skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

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). As a result, themore » method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

Authors:
 [1];  [1];  [2]; ORCiD logo [1]
  1. Centro de Investigaciones Biologicas, Madrid (Spain)
  2. Centro de Investigaciones Biologicas, Madrid (Spain); National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1378888
Report Number(s):
NREL/JA-5100-69122
Journal ID: ISSN 1422-0067; IJMCFK
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
International Journal of Molecular Sciences (Online)
Additional Journal Information:
Journal Volume: 18; Journal Issue: 8; Journal ID: ISSN 1422-0067
Publisher:
MDPI
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; laccase; lignosulfonate; high-throughput screening; phenolic content; enzyme directed evolution

Citation Formats

Rodriguez-Escribano, David, de Salas, Felipe, Pardo, Isabel, and Camarero, Susana. High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization. United States: N. p., 2017. Web. doi:10.3390/ijms18081793.
Rodriguez-Escribano, David, de Salas, Felipe, Pardo, Isabel, & Camarero, Susana. High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization. United States. https://doi.org/10.3390/ijms18081793
Rodriguez-Escribano, David, de Salas, Felipe, Pardo, Isabel, and Camarero, Susana. Fri . "High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization". United States. https://doi.org/10.3390/ijms18081793. https://www.osti.gov/servlets/purl/1378888.
@article{osti_1378888,
title = {High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization},
author = {Rodriguez-Escribano, David and de Salas, Felipe and Pardo, Isabel and Camarero, Susana},
abstractNote = {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). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.},
doi = {10.3390/ijms18081793},
url = {https://www.osti.gov/biblio/1378888}, journal = {International Journal of Molecular Sciences (Online)},
issn = {1422-0067},
number = 8,
volume = 18,
place = {United States},
year = {2017},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Degradation mechanisms of phenolic β-1 lignin substructure model compounds by laccase of Coriolus versicolor
journal, April 1988


Exploring the Oxidation of Lignin-Derived Phenols by a Library of Laccase Mutants
journal, September 2015


Laccase/mediated oxidation of a lignin model for improved delignification procedures
journal, November 2003


The pros and cons of lignin valorisation in an integrated biorefinery
journal, January 2014


Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis
journal, April 2010


Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY
journal, August 2015


Structure and Properties of Sodium Lignosulfonate with Different Molecular Weight Used as Dye Dispersant
journal, November 2014


Characterisation of enzymatically oxidised lignosulfonates and their application on lignocellulosic fabrics
journal, August 2009


Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus
journal, December 2011


Horseradish Peroxidase Modification of Sulfomethylated Wheat Straw Alkali Lignin To Improve Its Dispersion Performance
journal, February 2015


Enzymatic polymerization of sodium lignosulfonates: effect of catalysts, initial molecular weight, and mediators
journal, March 2013


Activation of Magnesium Lignosulfonate and Kraft Lignin: Influence on the Properties of Phenolic Resin-Based Composites for Potential Applications in Abrasive Materials
journal, June 2017


Computer-Aided Laccase Engineering: Toward Biological Oxidation of Arylamines
journal, July 2016


Polymerization of Various Lignins via Immobilized Myceliophthora thermophila Laccase (MtL)
journal, August 2016


Development of chimeric laccases by directed evolution
journal, July 2012


Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility
journal, July 2010


Influence of Oxygen and Mediators on Laccase-Catalyzed Polymerization of Lignosulfonate
journal, June 2016


    Works referencing / citing this record:

    A highly stable laccase obtained by swapping the second cupredoxin domain
    journal, October 2018