Author ORCID Identifier

https://orcid.org/0000-0002-2595-9256

Date Available

2-7-2021

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture; Engineering

Department/School/Program

Biosystems and Agricultural Engineering

First Advisor

Dr. Jian Shi

Second Advisor

Dr. Sue Nokes

Abstract

Lignin is one of the most abundant polymers found in nature, making up 15 – 40% of the weight of terrestrial biomass. Due to the structural and monomeric heterogeneity of lignin, it is recalcitrant thermochemical and biological valorization methods. Converting lignin to value-added products via sustainable and cost-effective pathways will reduce waste and add value to future cellulosic biorefineries. Biological methods for lignin valorization (e.g. lignin degrading enzymes or microbes) is limited by low lignin solubility in biocompatible solvents, resulting in low product yield. Recent reports on biocatalysts for lignin valorization have focused on the lignolytic multicopper oxidase laccase, which operates in a wide range of conditions, does not require additional solvents such as hydrogen peroxide, and can oxidize nonphenolic linkages when used alongside a small molecule mediator.

To improve the yield of products from biological lignin valorization, it is necessary to use a biocompatible solvent that is capable of solubilizing lignin in mild conditions alongside a biocatalyst capable of operating in high concentrations of the solvent. Ionic liquids (ILs) are molten salts that are liquid at temperatures < 100oC. The properties of ILs can be tuned by selecting the appropriate cation and anion. Many ILs have been developed for biomass pretreatment, for example 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), cholinium lysinate ([Ch][Lys]), and diethylamine hydrogen sulfate ([DEA][HSO4]). We screened the biocompatibility of these 3 ILs with the activity of a mesophilic fungal laccase from Trametes versicolor (TvL). [C2C1Im][OAc] and [Ch][Lys] severely inhibited TvL activity in low concentrations (TvL activity was observed in 10% [DEA][HSO4]. Treating Kraft lignin with the laccase-mediator-IL system resulted in low product yields and minimal changes to the lignin structure, but nonetheless served as a promising proof of concept upon which future studies can improve.

The extreme conditions (pH, temperature, salinity) in which thermophilic enzymes operate makes them desirable biocatalysts for use in many biotechnological applications, including lignin valorization. Thermophilic cellulases show minimal loss of activity in 20% [C2C1Im][OAc]. The hyperthermophilic bacterium Thermus thermophilus is reported to produce the most thermophilic laccase (TtL) to date, demonstrating optimal substrate oxidation at 92oC. Despite the thermophilicity of TtL, severe activity loss was observed in via a mixed competitive and noncompetitive inhibition mechanism. A rational design strategy, guided by previous enzyme engineering studies for improving laccase activity in ILs, produced 8 single amino acid mutants, none of which improved the activity of TtL in [C2C1Im][OAc] compared to the wild type. These results highlight the challenges faced when designing a biological lignin valorization system with a laccase and an aqueous ILs.

Recent reports show the IL 1-ethyl-3-methylimidazolium ethylsulfate ([C2C1Im][EtSO4]) is biocompatible with the activity of a laccase from the thermophilic fungus Myceliophthora thermophila (MtL) up to 25% IL concentration. When we screened the biocompatibility of [C2C1Im][OAc] with MtL, severe inhibition of MtL activity was observed in > 15% [C2C1Im][OAc]. The surface charges of MtL were modified to increase activity in [C2C1Im][OAc]. Increasing the acid or amine residues on the enzyme surface did not affect the activity or stability of MtL in [C2C1Im][OAc]. Docking simulations showed that the modifications do not significantly alter the interactions between the IL and the enzyme surface. These results suggest that IL inhibition of laccases is not due to IL docking on the enzyme surface, but rather destabilization of the coppers.

We applied a mixed technique approach using in vitro and in silico methods to understand the interactions between Toxicodendron vernicifluum laccase (RvL) and [Ch][Lys]. Biocompatibility, kinetics, and docking simulations support that [Ch][Lys] inhibits RvL through a mixed competitive and noncompetitive inhibition mechanism. Molecular dynamics (MD) simulations showed that the IL affects the stability of the substrate in the active site.

Collectively, these studies characterize the interactions between several mesophilic and thermophilic laccases and ILs used for biomass pretreatment with a variety of in vitro and in silico techniques. The information provided by these studies can be used to inform the design of future biological lignin valorization systems using laccase and a biocompatible, aqueous IL.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.346

Funding Information

National Science Foundation under Cooperative Agreement No. 1355438 and 1632854 (2016 - 2020) and the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch-Multistate project under accession numbers 1018315/1003563 (2018 - 2023). This material is based upon research supported by the Chateaubriand Fellowship of the Office for Science & Technology of the Embassy of France in the United States (2019).

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