18 /pmc/articles/PMC5578181/ /pubmed/28820431 http://dx.doi.org/10.3390/ijms18081793 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Communication Rodríguez-Escribano, David de Salas, Felipe Pardo, Isabel Camarero, Susana High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title | High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title_full | High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title_fullStr | High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title_full_unstemmed | High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title_short | High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization |
title_sort | high-throughput screening assay for laccase engineering toward lignosulfonate valorization |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578181/ https://www.ncbi.nlm.nih.gov/pubmed/28820431 http://dx.doi.org/10.3390/ijms18081793 |
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