High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization.

David Rodríguez-Escribano,Felipe De Salas,Susana Camarero,Isabel Pardo

doi:10.3390/ijms18081793

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.

Highlights

Conversion of waste products of the pulping processes, such as lignin, into valuable products is being considered as one of the main objectives to increase the economic viability of lignocellulosic biorefineries
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

Summary

Introduction

Conversion of waste products of the pulping processes, such as lignin, into valuable products is being considered as one of the main objectives to increase the economic viability of lignocellulosic biorefineries. Sulfite and kraft processes are the dominating pulping technologies to extract the cellulosic fibers from wood and annual plants. The sulfite process isolates cellulose by sulfonating the lignin with sulfourous acid and/or a sulfite salt, which solubilizes and extracts the lignin. The resulting technical lignins are denoted lignosulfonates (LS), which are water-soluble polyphenolic macromolecules with sulfonate groups on the aliphatic side chain (Figure 1). Due to their unique properties, LS have an established market as additives in concrete, but are used as road or animal feed binders or as dispersants in dyes, pesticides and other applications [1]. About 1 million tons of LS are produced annually, with Borregaard-LignoTech (Sarpsborg, Norway) and Tembec (Montréal, QC, Canada) the dominant producers [2]

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