skip to main content

DOE PAGESDOE PAGES

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

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., 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. doi:10.3390/ijms18081793.
Rodriguez-Escribano, David, de Salas, Felipe, Pardo, Isabel, and Camarero, Susana. 2017. "High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization". United States. doi: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},
journal = {International Journal of Molecular Sciences (Online)},
number = 8,
volume = 18,
place = {United States},
year = {2017},
month = {8}
}