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

Journal Article · · International Journal of Molecular Sciences (Online)
DOI:https://doi.org/10.3390/ijms18081793· OSTI ID:1378888
 [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)
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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.

Research Organization:
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Sponsoring Organization:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Grant/Contract Number:
AC36-08GO28308
OSTI ID:
1378888
Report Number(s):
NREL/JA-5100-69122; IJMCFK
Journal Information:
International Journal of Molecular Sciences (Online), Vol. 18, Issue 8; ISSN 1422-0067
Publisher:
MDPICopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 13 works
Citation information provided by
Web of Science

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Cited By (2)

A highly stable laccase obtained by swapping the second cupredoxin domain journal October 2018
Alternatives for Chemical and Biochemical Lignin Valorization: Hot Topics from a Bibliometric Analysis of the Research Published During the 2000–2016 Period journal July 2018

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09 BIOMASS FUELS
laccase
lignosulfonate
high-throughput screening
phenolic content
enzyme directed evolution