Characterization and Enzyme Engineering of Laccases Towards Lignin Valorization in Aqueous Ionic Liquids

University of Kentucky UKnowledge

Theses and Dissertations--Biosystems and Agricultural Engineering Biosystems and Agricultural Engineering

2020

CHARACTERIZATION AND ENZYME ENGINEERING OF LACCASES TOWARDS LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS

Joseph Stevens University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0002-2595-9256 Digital Object Identifier: https://doi.org/10.13023/etd.2020.346

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Recommended Citation Stevens, Joseph, "CHARACTERIZATION AND ENZYME ENGINEERING OF LACCASES TOWARDS LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS" (2020). Theses and Dissertations--Biosystems and Agricultural Engineering. 72. https://uknowledge.uky.edu/bae_etds/72

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The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Joseph Stevens, Student

Dr. Jian Shi, Major Professor

Dr. Donald Colliver, Director of Graduate Studies

CHARACTERIZATION AND ENZYME ENGINEERING OF LACCASES TOWARDS LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS

______

DISSERTATION ______A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Colleges of Agriculture and Engineering at the University of Kentucky

By Joseph Stevens Lexington, Kentucky Co- Directors: Dr. Jian Shi, Assistant Professor of Biosystems and Agricultural Engineering and Dr. Sue Nokes, Professor of Biosystems and Agricultural Engineering Lexington, Kentucky 2020

ABSTRACT OF DISSERTATION

CHARACTERIZATION AND ENZYME ENGINEERING OF LACCASES TOWARDS LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS

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 (<5%), whereas minimal loss of 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 <3% [C2C1Im][OAc] 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.

KEYWORDS: Lignin, biocatalysis, biorefinery, ionic liquids, enzyme engineering

Joseph Craig Stevens (Name of Student)

July 27, 2020 Date

CHARACTERIZATION AND ENZYME ENGINEERING OF LACCASES TOWARDS LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS

By Joseph Craig Stevens

Dr. Jian Shi Co- Director of Dissertation

Dr. Sue Nokes Co- Director of Dissertation

Dr. Donald Colliver Director of Graduate Studies

July 27, 2020 Date

ACKNOWLEDGMENTS

I would like to first and foremost thank my adviser of the last 4 years, Dr. Jian Shi.

Without his guidance I would not be the researcher I am today. Without his patience I would not understand the virtue of patience in research and understanding that good science takes time. Without his faith in me I would not have the confidence to pursue my future in research. I owe him a great debt of gratitude for helping shape my personal and professional future.

I would also like to thank Dr. David Rodgers for his instruction in my first biochemistry courses that influenced my research interests during my Ph.D. studies. You have shown me a great deal of patience, kindness, and insight while helping me with my research and serving on my Ph.D. committee. I would also like to thank my other committee members, Dr. Sue Nokes and Dr. Michael Montross, who provided me with valuable insight and helped me build confidence in myself during my Ph.D. I would also like to thank my committee for having faith in me to pursue a direct-to-Ph.D. pathway and to spend my 9 months in France wisely.

For my time in Toulouse I owe a great deal to my collaborators and friends at the

Toulouse Biotechnology Institute (TBI) in France and the Chateaubriand Fellowship from the Office for Science & Technology of the Embassy of France in the United States. By agreeing to be my research host for the fellowship, Dr. Claire Dumon has played a crucial role in shaping my confidence and abilities as a person and a researcher. The patience of

Dr. Isabelle André and Dr. Jérémy Esque knows no bounds and I thank them for spending many hours working with me on the bioinformatics portions of my later chapters. I also owe a deep debt of gratitude to the kindness and English-speaking abilities of my co-

iii workers at TBI, Dr. Cédric Montanier, Dr. Régis Fauré, Dr. Jiao Zhao, Dr. Tarun Narwani, and Thomas Enjalbert. I would also like to thank my host family in Toulouse, Dominique,

Nathalie, Lucy, and Jenny Bergonier for helping me navigate Toulouse upon my arrival and for sharing many Sunday lunches with me. To all of my friends in Toulouse: you made my 9 months there a once-in-a-lifetime experience and I promise that I will learn more French before I return.

I would like to extend a special thank you to Dr. Lalitendu Das, a former post-doc in Dr. Shi’s lab and a future co-worker (again), for lecturing and challenging me that first morning at the conference in San Francisco. I owe you a great deal for the experiences

I’ve had the last 4 years, not least of which are going to Spalding’s for an early morning doughnut, and you have played a critical role in shaping my future. I look forward to working with you again.

I would like to thank the friends I have made in Lexington and Toulouse these last

4 years: Ryan, Woomer, Wenqi shishou, Lalit, Jameson, Jérémy, and Tarun. You all mean more to me than I can express in words because I only know how to say how amazing everything is, how much I love the simplicity of Ramirez tacos and a burger from Five

Guys, and how that really was the best pizza we’ve ever made.

I would like to thank the rest of the Shi lab group, Enshi Liu, Luke Dodge, Truc

Nguyen, Siquan Xu, Makua Vin-Nnajiofor, Can Liu, Dr. Binling Ai and Yuxuan Zhang along with Dr. Udeep Chawla from Dr. Rodgers’ lab for providing me with a great deal of support and great memories the past several years. I look forward to hearing about all of your future research endeavors.

To all of the faculty, staff, and fellow graduate students in the BAE department, thank you for your kindness and for allowing a diehard Louisville Cardinal fan to work alongside you all. Maybe Cardinals and Wildcats aren’t so different after all.

I would like to thank my middle school science teacher, Mr. Fred Whittaker, for instilling in me a passion for science and, above all else, compassion for my fellow humans.

I would like to thank my parents, Barry and Mary Beth, and my brother, Porter, for pretending to know what a “lignin” is and for showing me unconditional love and support for the last 26 years.

A special, albeit strange, thank you to Malcolm James McCormick for helping me to appreciate the things I have yet to experience. One day I’ll be brave enough to listen to

Circles, but Swimming is still teaching me what it’s all about.

And lastly, I would like to thank Robert “Papa” Rowan for keeping me busy on

Sundays in Louisville. I sure hope San Francisco is as amazing as you made it out to be.

TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... III

LIST OF TABLES ...... XI

LIST OF FIGURES ...... XII

LIST OF ADDITIONAL FIGURES ...... XV

CHAPTER 1. BIOCATALYSIS IN AQUEOUS IONIC LIQUIDS FOR LIGNIN

VALORIZATION: OPPORTUNITIES AND RECENT DEVELOPMENTS1 ...... 1

1.1 INTRODUCTION ...... 1

1.2 LIGNIN...... 3

1.2.1 Lignin structure and characteristics ...... 3

1.2.2 Lignin depolymerization methods ...... 7

1.3 LIGNIN DEGRADING ENZYMES ...... 9

1.3.1 Peroxidases ...... 9

1.3.2 Laccases ...... 11

1.3.3 Other LDEs ...... 14

1.4 IONIC LIQUIDS ...... 18

1.4.1 IL properties...... 18

1.4.2 Biomass pretreatment with ILs ...... 21

1.4.3 Effect of IL treatment on lignin characteristics ...... 25

1.4.4 Rationale for pairing LDE and IL for lignin depolymerization ...... 26

1.5 ENZYME-IL INTERACTIONS ...... 27

1.5.1 Enzyme activity and stability in ILs ...... 27

1.5.2 Effect of IL on enzyme ...... 30

1.5.3 Methods for improving enzyme stability in ILs ...... 32

1.6 PROTEIN ENGINEERING TO IMPROVE IL COMPATIBILITY ...... 35

1.6.1 Structure-function relationship of proteins ...... 35

1.6.2 Protein engineering for improved function...... 38

1.6.3 Protein crystallization in ILs ...... 41

1.7 PRODUCT SELECTIVITY ...... 41

1.7.1 Controlling product formation via biocatalyst selectivity ...... 42

1.7.2 Controlling product formation via IL selectivity ...... 42

1.7.3 Preventing lignin repolymerization and improving yield ...... 43

1.8 CONCLUSIONS AND PERSPECTIVES ...... 45

1.9 ORGANIZATION OF CHAPTERS...... 46

CHAPTER 2. UNDERSTANDING LACCASE-IONIC LIQUID INTERACTIONS TOWARDS

BIOCATALYTIC LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS1 ...... 49

2.1 ABSTRACT ...... 49

2.2 INTRODUCTION ...... 50

2.3 METHODS AND MATERIALS ...... 53

2.3.1 Materials ...... 53

2.3.2 Biocompatibility screening ...... 54

2.3.3 Inhibition kinetics...... 54

2.3.4 Molecular docking simulations ...... 55

2.3.5 Lignin depolymerization in aqueous IL ...... 55

2.3.6 Lignin characterization ...... 57

2.4 RESULTS AND DISCUSSION ...... 60

2.4.1 Biocompatibility screening ...... 60

2.4.2 Inhibition mechanisms ...... 62

2.4.3 Docking simulations...... 63

2.4.4 GGE dimer transformation ...... 64

2.4.5 Depolymerization of alkaline lignin...... 68

2.5 CONCLUSIONS ...... 76

2.6 AUTHOR CONTRIBUTIONS ...... 76

vii

2.7 ACKNOWLEDGEMENTS ...... 77

2.8 SUPPLEMENTAL INFORMATION ...... 78

CHAPTER 3. CHARACTERIZATION AND ENZYME ENGINEERING OF A

HYPERTHERMOPHILIC LACCASE TOWARD IMPROVING ITS ACTIVITY IN IONIC

LIQUID1 83

3.1 ABSTRACT ...... 83

3.2 INTRODUCTION ...... 84

3.3 MATERIALS AND METHODS ...... 88

3.3.1 Materials ...... 88

3.3.2 Expression of T. thermophilus Laccase ...... 88

3.3.3 Purification and copper-induced laccase folding ...... 89

3.3.4 Biocompatibility screening ...... 89

3.3.5 Inhibition kinetics...... 90

3.3.6 Docking Simulations ...... 90

3.3.7 Site-directed mutagenesis of TtL ...... 91

3.4 RESULTS ...... 92

3.4.1 WT TtL Biocompatibility Screening ...... 92

3.4.2 Enzyme Kinetics ...... 93

3.4.3 Docking simulations...... 94

3.4.4 Rational Design of TtL ...... 96

3.5 DISCUSSION ...... 98

3.6 AUTHOR CONTRIBUTIONS ...... 99

3.7 ACKNOWLEDGEMENTS ...... 99

3.8 SUPPLEMENTAL INFORMATION ...... 100

CHAPTER 4. MODIFYING SURFACE CHARGES OF A THERMOPHILIC LACCASE

TOWARDS IMPROVING ACTIVITY AND STABILITY IN AQUEOUS IONIC LIQUID ...... 104

4.1 ABSTRACT ...... 104

viii

4.2 INTRODUCTION ...... 105

4.3 METHODS AND MATERIALS ...... 110

4.3.1 Materials ...... 110

4.3.2 Surface Charge Modifications ...... 110

4.3.3 Biocompatibility Screening ...... 111

4.3.4 MtL Thermostability...... 112

4.3.5 Docking Simulations ...... 112

4.4 RESULTS AND DISCUSSION ...... 113

4.4.1 Biocompatibility Screening ...... 113

4.4.2 Stability [C2C1Im][OAc] ...... 115

4.4.3 Docking Simulations ...... 117

4.5 CONCLUSIONS AND FUTURE WORK ...... 119

4.6 AUTHOR CONTRIBUTIONS ...... 123

4.7 ACKNOWLEDGEMENTS ...... 123

CHAPTER 5. A MIXED TECHNIQUE APPROACH TOWARDS UNDERSTANDING IONIC

LIQUID INHIBITION OF A PLANT LACCASE ...... 125

5.1 ABSTRACT ...... 125

5.2 INTRODUCTION ...... 126

5.3 METHODS AND MATERIALS ...... 129

5.3.1 Materials ...... 129

5.3.2 Plant laccase purification ...... 129

5.3.3 Biocompatibility screening ...... 130

5.3.4 Enzyme Kinetics ...... 130

5.3.5 Plant laccase modelling and copper parameterization ...... 131

5.3.6 Docking Simulations ...... 132

5.4 RESULTS AND DISCUSSION ...... 132

5.4.1 Biocompatibility of AILs with laccase activity...... 132

5.4.2 Inhibition Mechanisms ...... 134

5.4.3 Copper parameterization and stabilization during simulations ...... 135

5.4.4 IL Docking ...... 136

5.4.5 Steered Molecular Dynamics of DMP in [Ch][Lys]...... 137

5.5 AUTHOR CONTRIBUTIONS ...... 138

5.6 ACKNOWLEDGEMENTS ...... 139

5.7 SUPPLEMENTAL INFORMATION ...... 140

CHAPTER 6. CONCLUSIONS AND FUTURE WORK ...... 142

6.1 CONCLUSIONS ...... 142

6.2 FUTURE WORK ...... 144

BIBLIOGRAPHY ...... 145

VITA ...... 167

LIST OF TABLES

Table 1-1. Applications of lignolytic enzymes ...... 17

Table 1-2. Studies of enzyme activity and stability in ILs ...... 28

Table 2-1. Kinetic constants of ABTS oxidtion by T. versicolor laccase in AILs ...... 62

Table 2-2. Molecular weight distribution of GGE before (0 min), during (30 min), and after (120 min) treatment with TvL, ABTS, and ILs1...... 66

Table 2-3. Converstion and yield of alkaline lignin degradation products ...... 69

Table 2-4. Processed lignin molecular weight distribution and polydispersity of lignin streams1 ...... 71

Table 3-1. Kinetic constants of the WT TtL in different concentrations of [C2C1Im][OAc]. Values shown are mean ± standard error (SE) of 3 experiments...... 94

Table 3-2. Activity of WT and mutants relative to activity in buffer in different concentrations of [C2C1Im][OAc]. Values shown are mean ± standard deviation (SD) of 3 experiments1...... 97

Table 4-1. Summary of mutations to used mimic surface charge modifications in PyMol ...... 113

Table 4-2. Previous studies using various methods to improve enzyme activity in aqueous ILs ...... 123

Table 5-1. Kinetic constants of RvL in different concentrations of [Ch][Lys]. Values shown are mean ± standard error of 3 experiments ...... 134

LIST OF FIGURES

Figure 1-1. Representative lignin structure consisting syringyl and guaiacyl units (adapted from 42)...... 4

Figure 1-2. Formation of inter-unit linkages during polymerization of coniferyl alcohol monomers (adapted from 41)...... 5

Figure 1-3. a) Lignin peroxidase from Phanerochaete chrysosporium (PDBID: 1B82) 72, b) Manganese peroxidase from Phanerochaete chrysosporium (PDBID: 1MNP) 73 and c) Versatile peroxidase from Pleurotus eryngii (PDBID: 2BOQ) 74. Figures downloaded from the Research Collaboratory for Structural Bioinformatics’ Protein Data Bank (RCSB PDB)75 ...... 10

Figure 1-4. Laccase from Trametes versicolor (PDBID: 1GYC) 86. Figure downloaded from the Research Collaboratory for Structural Bioinformatics’ Protein Data Bank (RCSB PDB) 75...... 12

Figure 1-5. Lignolytic pathways of Sphingobium sp. strain SYK-6 121...... 15

Figure 1-6. a) [C2C1Im][OAc] has served as the benchmark IL for biomass pretreatment for several years. Recently, bioderived ILs like b) choline lysinate ([Ch][Lys]) and c) diethylamine hydrogen sulfate ([DEA][HSO4]) have attracted interest due to their low cost...... 21

Figure 1-7. Butylimidazolium ILs. These ILs have all been effectively utilized in biomass pretreatment strategies with different effects on downstream processing of biomass (adapted from 22)...... 23

Figure 1-8. Effect of surface charge modification on IL interaction with enzymes (Nordwald, 2015)...... 35

Figure 1-9. Project summary...... 46

Figure 2-1. Activity of T. versicolor laccase in AIL solutions. H = high laccase concentration (3.0 μg mL-1), M = medium laccase concentration (1.2 μg mL-1), L = low laccase concentration (0.6 μg mL-1). Values indicate the ratio of TvL activity in ILs relative to TvL activity in buffer (0% IL)...... 61

Figure 2-2. Results of simulations showing the docking of b) [C2C1Im][OAc], c) [Ch][Lys], and d) [DEA][HSO4] near the Type I copper of T. versicolor laccase. The red arrow in a) points to the Type I copper. The ILs are represented by the multi-colored stick models in panels b-d and the His residues that coordinate the coppers are colored blue...... 63

Figure 2-3. HPLC chromatograms of GGE incubated with laccase and different AILs a) without ABTS present during the reaction. b) ABTS present during the reaction. GGE elutes at ~23 min and reaction products elute in the 2-5 min range...... 65

xii

Figure 2-4. Molecular weight distributions of products after GGE reaction with TvL, ABTS, and a) buffer without IL, b) 2% [C2C1Im][OAc], c) 1% [Ch][Lys], d) 10% [DEA][HSO4] ...... 67

Figure 2-5. GC-MS chromatograms of alkaline lignin breakdown products after treatment with laccase, AILs, and ABTS. Major products identified were 1) guaiacol, 2) vanillin, 3) acetovanillone, 4) homovanillic acid, 5) syringaldehyde, and 6) acetosyringone...... 70

Figure 2-6. 13C-1H (HSQC) spectra of aliphatic (top) and aromatic (bottom) regions of a) raw alkaline lignin (KL), b) alkaline lignin treated with laccase and ABTS in buffer (KLA), and c) alkaline lignin treated with laccase and ABTS in 2% [C2C1Im][OAc] (EKLA). Figure S6 in the supplementary material contains all of the spectra from the study. The structures of side-chain linkages and lignin compositional units, shown in the middle of the figure, were coded with colors corresponding to the cross peaks in the spectra aliphatic and aromatic regions, respectively. Linkage percentages were calculated via volume integration of the Cα-H of the β-O-4', β-5', and β-β' linkages and treated as a relative ratio of the three. Volume integrations are intended to be only semi-quantitative due to problems inherent to HSQC experiments including a wide array of potential 1JC-H values and rates of T2 relaxation 260...... 73

Figure 3-1. Activity of the WT TtL in different concentrations of [C2C1Im][OAc] (A). Michaelis-Menten curves of TtL in different concentrations of [C2C1Im][OAc] (B). Values shown are mean with error bars representing the SD of 3 experiments...... 92

Figure 3-2. Top docking locations from AutoDock of [C2C1Im][OAc] to the surface of [E] (A) and [E][S] (B). The IL is shown as magenta spheres, the coppers are shown as brown spheres, and ABTS is shown as red sticks...... 95

Figure 4-1. Clustal Omega sequence alignment of laccases from a bacterium (T. thermophilus – 2XU9), a plant (T. vernicifluum - Plant), and fungi (T. versicolor – 1GYC, M. thermophila – 6F5K). Conserved HXH motifs and T1-coordinating methionine/phenylalanine/leucine are highlighted with thick blue bars...... 108

Figure 4-2. Heatmap showing the activity of MtL and the charge variants in [C2C1Im][OAc] relative to MtL activity in buffer...... 114

Figure 4-3. Heatmaps showing the residual activity of MtL charge variants after incubation in 50 mM citrate/100 mM phosphate buffer and 2.5 or 5% [C2C1Im][OAc] at pH 4.5 and 40oC...... 116

Figure 4-4. Major docking locations of [C2C1Im][OAc] to the surface of unmodified (a), succinylated/acetylated (b), and cationized/neutralized MtL (c). [C2C1Im]+ is shown as magenta spheres, [OAc]- is shown as red spheres, and coppers are shown as brown spheres ...... 118

Figure 5-1. Effect of aqueous [Ch][Lys] on the activity of RvL. Values shown are the mean ± SD of 3 experiments ...... 133

xiii

Figure 5-2. Distances between (top) T1 copper, (middle) T2 copper, and (bottom) T3 coppers and coordinating residues during 100 ns simulations...... 136

Figure 5-3. Major docking locations of [Ch][Lys] to the surface of [E] (panel a, green) and [E][S] (panel b, cyan). [Ch]+ is shown as red spheres, [Lys]+ is shown as magenta spheres, DMP is shown as yellow spheres, and coppers are shown as brown spheres...... 137

Figure 5-4. Free energy profile of DMP in 0 (blue line), 2 (green line), and 4% (red line) [Ch][Lys]. The measurement of the distance between the T1 copper of DMP hydroxyl group is also shown for reference...... 138

xiv

LIST OF ADDITIONAL FIGURES

Table S 3-1. List of primer names and sequences for making the mutants in this study. Mutated nucleotides are underlined...... 100

Figure S 2-1. Hanes-Woolf curves of T. versicolor laccase in a) [C2C1Im][OAc], b) [Ch][Lys], and c) [DEA][HSO4] ...... 78

Figure S 2-2. Structure of proposed a) coupled GGE dimers, b) three coupled GGE dimers, and c) addition of a G monomer unit to the S2b structure formed during GGE polymerization reaction with T. versicolor laccase, ABTS, and AILs...... 79

Figure S 2-3. a) FTIR spectra of alkaline lignin before and after treatment with laccase, AILs, and ABTS (The red arrow is indicating the corresponding S-lignin peak); b) Thermal profiles of alkaline lignin before and after treatment with laccase, AILs, and ABTS...... 80

Figure S 2-4. Molecular weight distribution of a) solid-phase and b) liqui-phase alkaline lignin after treatment with laccase, AILs, and ABTS ...... 81

Figure S 2-5. 13C-1H (HSQC) spectra of alkyl (top) and aromatic (bottom) regions of a) raw alkaline lignin (KL), b) alkaline lignin treated with laccase and ABTS, and alkaline lignin treated with laccase and ABTS in aqueous ionic liquids: c) [Ch][Lys], d) [DEA][HSO4], and e) [C2C1Im][OAc]. *The structures of side-chain linkages and lignin compositional units were coded with colors corresponding to the cross peaks in the spectra alkyl and aromatic regions, respectively...... 82

Figure S 3-1. Crystal structures of TtL (PDBID: 2XU9, left) and TvL (PDBID: 1GYC, right) showing domain 1 (green), domain 2 (orange), domain 3 (magenta), and the L1 loop (cyan) connecting domains 2 and 3 (A). Sequence alignment of TtL and B. subtilis CotA, homologous Glutamate residues are highlighted (B)...... 101

Figure S 3-2. Aromatic and anionic residues around E170 in TtL (A) and E188 in B. subtilis CotA (B). Glutamates targeted for mutations are shown in cyan while all other residues are shown in green. All residues shown are within 5Å of the respective Glutamate. ... 102

Figure S 3-3. Codon optimized sequence of TtL (NCBI Accession Number: I7AL37) . 103

Figure S 5-1. Michaelis-Menten curves of RvL in aqueous [Ch][Lys]. Data were plotted and models were fit in Sigma-Plot. Values plotted are mean ± SD of 3 experiments. .. 140

Figure S 5-2. Final charges and geometry of coppers and coordinating residues ...... 141

CHAPTER 1. BIOCATALYSIS IN AQUEOUS IONIC LIQUIDS FOR LIGNIN VALORIZATION: OPPORTUNITIES AND RECENT DEVELOPMENTS1

1.1 Introduction

Lignin is synthesized biologically by plants and is one of the most abundant biopolymers on the planet. Besides serving as one of the primary building blocks for plant cell walls, lignin plays important roles such as facilitating water transport and protecting the plant from pathogenic attack. As a result of human’s practice in utilizing plant materials

(i.e., paper and pulping industry), lignin is extracted from the cellulose portion and commonly treated as a low-value/waste stream. Due to the recent surge in cellulosic biofuel production, the volume of lignin from cellulosic biorefineries is predicted to reach

60 million tons annually by 2022, which will add on top of the lignin produced by the paper industry 1. Currently, lignin is primarily burned in boilers to generate heat and steam that power upstream processes 2, 3. Converting lignin to value-added chemicals will add revenue to the biorefineries and reduce emitted CO2 when burned 3, 4. Despite its great potential, lignin valorization is still challenging.

Lignin depolymerization is the first step toward chemical production since lignin in its polymeric form has limited uses. The breakdown of lignin to low molecular weight compounds helps to channel desirable functions and make suitable intermediates for upgrading to fuels and chemicals. Lignin depolymerization can be categorized into thermochemical routes such as high temperature pyrolysis, hydrogenolysis, and catalytic oxidation and biological routes using lignin degrading enzymes (LDEs) and/or microorganisms5-8. Tremendous efforts have been focused on improving the catalyst

1This chapter in its whole has been published in Biotechnology Advances. 2019, 37 (8), 107418. DOI: 10.1016/j.biotechadv.2019.107418.

1 efficiency and selectivity in a thermochemical conversion process 9, 10. However, the effectiveness and selectivity of the catalyst as developed on lignin model compounds often get compromised when applied to plant derived lignin due to its intrinsic structural and compositional complexity 11.

Biological lignin deconstruction can be performed at lower temperature and pressure than thermochemical methods while product selectivity can be potentially improved via the specificity of biocatalysts 12. The dominant lignolytic enzymes produced by white and brown rot fungi or some Actinobacteria are lignin peroxidases, versatile peroxidases, manganese peroxidases, and laccases. In addition, several unique pathways for the cleavage of lignin linkages by nicotinamide adenine dinucleotide (NAD) or glutathione dependent enzymes have been discovered in certain soil bacteria such as

Sphingobium sp. SYK-6 13-15. Application of these lignolytic enzymes in an industrially relevant lignin valorization process is still hindered due to the high cost of enzyme production and by the very low lignin solubility (for many industrial relevant lignin streams) in the aqueous environment compatible with enzyme activity 16, 17. However, most of the known effective lignin solvents such as hexane, dimethylsulfoxide (DMSO), acetone, and alkalis are detrimental to enzyme activity in their pristine form or aqueous solutions 18, 19 and often require high temperature for complete lignin dissolution especially when dealing with plant derived native lignins 20. Thus, a better solvent system with both high lignin solubility and enzyme compatibility is essential for developing a better biological lignin depolymerization process.

As alternatives to common organic solvents, certain ionic liquids (ILs) have demonstrated a remarkable ability to solubilize and depolymerize lignin during biomass

2 pretreatment 21-23. The cellulose portion recovered from IL pretreatment is highly digestible by using cellulolytic enzymes while lignin in the liquid stream can undergo additional processing and be converted to value-added chemicals 21. Taking advantage of

IL solvent systems, a number of base, acid and metal catalysts have been tested previously for catalytically depolymerizing lignin in IL media 10, 24-28. Meanwhile the use of biocatalysts in IL, i.e. LDEs has attracted increasing interest in recent years due to potential advantages such as better selectivity and affinity, in milder reaction conditions. The toxicity of certain ILs to enzymes and microbes however hinders direct product formation via biological lignin depolymerization in pure IL or its aqueous solution.

There have been several articles reviewing the effects of ILs on enzymes such as lipase, xylanase, cellulase, and laccase, etc. 29-37. To the best of our knowledge, however, there has not been a review of the effects of ILs on LDEs in the context of lignin valorization. This paper emphasizes the effects of IL on lignin characteristics, the relationships between LDE protein structure and function and ILs (enzyme-IL interactions), along with strategies for improving the efficiency and product selectivity of

LDEs in ILs for lignin depolymerization. Finally, possible pathways toward a lignin valorization system using a biocompatible IL and LDE are proposed; challenges and perspectives associated with designing such a system are also discussed.

1.2 Lignin

1.2.1 Lignin structure and characteristics

Lignin is a heterogeneous biopolymer, responsible for providing plants with structural support, microbial resistance and channels for water and nutrient transport 38.

Lignin is constructed of three major subunits, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol 39. These monolignols are differentiated based on the degree of methoxylation of their phenyl rings; p-coumaryl alcohol has no methoxy group, coniferyl alcohol has one methoxy group (2-methoxyphenol), and sinapyl alcohol has two methoxy groups (2,6-dimethoxyphenol). These heterogeneous building blocks are the precursors to what will ultimately become lignin. During lignin biosynthesis, the monolignols undergo slight structural changes as they are converted to phenylpropanoids 39. The p-coumaryl alcohol is converted to hydroxyphenol (H-lignin), coniferyl alcohol is converted to guaiacyl (G-lignin), and sinapyl alcohol is converted to syringyl (S-lignin) 40, 41. Figure 1-

1 highlights the structural differences between the monolignols before and after lignin biosynthesis; most notable is the oxidation of the double bond between the  and  tail carbons.

Figure 1-1. Representative lignin structure consisting syringyl and guaiacyl units (adapted from 42).

Monolignols are linked via a heterogeneous network of inter-unit linkages. The inter-unit linkages seen in lignin are largely dictated by the monolignols present. The most common linkage is the β-O-4’ linkage between H-, G-, or S-lignin 39, 41, 43. G- and H-lignin

4 can be linked together via β-5’, 5-5’-O-4, or 4-O-5’ linkages. H-, G-, and S-lignin can further be linked together via β-β’ or β-1’ linkages 43. Figure 1-2 highlights the complexity of the most common inter-unit linkages. The combination of different monolignols alongside the network of inter-unit linkages, contributes to lignin’s inherent heterogeneity.

Figure 1-2. Formation of inter-unit linkages during polymerization of coniferyl alcohol monomers (adapted from 41).

The source of the biomass determines important properties of lignin. The fraction of lignin present in the biomass is dependent upon the source; softwoods contain 25-31% lignin, while hardwoods contain 16-24% lignin 44. Additionally, the ratio of monolignols present depends upon the source of biomass: softwoods primarily contain G-lignin, hardwoods primarily contain G- and S-lignin, and grasses contain different ratios of H-, G-

, and S-lignin 44. It is this heterogeneity present at all levels of lignin that is responsible for the challenges in selectively depolymerizing these molecules 45.

Plant cell wall engineering has attracted increasing interest in recent years. Genetic modification/engineering the monolignol composition and/or inter-unit linkages present in the native lignin, such as β-O-4’ linkages, could make lignin decomposition processes more

5 efficient. For example, Wilkerson et al. genetically modified poplar trees to synthesize lignin from monolignol ferulate conjugates and demonstrated improved cell wall digestibility after mild alkaline pretreatment of the engineered plants 46. In a follow-up study, Parsell et al. applied a bimetallic Zn/Pd/C catalyst to breakdown engineered poplar trees that contained higher levels of S-lignin; results suggest that the ester linkages present in the genetically modified poplar resulted in higher levels of lignin digestion to a single product (dihydroeugenol) when compared to native poplar 47.

Since lignin is such a vital component for plant growth, altering lignin biosynthesis may have deleterious effects. Repression of a gene responsible for lignin biosynthesis

(hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase, HCT or p-coumaroyl shikimate 3'-hydroxylase, C3'H) in Arabidopsis thaliana led to stunted growth of the plant, a result of reduced auxin transport throughout the plant 48. Bonawitz et al. were able to engineer an Arabidopsis mutant and simultaneously retain its growth and engineered lignin properties 49. It was shown that disruption of a transcriptional regulatory complex, complex subunits MED5a and MED5b restored lignin levels to those found in wild type

Arabidopsis. Interestingly, the resulting lignin was primarily made of H-lignin (~95%), with trace amounts of G- and S- lignin, a trait greatly improving enzymatic digestibility of this mutant after pretreatment 50. Engineering plants with lower lignin content, different lignin composition and inter-unit linkages, and lignin deposition creates opportunities to design better energy crops with traits suitable for bioconversion to biofuels and chemicals

40, 51.

1.2.2 Lignin depolymerization methods

Lignin is often underutilized in its polymeric form. Most of the lignin is burned to produce heat and power; however, a small portion finds its applications as dispersants and binders 52. The breakdown of lignin opens access to desirable functions that are not achievable when lignin is a polymer; while the resulting low molecular weight compounds become suitable for upgrading to fuel and chemicals. Several lignin depolymerization pathways are being developed, including catalytic oxidation, pyrolysis, gasification, and hydrogenolysis, etc. These methods utilize a suite of tools like high temperature, chemical catalysts, and solvents to break down lignin. For example, Alkali processes typically use sodium hydroxide (NaOH) in combination with an oxidative agent for lignin decomposition, which are the most common methods in the paper industry. The two most common alkali pretreatment methods are the Soda and Kraft processes, both of which use

NaOH along with other chemicals such as sodium sulfide and anthraquinone 53.

Ammonium hydroxide has also been shown to be effective for lignin decomposition 21.

While use of high pH methods is effective, chemistry was shown to be effective in depolymerizing lignin in acid, alkaline, or organic solvents. Acid-catalyzed lignin decomposition uses a variety of catalysts including mineral acids, Lewis acids, and ILs 54, most notably through the use of dilute sulfuric acid (H2SO4). Lignin extracted from dilute

H2SO4 pretreated switchgrass was found to have a reduced molecular weight compared to untreated lignin 21. One marker of lignin degradation is the formation of aromatic products due to the aromatic nature of monolignols. Rahimi et al 55 used formic acid for lignin decomposition, resulting in a yield of over 60 wt% aromatics under mild reaction conditions. While oxidative chemistry has been shown to be effective to depolymerize

7 lignin in acid, alkaline or solvents, reductive chemistry using hydrogen molecules or hydrogen donor provides another pathway.

Hydrogenolysis breaks down lignin using hydrogen with help from metal catalysts.

During hydrogenolysis, reductive bond cleavage takes place within lignin and/or lignin model compounds in the presence of hydrogen as a reducing agent 56. A range of catalysts, including noble metals (such as platinum), transition metals, and bimetallic catalysts, such as Zn/Pd/C and Pd/CeO2, were reported to facilitate the cleavage of lignin into aromatic products 47, 57. Alternatively, catalytic transfer hydrogenolysis (CTH) has shown great promise 58. In the CTH reaction scheme, an equivalent of H2 is transferred from a donor molecule to the acceptor molecule. Hydrogen donor molecules are often inexpensive organic alcohols capable of readily generating hydrogen molecules and the same time serving as solvents for lignin 59. A variety of hydrogen donating agents have been tested including formic acid, methanol, ethanol, tetralin etc., among which isopropyl alcohol remains a popular choice due to its relative low cost and easy subsequent separation from the reaction mixture 60.

Pyrolysis is the breakdown of large molecules into smaller ones by the application of heat in the absence of oxygen. During pyrolysis, lignin is heated to temperatures between 160-900 °C where cleavage of the ether (C-O) and C-C linkages takes place 61.

Lignin pyrolysis produces a range of pyrolytic aromatic compounds in oil form in addition to gas products and residual char 62, 63. The yield and composition of pyrolytic oil are influenced by many factors, including lignin type and operation conditions 64.

As opposed to thermochemical lignin depolymerization methods, which require intense energy input and/or harsh chemicals and costly catalysts, biological lignin

8 depolymerization takes advantage of microorganisms and their enzymatic excreta to cleavage the inter-unit lignin linkages and lignin-carbohydrate linkages. The LDEs play key roles in biological lignin depolymerization in nature. The next section summarizes the key LDEs with emphasis on their temperature and pH optima (parameters relevant to IL tolerance) and potential applications in lignin depolymerization.

1.3 Lignin degrading enzymes

1.3.1 Peroxidases

Lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase

(VP) are a series of iron-based LDEs present in nature 65-67. Numerous forms of these

LDEs have been isolated from a variety of lignolytic fungi and bacteria 65, 68-71.

Additionally, isozymes have been identified within individual species. For instance, 16 forms of LiP and 5 forms of MnP were isolated from liquid culture containing Trametes versicolor 69. While these enzymes serve a similar purpose in nature, many have been found to have unique properties.

Peroxidases can retain activity in a wide range of pH and temperature. The optima are dependent upon the enzyme’s source and the oxidation substrate. It was found that LiP and MnP from a soil fungus Deuteromycete retain activity over a pH range of 4-11 and a temperature range of 30-55 ºC 68. Fourteen forms of MnP and LiP from Pleurotus ostreatus and Ceriporiopsis subvermispora were expressed in Escherichia coli; remarkably, an isozyme of MnP from C. subvermispora retained over 80% activity at pH 2.0 76.

Recombinant VP from P. eryngii expressed in P. chrysosporium was found to have optimal activity at pH 3.0, while retaining activity at temperatures up to 40 ºC 77. Fujii et al. observed different pH optima for the LDEs collected at different depths from the forest

10 floor of regions in Japan, Thailand, and Indonesia. It was shown that MnP activity was detected at pH greater than 5.0, while LiP activity was detected at pH less than 5.0 78. The different pH optima suggest that LiP and MnP fulfill important roles in biodegradation of plant matter at different depths of the forest floor. The optimal temperature for LDE activity has also been observed to vary with respect to the substrate present. A peroxidase from Bacillus subtilis had an optimal pH of 3.0, while maximum substrate oxidation was observed at different temperatures from 30-50 ºC 79. LiP displayed optimal removal of a nonylphenol at pH range 4.0-7.0 and temperature range of 25-50 ºC 66. Evidence has suggested that the difference in pH and temperature optima between LDEs serves important functions for the microbes to adapt to various environments in nature.

LiP, MnP, and VP have a wide range of applications beside biodegradation of lignin. This is due to the capability of LDEs to oxidize a wide variety of phenolic substrates

66, 71, 80, 81. For example, LiP was shown to remove a nonylphenol from wastewater 66, while a peroxidase expressed in B. subtilis can be used in dye-decolorizing operations 79. MnP from Bjerkandera adusta was used to depolymerize chemically-pretreated coal, forming aromatic compounds in the process 82. Also reported is a MnP from Ganoderma lucidum for use in reducing both color and turbidity of apple and orange juices 83. The oxidation potential of peroxidases allows for applications besides oxidizing phenolic substrates

(Table 1-1).

1.3.2 Laccases

First discovered in 1883 in an extract from the tree Rhus vernicifera, laccases are a member of the superfamily of multicopper oxidases 84, 85. Laccases utilize four coppers to reduce O2 to H2O and, in the process, oxidize the substrate 86, 87. This oxidative mechanism

11 stands in stark contrast to the mechanism of the peroxidase LDEs; the use of O2 in place of

H2O2 results in a milder chemical environment. This unique trait has led laccases to attract interest in the lignin valorization community. The most commonly investigated laccases are produced commercially from a polypore basidiomycete, T. versicolor (Figure 1-4). The laccases secreted by T. versicolor have moderate degrees of stability at pH from 5 to 8 and at temperatures from 10 to 45 °C 86, 88, 89.

Figure 1-4. Laccase from Trametes versicolor (PDBID: 1GYC) 86. Figure downloaded from the Research Collaboratory for Structural Bioinformatics’ Protein Data Bank (RCSB PDB) 75.

1.3.2.1 Thermophilic laccases

Laccases from thermophilic microorganisms have received particular interest for use in lignin valorization and other applications because of the improved thermostability.

These laccases have been shown to oxidize a wide variety of phenolic substrates. Kiiskinen et al. expressed a thermophilic laccase from Melanocarpus albomyces in Trichoderma reesei. This laccase is capable of oxidizing 2,6-dimethoxyphenol (DMP), syringaldazine, and guaiacol at temperatures up to 50 oC 90. Recently, An et al. expressed a thermostable fungal laccase (PPLCC2) from Postia placenta in Pichia pastoris 85. PPLCC2 displayed

12 optimum oxidation of 2,2’-azino-bis(3-ethylbenzthioazoline-6-sulphonic acid) (ABTS) and DMP at 60 ºC. While continuing to search for thermophilic laccases in fungi, researchers have expanded their search to bacterial and archaeal laccases.

There have been several thermophilic laccases and laccase-like enzymes discovered in bacteria in the past decades. B. subtilis was found to produce a thermostable form of a laccase-like enzyme, CotA 91, which has maximum enzyme activity at 75 °C with

ABTS as substrate 92. Laccase from the bacterium Thermus thermophilus (TtL) is the most thermostable laccase to date 93; optimal substrate oxidation was observed at 92 °C. A recent work reported a thermostable laccase (LccA) isolated from the Dead Sea archaeon

Haloferax volcanii 94. It was found that LccA oxidized the phenolic substrates DMP and syringaldazine along with the nonphenolic substrate ABTS. Remarkably, LccA retained significant activity at 50 °C and in salt concentrations up to 1.4 M.

These laccases have been observed to be reliably thermophilic; however, they have been observed to vary in pH optima. The pH optima of TtL and PPLCC2 change depending upon which oxidation substrate is present. PPLCC2 exhibits optimal oxidation of ABTS and DMP at pH 3.5 and 5.0, respectively 85. TtL exhibits optimal oxidation of ABTS and syringaldazine at pH 4.5 and 5.5, respectively 93. The diverse temperature, pH, and substrate affinity exhibited by laccases enable their use in a number of occasions.

1.3.2.2 Laccase applications

Laccases have a wide variety of applications with a few examples listed in Table 1-

1 95, 96. In the pulp and paper industry, laccases have been used to reduce the lignin content of wood pulp 97. In the textile industry, laccases have been used to decolorize dyes and

13 treat a number of fabrics 98-101. Laccases can also be used to remove chemicals from wastewater and contaminated soil 102-106. In food industry, laccase can be used in processing fruit juice and treating wastewater from various food processes 95, 107-109. In addition, laccases have found use in the pharmaceutical industry, producing compounds that have various medical applications 110, 111. The diverse list of laccase applications continues to expand with research into bioenergy.

The primary application of laccases for bioenergy is for biomass deconstruction.

Laccase is capable of removing lignin from the biomass, resulting in increased digestibility of the cellulose and hemicellulose fractions 112. Laccase-mediator systems have been reported to fractionate and modify lignin to produce quality carbon fibers 113; such a system converts lignin to a high molecular weight fraction suitable for carbon fiber and a soluble low molecular weight fraction suitable for biological conversion to lipids 114. Laccases have also been used in microbial fuel cells (MFCs), in such cases laccases function as a biocathode, providing a cheaper alternative to platinum 115. Immobilization of laccase on carbon nanotubes further improves laccase activity in MFCs over time 116.

1.3.3 Other LDEs

In addition to the peroxidases and laccases, the search for LDEs led to the discovery of novel enzyme consortia in bacteria. Several classes of the bacterial dye-decolorizing peroxidases, multi-copper oxidases and β-etherases have been reviewed previously 16, 117,

118. The soil bacterium Sphingobium sp. SYK-6 is capable of growing on lignin-derived monoaryls and biaryls substrates via expression of several LDEs as shown in Figure 1-5

119-121. SYK-6 has been shown to be extremely tolerant of high pH conditions, a desirable characteristic for lignin valorization 14. Additionally, SYK-6 was found to prefer vanillin

14 when grown in the presence of several carbon sources; through further observations, it was determined that ligninolysis is a mechanism required by SYK-6 for survival 14. The vanillin catabolic pathway is responsible for providing SYK-6 with sufficient concentrations of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), important reducing species vital for microbial growth.

These unique lignolytic characteristics of SYK-6 have made it an attractive target for designing lignin valorization strategies. The complete genome of SYK-6 has been sequenced, which consists of a 4,199,332-bp-long chromosome and a 148,801-bp-long plasmid 122.

Figure 1-5. Lignolytic pathways of Sphingobium sp. strain SYK-6 121.

The lignolytic capabilities of Sphingobium have been leveraged in plant engineering for lignin valorization. A family of LDEs from Sphingobium, LigDFG, were

15 expressed in A. thaliana, resulting in high levels of oxidized G- and S-lignin subunits in plants 123. Furthermore, E. coli was engineered by combining vanillin degradation pathways (LigV, LigM) from SYK-6 and a protocheuate decarboxylase (aroY) and a catechol dioxygenase (CatA) from tobacco plant 124. In a follow-up study, an autoregulatory system was constructed into the engineered E. coli chassis and the new strategy demonstrated bioconversion of the central intermediates such as vanillin into cis, cis-muconic acid at high yield in E. coli 125. Such approaches highlight the opportunities of developing novel applications of bacterial LDEs for lignin depolymerization.

Table 1-1. Applications of lignolytic enzymes

Enzyme Source Application References

Peroxidase B. subtilis Dye decolorization 79

MnP Bjerkandera adusta Coal treatment 82

MnP G. lucidum 107 Food industry Laccase Trametes hirsuta 95

108

Lignin removal from wood Laccase T. versicolor 97 pulp

T. hirsute 98

Trametes villosa 99 Laccase Dye decolorizing in textiles Sclerotium rolfsii 100

T. versicolor 101

Trametes hirsute 66 Various lignolytic 102, 103 microbes Laccase 104 T. versicolor Wastewater treatment LiP 105 T. villosa

106 Lentimula edodes

109 Botrytis cinerea

Myceliophtora thermophyla 110 Laccase Trametes pubescens Production of pharmaceuticals 111 Saccharomyces cerevisiae

T. versicolor 115 Laccase Biocathodes T. hirusta 116

1.4 Ionic liquids

ILs are pairs of ions that exist in liquid form, often below 100 ℃ (also called room temperature ILs). Given the enormous selections of cations and anions, a large number

(1018 theoretically) of ILs can be synthesized. For the same reason, ILs can be designed with fine-tuned properties to suit the needs of a process in a highly specific manner. For instance, by selecting environmentally friendly ion pairs, ILs can be synthesized to replace environmentally harmful volatile organic solvents commonly used in industry 126, 127.

Many ILs have little to no vapor pressure, minimizing harmful emissions that are associated with more common organic solvents 128. There have been, however, some circumstances that challenge this notion 129. Many alkylimidazolium ILs were shown to be flammable when exposed to a heat source 130. ILs have a wide range of applications, including biomass processing, CO2 capture, compound extraction, thermostable lubricant, reaction medium/catalyst for catalysis, pharmaceuticals, etc. which can be seen from several review articles 126, 131, 132. The foci of this section are to review the properties of

ILs that are relevant to lignin fractionation and enzyme activity/stability and the effect of

IL treatment on lignin characteristics.

1.4.1 IL properties

Solvent properties, hydrophobicity, and biocompatibility/toxicity are important properties of ILs that determine their potential applications. Those properties are directly influenced by the cation and anion species. In other words, it is possible to tune the IL properties by changing the anion/cation combinations 133. The Kamlet-Taft system is commonly used to describe IL’s solvent properties in three separate terms/parameters: polarizability (π*), hydrogen bond donator capacity (α) and hydrogen bond acceptor

18 capacity (β) 134. Kamlet-Taft parameters were not only used for measuring solvent properties of a single IL but also applied to describing the average or bulk solvent properties of binary and ternary IL/H2O mixtures 135, 136. Primarily determined by the anion, the β value has been used to predict ILs’ capacity to disrupt cellulose 137, 138. As proposed by Doherty et al., higher β values indicate that ILs tend to form strong attractions with the hydroxyl protons of cellulose; this interaction leads to disruption of the crystalline lattice 139. More computational-based evidence was reported by Sun and co-workers linking the predicted interaction energies with experimentally determined Kamlet-Taft parameters140. A positive correlation was observed between cellulose conversion and β values. ILs with larger differences between β and α, net basicity (β-α), tend to dissolve cellulose more efficiently141, 142.

The hydrophobicity of IL is relevant to enzyme stability/activity in IL; some ILs have been observed to deactivate enzymes with decreasing hydrophobicity. When a series of alkylimidazolium ILs were screened for hydrophobicity, the alkyl chain length of the cation was well correlated with the hydrophobicity of the ILs 143. In addition, hydrophobicity of the anion also contributes largely to the IL’s hydrophobicity. For example, ILs with a bis(trifluoromethylsulfonyl)amide ([NTf2]-) anion have been used to stabilize lithium electrochemistry due to the anion’s hydrophobic nature 144-147. It has been reported that alkylimidazolium ILs with alkyl sulfate anions, with decreasing anion chain length, resulted in higher T. versicolor laccase deactivation 148, 149. Another predictor of the interaction between an IL and an enzyme is the Hofmeister series (aka. lyotropic series), a classification of ions in order of their ability to salt out or salt in proteins. It has been shown that kosmotropic anions and chaotropic cations are preferable for enzyme stability,

19 with the anion’s properties largely determining enzyme-IL interactions 33, 150. Molecular dynamics simulations of a lipase in the ILs 1-butyl-3-methylimidazolium hexafluorophosphate ([C4C1Im][PF6]) and 1-butyl-3-methylimidazolium nitrate

([C4C1Im][NO3]) showed that IL-enzyme interactions are dominated by the anion 151.

Collection of these properties leads to a more generic definition of IL’s biocompatibility/toxicity.

Quantitative structure–property relationship (QSPR) modeling is a powerful tool predicting IL toxicity. Results from Rank et al. show strong correlation between lipophilicity of the cations and IL cytotoxicity by comparing experimental with computational data over a set of 74 ILs with imidazolium, pyrrolidinium, pyridinium, quinolinium, quaternary phosphonium, and quaternary ammonium cations and the small anions such as Cl-, Br-, BF4- or PF6- 152. Results from the same group suggest that anion’s lipophilicity and/or vulnerability to hydrolytic cleavage also appeared to be a crucial structural feature leading to cytotoxicity 153. Using a set of electronic, spatial, structural, thermodynamic, and topological descriptors, Couling et al. constructed QSPR models to predict IL toxicity; results show an increasing trend of toxicity based on cation type of ammonium, pyridinium, imidazolium, triazolium, tetrazolium and decreasing trend of toxicity with increasing number of negatively charged atoms and ring methylation on the cation 154. A more recent work from Yoo et al. 155, adopted an integrated experimental and computational approach to explore the molecular-level details of IL’s cytotoxicity.

Molecular dynamics (MD) simulations based on 1-palmiltoyl-2-oleoyl- phosphatidylcholine lipid bilayer suggest that cytotoxicity increases with increasing alkyl chain length of the cation, due to the ability of the longer alkyl chain to insert in and disrupt

20 the cell membrane 155. Collectively, it is concluded that both the cation and anion play important roles in determining IL’s toxicity/biocompatibility through their interactions with biomolecules.

1.4.2 Biomass pretreatment with ILs

The ability of certain ILs to solubilize one or all three major components of lignocellulose has made them attractive solvents for biomass pretreatment 156. This capability is largely attributed to IL’s ability to form strong hydrogen bonds with the cellulose, hemicellulose, and lignin polymers present in biomass 157, 158. A computational approach was used to predict the ability of 750 ILs to solubilize cellulose and lignin using p-coumaryl alcohol as a lignin model compound; it was predicted that a very wide range of anion/cation combinations can form ILs capable of solubilizing lignin 159. The factors affecting IL pretreatment performance and challenges facing this pretreatment technology are discussed below.

Effect of cation/anion on pretreatment

The properties of ILs are sensitive to the cation and anion species present in the IL.

As a result, these species can significantly alter the products obtained from biomass after

IL pretreatment. The ability of ILs to dissolve cellulose is dependent upon the anion and cation species. ILs with the 1-butyl-3-methylimidazolium ([C4C1Im]) cation are capable of dissolving cellulose when paired with a chloride anion ([Cl]) 160. When the same cation is paired with [NTf2] anion, the IL can no longer dissolve cellulose. Furthermore, when

[Cl] is paired with a pyrrolidinium or piperidinium cation, the IL can no longer dissolve cellulose 161. The ability of choline amino acid ([Ch][AA]) ILs to solubilize the components of biomass have been shown to be sensitive to the choice of amino acid. Liu et al. screened a series of [Ch][AA] ILs for lignin, cellulose, and hemicellulose solubility and found hemicellulose solubility was heavily dependent upon the amino acid anions 162.

When pretreating wheat straw with dilute [Ch][AA] ILs, it was found that glutamic acid significantly reduced lignin removal during pretreatment 163. In a follow up study, Hou et al. 164 established a positive correlation between the pKa of the anion and the delignification observed during pretreatment.

Alkylimidazolium ILs have served as benchmark solvents for pretreatment of lignocellulosic biomass (Figure 1-6). These ILs have been shown to improve cellulose and hemicellulose digestibility after pretreatment, via lignin removal and reduction or change in the crystallinity of cellulose. Enzymatic hydrolysis sugar yields can be greatly improved after pretreating a variety of biomass feedstocks with alkylimidazolium ILs 22, 165. In one example, it is shown that [C2C1Im][OAc] reduces the crystallinity of the cellulose in wood flour and removed 40% of lignin, leading to over 90% glucose yield from enzymatic hydrolysis of the pretreated biomass 166. Despite their effectiveness in pretreatment strategies, the high cost of alkyl imidazolium ILs has hindered their widespread adoption in biorefineries.

Figure 1-7. Butylimidazolium ILs. These ILs have all been effectively utilized in biomass pretreatment strategies with different effects on downstream processing of biomass (adapted from 22).

Cost reduction strategies to enable IL pretreatment

Three primary avenues for IL cost reduction are being explored: the use of inexpensive feedstocks for IL synthesis, use of aqueous ILs, and reusing or recovering ILs for repeating pretreatment cycles 23. a. Inexpensive feedstock for IL synthesis

There are a number of low-cost precursors that can be used for IL synthesis. To serve as viable replacements, however, these new ILs must match the pretreatment efficacy of alkylimidazolium ILs. Cations and anions derived from renewable resources such as protein, sugars, and biomass-derived carboxylic acids are of particular interest. George et al. synthesized low-cost ILs from ammonium cations and the [HSO4]- anion and tested their effectiveness in pretreating switchgrass; results show that sugar yields and lignin removal using the low-cost ILs were comparable to 80% [C2C1Im][OAc] 167. ILs synthesized from choline chloride and bio-derived amino acids have been shown to be highly effective in solubilizing hemicellulose and lignin without solubilizing cellulose 168.

Eight choline amino acid ([Ch][AA]) ILs were screened for their ability to remove lignin during pretreatment of rice straw. The majority of the ILs were shown to remove over 50%

23 of lignin during pretreatment, with [Ch][Lys] removing 60.4% of lignin during pretreatment 169. These new ILs are potentially more biocompatible and cheaper compared to petroleum based alkylimidazolium ILs 170. In addition to lowering the cost of IL itself, reducing the dosage of IL by using IL-water mixtures (aqueous IL) will further lower the chemical cost of pretreatment. b. Aqueous ILs

Instead of using pure, or “neat” ILs, using IL-water mixtures reduces the amount of IL needed to achieve a desirable pretreatment outcome, thus reducing the overall cost.

Elucidating the role of water on the IL pretreatment efficiency helps to design a better pretreatment process using aqueous IL. In a previous study, the effect of water on IL pretreatment was investigated by pretreating switchgrass with a series of [C2C1Im][OAc]- water mixtures. It was found that pretreatment with dilute [C2C1Im][OAc] at 50-80% IL concentration led to comparable glucose yields and lignin removal to those obtained with

100% [C2C1Im][OAc] 171. Pretreatment with dilute [Ch][AA] ILs was also shown to improve sugar yields and lignin removal when compared to pretreatment using pure ILs

163. These findings provide insights into the interplay of water as a co- and anti-solvent in aqueous IL system. Using aqueous IL as pretreatment agents offers additional benefits including reduced viscosity of the IL medium, less gel formation, and lower energy requirements and costs associated with IL recycling 171, 172. c. IL reuse and lignin recovery

Recovery and reuse of ILs following pretreatment can reduce the demand for purchasing or synthesizing nascent ILs. Oftentimes these ILs can be reused several times

24 without a significant reduction in pretreatment efficacy. For instance, reuse of

[C2C1Im][OAc] for 5 times was possible without observing a significant change in the pretreatment maple wood flour 166. [Ch][Lys] can be recovered and reused for pretreatment of rice straw 5 times until a significant reduction in pretreatment efficacy was observed 169.

Separation technologies such as evaporation/distillation, electrodialysis, reverse osmosis, and pervaporation have been investigated for concentrating ILs for reuse 173-175. However, sugars and lignin need to be separated from the IL before sending the IL back for the next batch; the cost associated with this step can be prohibitive.

The use of IL-based solvents for biomass dissolution and lignin depolymerization is relatively new and much needs to be learned before an industrial scale process can be implemented. System complexities associated with IL recycling, biomass-solute separation and downstream processing remain the key challenges in the commercialization

176. Recent studies have identified some key areas that influence the overall process economics of IL-based biorefinery processes 175, 177, 178. These areas include the use of low- cost IL at high solid loading in the reaction, development of biocompatible ILs or low price

IL-tolerant enzyme mixtures, development of efficient IL recycle and product recovery technologies, and system intensification and optimization. In-situ lignin valorization is one of the possible ways to lower the product recovery cost and in turn improve the overall economics.

1.4.3 Effect of IL treatment on lignin characteristics

The structure of lignin undergoes numerous changes during IL pretreatment.

Molecular weight distribution can be quantified with gel permeation chromatography

(GPC), while linkage frequency can be quantified with Heteronuclear Single Quantum

Coherence Nuclear Magnetic Resonance (HSQC NMR). Changes in lignin structure as a result of pretreatment with [C2C1Im][OAc] were investigated at different treatment temperatures and times. Changes in β-O-4’, β-5’, and β-β’ linkage frequencies were observed, along with a reduction in the molecular weight distribution 179. GPC analysis of lignin extracted from switchgrass with dilute [Ch][Lys] found lignin in the liquid stream had a lower molecular weight than lignin found in the liquid stream after dilute acid and ammonium hydrolysis pretreatment 21. GPC and HSQC NMR provide helpful information regarding changes to the lignin structure. Due to the heterogeneous nature of lignin, it is challenging to elucidate lignin depolymerization mechanisms in ILs. β-O-4’ linked lignin model compounds have been used to elucidate probable depolymerization mechanisms of methylimidazolium ILs 180. Model lignin compounds enable researchers to better investigate the effects of IL treatment on common aryl ether or carbon-carbon linkages found in real lignin 25.

1.4.4 Rationale for pairing LDE and IL for lignin depolymerization

Taken together, given the unique properties of certain ILs that provide for lignin solubility and biocompatibility to enzymes and microbes, there is a great opportunity to develop a new strategy for lignin extraction, depolymerization, and upgrading via biocatalysis in ILs. Instead of using harsh thermochemical processes, the relatively mild biochemical process facilitates the selective deconstruction of lignin and retains the functionality of the oligomeric lignols, thus helping to channel desirable aromatic products.

Despite previous exploration of catalysis and enzyme activity in ILs, there is a gap in our fundamental understanding of the mechanisms determining the interactions of lignolytic enzyme and these solvents. Further exploration is needed into how the solvent properties

(acidity, basicity and polarity) as determined by the cation and anion pairs impact the lignin solubility and enzyme activity in aqueous IL systems. Furthermore, in order to enable enzymatic reactions in IL, two aspects need further investigation: the development of biocompatible ILs with improved lignin solubility, and the selection of IL tolerant enzymes. These developments rely on a better understanding of the enzyme-IL interactions, such as the structural changes, surface charges, inhibition kinetics, enzyme stability and the structure-function relationship of enzyme in IL media. Based on that, the

LDE-IL system can be tuned to improve the product yield and selectivity.

1.5 Enzyme-IL interactions

1.5.1 Enzyme activity and stability in ILs

Certain ILs, as part of their highly tunable nature, can provide an environment suitable for enzymatic activity. Cellulase 181, laccase 150, 182, 183, lipase 184, 185, α- chymotrypsin 185-187, tyrosinase 185, and lysozyme 188 activity have all been tested in the presence of IL (Table 1-2). Numerous studies have attempted to elucidate the IL characteristics affecting enzyme’s activity and stability in IL. Efforts have also been made to identify enzymes with improved resistance to enzymatic inhibition caused by IL.

Table 1-2. Studies of enzyme activity and stability in ILs

Enzyme Source Ionic Liquid Reference

182 Laccase T. versicolor Alkyl imidazolium 183

Laccase T. versicolor 56 ILs 189

Cellulase T. reesei [C2C1Im][OAc] 190

β-Glucosidase A. niger [C2C1Im][OAc] 190

Laccase T. versicolor [Ch][H2PO4] 189

Aspergillus (DeniLite Laccase [C2C1Im][MDEGSO4] 191 Base)

Ethanolammonium formate

Ethylammonium nitrate

Lysozyme Chicken egg white Bis(2-methoxyethyl)ammonium acetate 188

N,N-dimethylethanolammonium glycolate

[Ch][H2PO4]

[C2C1Im][EtSO4] Laccase T. versicolor 183 [C2C1Im][OAc]

Laccase T. versicolor [Ch][H2PO4] 189

Triethylammonium acetate

Triethylammonium phosphate

Bovine pancreas type 1-benzyl-3-methylimidazolium chloride [C6C1Im][Cl] α-Chymotrypsin 186 II 1-benzyl-3-methylimidazolium tetrafluoroborate [C6C1Im][BF4]

Tetra-butylphosphonium bromide

[Ch][H2PO4] RNase A N/A 192 [C2C1Im][DCA]

1.5.1.1 Enzyme activity in ILs

A wide variety of factors are thought to play a role in enzyme activity in ILs. There is a clear trend that persists among studies performed: the higher the IL concentration, the more severe the inhibition observed. Laccase from T. versicolor was screened for activity

28 and stability in a series of methylimidazolium ILs 193. Increasing concentrations of ILs resulted in increased levels of enzymatic inhibition; while 1-ethyl-3-methylimidazolum ethylsulfate ([C2C1Im][EtSO4]) caused less inhibition to laccase activity as compared to

[C4C1Im][Cl]. Similarly, as shown in another study, T. versicolor laccase activity decreased with increasing IL concentration of the two tested alkylimidazolium ILs,

[C2C1Im][EtSO4] and [C2C1Im][OAc] 183. This trend was not always true as improved laccase activity has been observed in some ILs. Galai et al. screened T. versicolor laccase activity in 56 ILs and found that 13 of the ILs improved laccase activity; the most notable improvement was seen in choline dihydrogenphosphate ([Ch][H2PO4]) which increased laccase activity 451% 150. In addition to the enzyme activity, which is determined by short- term inhibitory effect, the long-term effect of IL on enzyme, in other words, the enzyme stability in IL, is equally important.

1.5.1.2 Enzyme stability in ILs

ILs have been shown to stabilize enzymatic activity over varying periods of time.

The long-term effects vary with respect to the IL-enzyme pair and IL concentration. Some

ILs, like [Ch][H2PO4], continue to positively impact enzymatic activity over time 150, 188.

However, the long-term effects of several alkylimidazolium ILs stands in contrast to their short-term effects. T. versicolor laccase in [C4C1Im][Cl], [C2C1Im][OAc], 1-ethyl-3- methylimidazolium MDEGSO4 ([C2C1Im][MDEGSO4]), and [C2C1Im][EtSO4] exhibited improved stability over varying periods of time, however, all ILs caused inhibition to enzyme activity in short term 193.

Certain ILs have also been shown to improve enzymatic resistance to inhibition caused by pH and thermal deactivation. For example, T. versicolor laccase in 10 mM

[Ch][H2PO4] retained activity up to pH 12, compared to pH 9 without IL 150. Short alkyl chain ammonium ILs, triethyl ammonium acetate ([TEA][OAc]) and triethyl ammonium phosphate ([TEA][PO4]) were shown to improve the thermal stability of α-chymotrypsin when compared to enzyme activity in 0.05 M pH 8.2 Tris-HCl buffer 186. Not surprisingly, the ability of ILs to stabilize enzymes is sensitive to the cation and anion present; the thermostability of RNase A was improved in [Ch][H2PO4] whereas choline chloride

([Ch][Cl]) did little to enhance thermostability 192. This anion-specific effect can be partially explained by the Hofmeister series: H2PO4- has been observed to be less chaotropic than Cl- when paired with the same cation 37, 148, 194. In spite of the numerous studies of IL-enzyme interactions, the exact mechanisms by which ILs affect enzyme stability are still largely uncertain.

1.5.2 Effect of IL on enzyme

Investigating the changes of enzyme structure provides important information about enzyme stability in different ILs with varying properties. The structure of an enzyme is critical for its function, whereas enzyme-IL interactions may cause structural changes of the enzyme that further impact its activity and stability. The enzyme-IL interactions are dependent on IL properties and the enzyme sources. Thus, an in-depth understanding of enzyme structure and enzyme-IL interactions is critical for designing future biocatalytic systems in ILs.

1.5.2.1 Enzyme structure

Circular Dichroism (CD) spectroscopy can help understand changes made to enzyme structure in ILs by highlighting secondary structures of the enzyme. CD

30 spectroscopy of T. versicolor in [Ch][H2PO4] revealed that addition of IL resulted in a conformation change around the active site, along with a shift in the ratio of α/β structures

150. Enzyme crystallization in IL and resolving the structure could provide a more comprehensive view of structural changes; this approach will be discussed later in the review.

1.5.2.2 IL properties affecting enzymatic activity

There have been a myriad of in-depth reviews discussing the different IL properties responsible for enzymatic inhibition and stability 29-37. Each of the reviews contains tables summarizing studies examining different IL properties and their effect on enzymatic activity and stability. Polarity, alkyl chain length of the cation, hydrophobicity, anion, and viscosity are common factors discussed in each of the reviews. Increased cation chain length, increased anion size, and decreased polarity (a consequence of chain length and anion size) were all factors associated with reduced α-chymotrypsin activity 184.

Thermostability of α-chymotrypsin in ILs revealed that ILs with imidazolium and phosphonium-based cations were weak stabilizers, while ILs with ammonium-based cations were strong stabilizers 186.

Increasing cation hydrophobicity and decreasing anion nucleophilicity has been shown to increase lipase activity 195. Two enzymes, a lipase and a tyrosinase, were shown to be biocompatible with aqueous IL made of chaotropic cations and kosmotropic anions

196. Results indicate that in general ILs with choline or ammonium cations activated lipase while imidazolium ILs deactivated the enzyme. More specifically, ammonium ILs composed of chaotropic cations (favorably with H-bonding capability) and kosmotropic anions are favored for enzyme catalysis 196. The interactions between a lipase and several

ILs were simulated with molecular dynamics (MD) simulations, revealing dominant

Coulombic and van der Waals interactions 29. The same simulations also showed some

[C4C1Im] cations diffused into the active site, implicating competitive inhibition. There is no consensus as to which, if any, how IL property dominates the impact on enzyme activity and stability. Regardless, there have been numerous efforts focused on improving enzyme activity in ILs.

1.5.3 Methods for improving enzyme stability in ILs

In an effort to combat the inhibitory effects of ILs on enzymatic activity, a number of methods have been explored. Most predominant of these approaches are immobilization of the enzyme on a substrate, screening enzymes from thermophilic and halotolerant organisms, and engineering the enzyme surface residues. Each of these methods has been successfully demonstrated with different enzymes.

1.5.3.1 Immobilization

Enzymes can be attached to an inert, insoluble material via different mechanisms such as adsorption, entrapment, cross-linking, etc. Enzyme immobilization has proven to be an effective method of improving enzyme activity and/or stability in less than optimal conditions 197. Furthermore, immobilized enzymes have better recyclability than free enzymes. Immobilization strategies have been successfully employed to improve enzyme activity in ILs. In one study, immobilization of lipase on acrylic resins improved yields of biodiesel in 1-butyl-3-methylimidazolium hexafluorophosphate ([C4C1Im][PF6]) 185.

Laccase from Myceliophthora thermophila was immobilized on glyoxyl-agarose beads, improving thermostability in [C2C1Im][EtSO4] at 70 °C 198. Several other enzyme

32 immobilization strategies (on solid support, sol–gel, and cross-linked enzyme aggregate,

CLEA), have been reviewed previously and will not be discussed here 30.

1.5.3.2 Thermophilic and halotolerant enzymes

Thermotolerant and halophilic organisms have evolved to produce enzymes with resistance to harsh environments. This selective pressure from the environment has enabled these enzymes to potentially resist the inhibitory effects of ILs. Cellulases from a hyperthermophilic bacterium and archaeon retained activity in two-fold higher

[C2C1Im][OAc] concentration than their mesophilic counterparts 199. In a follow-up study,

Zhang et al. identified a halophilic cellulase from a halophilic archaeon and found it can tolerate relatively high concentrations (20% w/w) of several alkylimdazolium ILs 200. The halophilic and IL-tolerant cellulase identified by Zhang et al. was found to have a large number of negatively-charged surface residues 200. Similarly, cellulases from the halophilic Aspergillus terreus strains displayed improved activity and stability in high salt conditions and a series of alkylimidazolium ILs, most notably [C2C1Im][OAc] 201.

Although, limited number of structural evidences exist to explain what causes the improved resistance of those enzymes to IL, it’s possible that the thermophilic cellulases evolved to have more negatively-charged surface residues than the mesophilic cellulase. Several studies have suggested that reducing the number of positively-charged surface residues and increasing the number of negatively-charged surface residues improve enzyme resistance to ILs 202, 203. The surface characteristics enabling enzyme tolerance to IL have inspired artificial ways to modify enzyme surfaces.

1.5.3.3 Charge engineering

Altering the charged residues on the surface of the enzyme could affect the ability of the ILs to bind to the enzyme (Figure 1-7). This reduction in binding affinity would reduce IL inhibition. Reducing the amine:acid ratio of surface residues has been shown to improve activity of several enzymes in [C4C1Im][Cl] 202. Later molecular dynamics simulations of modified lipase and α-chymotrypsin showed reduced anion binding and increased cation binding on the enzyme surface 203. Surface modification of enzyme by introducing protein–polymer surfactant nanoconstructs was shown to greatly improve enzyme solubility in hydrophilic and hydrophobic ILs 204. Applying this method, Brogan et al. 205 recently demonstrated that chemically modified glucosidase was thermally stable at up to 137 °C and 30 times more active towards cellulose in [C2C1Im][EtSO4] as compared to reactions in aqueous media due to solvent-induced substrate promiscuity of glucosidase.

Figure 1-8. Effect of surface charge modification on IL interaction with enzymes (Nordwald, 2015).

1.6 Protein engineering to improve IL compatibility

1.6.1 Structure-function relationship of proteins

The structural hierarchy of proteins can be broken down into 4 levels: primary, secondary, tertiary, and quaternary. The primary structure of a protein is the sequence of amino acids. The secondary structure captures how those amino acids are folded into α- helices, β-sheets, or loops. The tertiary structure captures how the secondary structures fold together to form the final protein structure; oftentimes this is accomplished by an intricate network of hydrogen bonds, salt bridges, and disulfide bonds 206, 207. The quaternary structure is defined by tertiary structures forming higher-order structures such as dimers or trimers. Each of these structural levels plays a key role in the function of proteins.

1.6.1.1 Primary structure

Directed mutagenesis of key residues in a variety of enzymes has implicated the ubiquitous role primary structure plays in protein function. These residues have been shown to be responsible for a variety of key functions of the protein.

Substrate recognition and binding

In many proteins, key amino acids have been implicated in the binding of the substrate. Mutations of 2 residues near the heme access channel, H82A and Q222A, and

2 residues near the surface of the protein, W171A and F267L, revealed the importance of each residue for LiP substrate binding and function 208. Mutation of Asp376 to Asn, Glu, or Thr at the catalytic phosphorylation site of the α subunit leads to loss of ATPase activity

209. It was found that leptin binding to the leptin receptor of a variety of species is highly specific to the residues present on both the receptor and leptin 210. Mutations of residues near the surface of Type III antifreeze protein (AFP) resulted in loss of activity 211.

Oftentimes, the residues responsible for enzymatic activity have a net charge at physiological pH.

Altering the primary structure of an inactive enzyme can alter the enzyme activity and substrate affinity. A heme peroxidase from Coprinus cinereus was unable to catalyze the oxidation of veratryl oxidase, a common substrate used for screening lignolytic enzymes. The inactive heme peroxidase has a structure similar to LiP, so a number of mutations were made using the active LiP as a template 212. A Trp residue (Trp179) was introduced to interact with veratryl alcohol, while D179W, R258E, and R272D mutations created an environment mimicking that found around the catalytic Trp residue in the active

LiP 212. All of these mutations enabled the previously inactive heme peroxidase to oxidize veratryl alcohol. This approach allows identification of primary structure that are crucial for proper enzyme function.

Charged residues

Charged residues on the surface of proteins can be implicated when binding to a charged substrate. For instance, alanine mutations of a series of positively-charged Lys and Arg residues resulted in decreased activity in proliferating cell nuclear antigen (PCNA)

213. Key mutations of charged residues of TRPV1 and TRPV4 dramatically reduce the permeability of calcium and magnesium ions 214. TRPC1 and TRPC5 channel permeation properties have been shown to be highly dependent upon negatively charged residues close to the pore region 214. As discussed in section 5.3, modification to the charged residues on surface of the enzyme has proven effective to reduce IL inhibition by altering IL binding affinity to enzymes.

1.6.1.2 Secondary structure

As amino acids begin interacting with each other, they start forming higher-order structures. The presence of these secondary structures can impact the function and stability of a protein. T. versicolor laccase experienced a shift in the ratio of α-helices and β-sheets in the presence of [Ch][H2PO4], likely due to anion-peptide interactions, though the exact reason why this shift occurred was not confirmed 150. The β-sheet near the C-terminus of a Type III antifreeze protein is thought to be responsible for antifreeze activity 211. The secondary structure of a laccase is related to its thermostability. For instance, bacterial laccase has a coiled section connecting 2 domains, whereas plant and fungal laccases do

37 not; for that reason, among many others, bacterial laccases are typically more thermostable than fungal and plant laccases 206, 207.

1.6.1.3 Tertiary and quaternary structure

Tertiary and quaternary structure have been shown to affect a wide variety of protein functions. Changes to the tertiary and quaternary structure of the Tobacco Mosaic

Virus Coat Protein have been shown to significantly affect the immune system response of the host 215. An N-terminus domain of a protein from Bacillus stearothermophilus is responsible for anchoring the protein to the cell wall of the organism 216. The structure of

G-protein coupled receptors (GPCRs) is critical in determining their conformation and, as a result, their function 217. When a GPCR receptor is unbound, it adopts an inactive conformation; once it is bound by an agonist, the structure of the GPCR receptor changes to an active form 217. Although few studies have examined IL effects on tertiary structure, it is reported that the tertiary structure of the immunoglobulin binding domain of streptococcal protein B disrupted in high concentrations (60% v/v) of [C4C1Im][Br] using high-resolution magic-angle-spinning NMR spectroscopy 218. Knowledge of IL’s impact on protein structure provides vital information for protein engineering strategies to improve enzymes’ IL tolerance.

1.6.2 Protein engineering for improved function

To improve function of proteins under environmental stresses (high pH, high temperature, etc.), key residues can be identified and mutated. The library obtained from screening mutations of residues grows at an enormous rate as more sites are mutated, a consequence of the number of possible amino acids at each position (20n, n = number of

38 sites mutated). When screening mutations at 4 sites, the library will contain 204 = 160,000 entries. Addition of just 1 more site mutation increases the size of the library to over 3 million entries. It’s for this reason that most mutation studies are limited to just a few sites.

Despite this limitation, impressive results have been obtained by mutations of just a few sites on many proteins. Two protein engineering approaches are commonly used for improving protein function: directed evolution and rational design 219. The ultimate goal of both approaches is the same, which is a protein with improved stability in a harsh environment. What sets the approaches apart is the size of the generated library of mutants.

Directed evolution usually generates a much larger library of mutants than rational design.

1.6.2.1 Directed evolution

Directed evolution mimics the environmental stresses to an organism which is performed in lab using methods like error-prone PCR for diversity generation (random mutagenesis) and screenings in iterative cycles 220. The organisms producing the best performing enzymes are subjected to another round of, and in most cases more severe, environmental stress. The screening procedure is repeated until a desired optimal protein is obtained. Throughout the screenings the proteins are sequenced and key residues that improve performance are identified. In one example, Martin et al. generated a thermostable mutant of a mesophilic cold shock protein through the use of directed evolution 221. Through application of high heat and high salt stresses, a mutant was produced that outperformed a thermophilic cold shock protein. By sequencing the mutants, key residues responsible for improving the thermostability of the protein can be identified.

A thermophilic fungal laccase from M. thermophila was subjected to repeated rounds of directed evolution in organic solvents, in the process developing an affinity for higher pH

39 environments 106. Other directed evolution strategies involving laccases are primarily focused on improving expression in non-native organisms, i.e. fungal laccase expression in yeast 222.

1.6.2.2 Rational design

Rational design requires the knowledge of the residues thought to be responsible for improving a protein’s tolerance to environmental stresses. These residues are often identified through the use of a homolog. The crystal structure of MnP from C. subvermispora was used as a template for directed mutagenesis of a P. ostreatus versatile peroxidase (VP), producing 2 variants with improved resistance to a low pH environment

223. Identifying residues in ancestral forms of the enzyme, a triple mutant

(H239F/T240L/I241L) LiP from P. chrysosporium was engineered for improved activity at high temperatures and stability in the presence of H2O2 80. Threonine residues surrounding the type I copper in B. subtilis CotA, T415 and T418, were the focus of a rational design strategy by Khodakarami et al. 224 with the goal of improving activity and substrate selectivity. The T415I mutation improved activity with ABTS 4-fold, whereas selectivity towards ABTS was 11 times higher than syringaldazine in the same mutant 224.

A fungal laccase from T. versicolor exhibited improved resistance to the IL

[C2C1Im][EtSO4] when mutations were made to loop residues connecting domains 2 and 3

225. While rational design can be a powerful tool for improving enzyme function in stressful environments, it can be limited by lack of knowledge about the protein’s sequence and structure.

1.6.3 Protein crystallization in ILs

Crystallizing proteins in ILs could provide more detailed insight into the binding locations of ILs on the surface of proteins. Crystallization of proteins in ILs has been shown to, in some cases, improve crystal quality, size, and number. When trypsin and lysozyme were crystallized in ILs, crystal number and quality were improved; resolution in [C4C1Im][BF4] was improved from 2.8 Å to 2.1 Å 226. Addition of ILs resulted in formation of larger lysozyme crystals 188. When trypsin was crystallized in ILs, some fragments of the ILs were found to have been incorporated as part of the protein structure

227. Numerous studies have highlighted the benefits of using ILs for protein crystallization, though none have identified IL binding sites on the protein’s surface. Docking simulations can be used to identify IL binding sites, as seen in the 2016 study by Anbarasan et al. with a thermophilic xylanase in biomass-dissolving ILs 228. To get a clearer picture, however, crystallization of proteins in ILs is critical for elucidating the effects of enzyme-IL interactions.

1.7 Product selectivity

Due to the complex nature of lignin, controlling product formation from lignin depolymerization proves difficult. A biocatalysis system coupling the LDEs and ILs opens opportunities in directing product formation from lignin degradation. The selectivity is rooted in both the tunable properties of ILs and the specificity of LDEs. Additionally, use of a mediator alongside LDEs (e.g. laccase) can increase the oxidation window thus extending the suite of products obtained from lignin. Use of capping agents has also been shown to prevent condensation of lignin depolymerization products.

1.7.1 Controlling product formation via biocatalyst selectivity

The subject of directing formation of products from lignin has already been explored through the use of a number of base, acid, and metal catalysts for catalytically depolymerizing lignin in IL media 10, 24-28. The use of a biocatalyst, however, could potentially offer improved selectivity at less severe reaction conditions. Laccase uses O2 as the final electron acceptor in its oxidative mechanism, and there is no need for harsh temperature/pressure and costly catalysts. In addition, a number of ways can be used to tune the biocatalyst for different products. One way is via the use of a mediator such as 1- hydroxybenzotriazole (HBT) and ABTS. Introduction of a mediator helps increase the oxidative potential of laccase, thus increasing the number of products obtained by enabling laccase to oxidize non-phenolic compounds 229, 230. Furthermore, the source of enzymes could potentially affect the products obtained from lignin as a consequence of the oxidative mechanisms. For instance, white rot fungi typically utilize several oxidative enzymes, such as LiP, MnP, and laccases 17 while brown rot fungi utilize a Fenton redox reaction when degrading lignin 231, 232. Recent discoveries of new classes of LDEs that selectively cleave

C-C bonds, hold great promise since this capacity is unrivalled by common metal catalysts

233, 234.

1.7.2 Controlling product formation via IL selectivity

ILs are capable of directly influencing the products obtained from lignin degradation. It has been demonstrated that acidic ILs are capable of cleaving the β-O-4’ linkage 180. Additionally, Dutta et al. 235 demonstrated that product formation from lignin degradation was a function of the IL used and the treatment temperature. At high

42 temperatures [Ch][Lys], [TEA][HSO4], and [C2C1Im][OAc] were shown to favor formation of guaiacol, guaiacylacetone, and both guaiacol and guaiacylacetone, respectively. This product selectivity was only observed at high temperatures and high IL concentrations.

Several ILs have been shown to be capable of shifting product formation of biocatalysts. Increasing concentration of ILs was correlated with an increase in the monophenolase:diphenolase activity ratio of a tyrosinase 236. A protic IL, triethylammonium mesylate increased the selectivity of lipase towards omega-3 fatty acids

237. Product selectivity of a sialidase was shown to be highly sensitive to the amount and the type of IL present in the system 238. Pairing the product selectivity offered by both the

LDEs and ILs could potentially lead to more effective lignin depolymerization pathways.

1.7.3 Preventing lignin repolymerization and improving yield

One of the primary issues encountered when dealing with lignin decomposition, along with directing product formation, is that of lignin repolymerization. The intermediates formed during lignin decomposition are susceptible to repolymerizing rather than forming monomeric, dimeric, or oligomeric products. Under acid and/or high temperatures conditions commonly used for lignin extraction, stable carbon-carbon (C–C) bonds are formed once the lignin ether bonds are cleaved. This leads to severe and irreversible condensation that dramatically affects further upgrading of lignin. When examining depolymerization of organosolv lignin with base catalysts, it also was found that lignin repolymerization occurred; S-lignin was the primary subunit involved in the repolymerization reactions 239. Condensation has also been observed in enzymatic depolymerization strategies. Lignin model compounds and dimers underwent

43 condensation when treated with fungal laccases from Melanocarpus albomyces and

Trametes hirsuta 240. Interestingly, formation of some condensed lignin structures appeared at different times dependent upon the redox potential of the laccase. Preventing repolymerization could improve product yields from lignin depolymerization.

Perhaps the most promising and widely researched method for preventing lignin repolymerization is the use of capping agents. Several capping agents have been demonstrated to improve lignin product yield. Boric acid and phenol were used as capping agents, with phenol improving product formation from organosolv olive tree pruning lignin

241. A phenol:lignin ratio of 2:1 resulted in the highest oil yield and lowest repolymerization. Phenol as a capping agent is not without its limitations, however; increasing the phenol:lignin ratio beyond 2:1 resulted in the formation of higher molecular weight lignin, which is likely a result of the excess phenol reacting with products and residual lignin. Formaldehyde has also been used as a capping agent for stabilizing lignin during biomass pretreatment, greatly improving yields of guaiacyl and syringyl monomers close to the their theoretical limits 242. Using a capping agent alongside an enzyme-IL pair could improve product yields while enabling selective product formation.

The time and enzyme quantity required for significant biocatalytic lignin valorization will also need to be investigated as a potential roadblock towards improving yield. Most studies reported hours to days of reaction time used for enzymatic lignin depolymerization in ILs. These reaction times are generally comparable to enzymatic depolymerization of other biopolymers in IL, such as cellulose and hemicellulose. Studies investigating the sugar (xylose and glucose) yields from enzymatic saccharification after pretreatment of biomass with dilute [TEA][HSO4] or [C2C1Im][OAc] required 24-72h and

44 several mg protein/g biomass to achieve the maximum sugar yields 171, 243. Given the heterogeneous nature of lignin it’s likely that the reaction times and enzyme quantities required for enzymatic lignin depolymerization, prior to enzyme engineering, will be similar or even greater. However, it is projected that the efficacy can be improved by enzyme engineering for better enzymes and/or by process intensification and optimization

(i.e. immobilized enzymes) for better enzyme stability and recyclability.

1.8 Conclusions and perspectives

The great potential for lignin to be a renewable feedstock for commercial chemicals is hindered by its structural heterogeneity and recalcitrance. With such an intense research focus in recent years, great strides have been made in developing lignin valorization strategies. ILs have been proven to be a capable class of lignin solvents and guiding principles of designing biocompatible ILs have been proposed based on both experimental and computational studies. Thermophilic and IL-tolerant LDEs have been explored for increased resistance to harsh environments, highlighting their potential to be used alongside biocompatible ILs. However, further research is needed to elucidate the LDE-

IL interactions for the purpose of lignin valorization. Toward this end, several research areas appear important: 1) utilizing the suite of structural biology tools to explore the LDE-

IL interactions and how they affect the lignolytic capabilities of the biocatalysts, 2) protein engineering strategies can be applied to create novel LDEs that can withstand high concentrations of ILs while maintaining their lignolytic properties, 3) understanding the interaction between IL and LDEs for better product selectivity and developing strategies to prevent lignin condensation in IL; and 4) testing the effectiveness of lignin-IL-enzyme systems on industrial relevant polymeric lignin streams on top of model compounds.

Collectively, this review highlights the tremendous potential of designing LDE-IL pairs for lignin valorization in the IL medium.

1.9 Organization of Chapters

Lignin Ionic Liquids Biocatalyst

Chemicals

Figure 1-9. Project summary

The project summary in Figure 1-9 provides a general overview of the goal of this dissertation. This dissertation seeks to design a system for biological lignin valorization through the use of an aqueous IL and a laccase that can retain high levels of activity in the aqueous IL. The aqueous IL will be used to pretreat biomass and extract lignin into the aqueous phase during pretreatment. The laccase will then be introduced into the lignin- containing aqueous phase to convert the extracted lignin into value-added products.

Chapter 1 of this dissertation provides a general overview of several topics as they relate to the goal of this dissertation.

Chapter 2 of this dissertation seeks to establish a framework that subsequent chapters can follow. This chapter also provides a proof-of-concept biocatalytic lignin

46 valorization system. The interactions between 3 aqueous ILs and a fungal laccase are characterized using several techniques (biocompatibility screening, enzyme kinetics, and molecular docking simulations) that will be used in subsequent chapters. The effect of the

ILs on lignin oxidation are also assessed to provide a foundation upon which the subsequent chapters can improve.

Chapters 3 and 4 of this dissertation seek to produce a laccase that can retain high levels of activity in concentrations of aqueous IL such that the aqueous IL is effective for biomass pretreatment. Chapter 3 characterizes the interactions between aqueous ILs and a hyperthermophilic bacterial laccase. Several single amino acid variants of this laccase are produced in an effort to improve activity in the aqueous IL. Chapter 4 characterizes the interactions between an aqueous IL and a thermophilic fungal laccase. Once again, several variants of this laccase are produced, this time by engineering the enzyme surface charge, in an effort to improve its activity in the aqueous IL.

Chapter 5 of this dissertation focuses on characterizing the interactions between a mesophilic plant laccase and an ionic liquid. The interactions are characterized using the same framework established in the Chapter 1 (biocompatibility screening, kinetics, and molecular docking simulations) along with molecular dynamics (MD) simulations. The goal of the MD simulations is to improve the understanding of the laccase-IL interactions using the atomistic resolution afforded by MD simulations. This final chapter further expands upon the framework used in previous chapters to understand laccase-IL interactions.

The final chapter summarizes all of the work contained within the dissertation and discusses many of the challenges faced during the experiments. This chapter also discusses

47 many of the challenges that could be faced in future studies that seek to achieve a similar goal as this dissertation: characterizing the complex laccase-IL interactions and improving laccase activity in aqueous ILs. Future work, both based on the results of this dissertations and recent developments in the field of biomass pretreatment and lignin valorization, is proposed and discussed as well.

CHAPTER 2. UNDERSTANDING LACCASE-IONIC LIQUID INTERACTIONS TOWARDS BIOCATALYTIC LIGNIN VALORIZATION IN AQUEOUS IONIC LIQUIDS1

2.1 Abstract

Lignin is a crucial component of plant matter; however, it is also largely responsible for the recalcitrance of lignocellulosic biomass when subjected to pretreatment processes.

Lignin is generated in large volumes as a waste product from paper and pulping industry as well as cellulosic biorefineries. As a result, lignin valorization is critical to the successful implementation of cellulosic biofuels. To this end, we investigated interactions between three ionic liquids (AILs) and the lignolytic enzyme laccase toward biocatalytic lignin conversion to aromatic monomers. Laccase exhibited minimal loss of activity in

10% [DEA][HSO4]. Changes in Vmax and Km of laccase with respect to IL concentration indicate [DEA][HSO4] is a non-competitive inhibitor, whereas [Ch][Lys] and

[C2C1Im][OAc] are mixed inhibitors. Docking simulations suggested that [Ch][Lys] and

[C2C1Im][OAc] disrupt residues leading to the active site. Experiments with a β-O-4' linked model dimer revealed laccase in [C2C1Im][OAc] and [Ch][Lys] requires the presence of ABTS to oxidize the β-O-4' linkage, leading to polymerization of the model dimer. Alkaline lignin treated with laccase, ABTS, and the AILs showed few structural changes, although the lignin was partially solubilized and converted to degradation products. The major products obtained from alkaline lignin were vanillin, acetosyringone, syringaldehyde, and acetovanillone. The results of this study provide, for the first time, an in-depth explanation of the interactions between laccase and AILs for the purpose of lignin valorization.

1This chapter in its whole has been published in ACS Sustainable Chemistry & Engineering, 2019, 7 (19), 15928-15938. DOI: 10.1021/acssuschemeng.9b02151.

2.2 Introduction

Lignin is among the most abundant biopolymers found in nature and makes up around one quarter of lignocellulosic biomass 244-246. Lignin is primarily made of three phenylpropanoid subunits synthesized from corresponding alcohol precursors: p-coumaryl alcohol (H), coniferyl alcohol (G), sinapyl alcohol (S) 40, 41. These monolignols are joined via a complex network of linkages including β-aryl ether (β-O-4'), phenylcoumaran (β-5'), and resinol (β-β') as the dominant linkages 43, 45. The nature of the linkages is largely determined by the monolignol content of the biomass which, in turn, is dependent upon the source of the biomass 44. The compositional and structural heterogeneity contributes largely to lignin’s recalcitrance to biological or chemical decomposition. Currently, lignin is generated in large volume as a waste product from the paper and pulping industry as well as a byproduct of the cellulosic biorefinery 1, 247. Developing technologies to convert lignin to value-added products will not only reduce waste generation but also increase revenue for the paper industry as well as future cellulosic biorefineries 3, 4.

Lignin has limited use in its polymeric form. In contrast, the phenolic compounds derived from lignin depolymerization can be used as fuels or building blocks to synthesize chemicals for the food, plastic, and pharmaceutical industries, which are, at present, primarily derived from petroleum 248-250. Lignin deploymerization methods can be categorized into thermochemical, such as high temperature pyrolysis, hydrogenolysis, catalytic oxidation, and biological, using lignolytic enzymes or microbes 5, 6, 8, 78. In general, biological lignin depolymerization can be performed at milder temperatures and pressures compared to thermochemical methods, potentially improving product selectivity via the specificity of the biocatalysts 6, 12. A range of lignin degrading enzymes (LDEs)

50 have been investigated including lignin peroxidases, versatile peroxidases, manganese peroxidases, laccases from white and brown rot fungi, and the newly discovered NAD or glutathione dependent enzymes from certain soil bacteria, such as Sphingobium SYK-6 13-

15. However, application of these lignolytic enzymes in large-scale operations valorizing lignin to aromatic monomers is still hindered due to the high cost of enzyme production and poor yield of aromatic monomers due to the very low lignin solubility (for many of the industrially relevant lignin streams) in the aqueous environment compatible with enzyme activity as well as the poor selectivity of the lignolytic enzymes to produce defined monomers.16, 17.

Most of the known effective lignin solvents (e.g., DMSO, acetone, or alkaline solutions) are detrimental to enzyme activity when added to aqueous solutions 18, 19. In addition, they often require high temperature for complete lignin dissolution, especially when dealing with plant derived native lignins 20, which again can be incompatible with the use of enzymes. Thus, a solvent system with both high lignin solubility and enzyme compatibility is preferred for developing a better biological lignin depolymerization process. Ionic liquids (ILs) are molten salts made of organic cations and anions that are liquid at temperatures <100 oC 127. By selecting the cation and anion, the properties of an

IL can be tuned for a wide range of applications128, 131, 133. Alkylimidazolium based ILs have been extensively studied for biomass pretreatment and lignin fractionation251-253.

Recently, ILs from bio-derived cations (such as choline and ammonium) and anions (such as amino acids and carboxylic acids) have been investigated as lower-cost alternatives to imidazolium-based ILs 167. For example, pretreating switchgrass with 10% cholinium lysinate ([Ch][Lys]) in water resulted in >80% of the lignin being solubilized into the liquid

51 stream 254. Results also suggest that tuning aqueous IL (AIL) mixtures can favor specific products during a catalytic oxidation process 255.

Laccases are a member of the superfamily of multi-copper oxidases (E.C. 1.10.3.2).

First discovered in extracts from the Chinese lacquer tree (Toxicodendron vernicifluum, previously named Rhus vernicifera), laccases have since been found in a variety of organisms including plants, fungi, bacteria, and archaea 84, 85, 90, 91. Laccases utilize a multicopper cofactor for substrate oxidation 86. Rather than requiring H2O2 as in other lignolytic enzymes, laccases use O2 as the final electron acceptor 66, 71, 80. Furthermore, oxidation of small-molecule mediators by laccase, such as 1-hydroxybenzotriazole (HBT) or 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), enables the oxidation of nonphenolic lignin compounds 229, 230. This feature, alongside their inherent lignolytic capabilities and their activity under milder reaction conditions, makes laccases a desirable biocatalyst for lignin upgrading strategies 97, 256. ILs have been found to be capable of supporting and, in a few cases, improving activity and stability of enzymes including laccases 31, 32, 186, 257. T. versicolor laccase activity decreased with increasing IL concentration of the two alkylimidazolium ILs tested, [C2C1Im][EtSO4] and

[C2C1Im][OAc] 183. However, Galai et al. screened T. versicolor laccase activity in 56

AILs and found that 13 of the AILs improved laccase activity; the most notable improvement was seen in choline dihydrogenphosphate ([Ch][H2PO4]) which increased laccase activity 451% 150.

Given the unique properties that certain AILs provide regarding lignin solubility and biocompatibility, the utilization of AIL and LDEs confluently holds great potential for developing a new strategy for lignin extraction and depolymerization. Despite previous

52 exploration of catalysis and enzyme activity in AILs, there is a gap in our understanding of the mechanisms underlying the interactions between lignolytic enzymes and these solvents. This study aims to elucidate the interactions between laccase and ILs, inhibition mechanism and product profiling on such a biocatalytic system with laccase and three

AILs. To achieve this goal, we screened the ILs 1-ethyl-3-methylimidazolium acetate

([C2C1Im][OAc]), [Ch][Lys], and diethylamine hydrogensulfate ([DEA][HSO4]) for their biocompatibility with a fungal laccase. We then investigated enzyme kinetics and the inhibition mechanism using docking simulations with the ILs and the active site. Finally, the IL-laccase system was tested on a β-O-4' linked model dimer (GGE) and commercial kraft lignin in the presence of the mediator ABTS; the resulting residual lignin and lignin- derived products were then characterized. This work provides a better understanding of the inhibition mechanisms of ILs on laccase for developing a biocatalytic lignin valorization process using AILs and LDEs.

2.3 Methods and Materials

2.3.1 Materials Laccase from T. versicolor (0.66 U/mg), 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) (ABTS), alkaline lignin (lot# MKBV5831V), choline hydroxide, L-lysine, and the IL [C2C1Im][OAc] were purchased from MilliporeSigma (St. Louis, MO, USA).

Diethylamine (DEA), sulfuric acid (H2SO4), guaiacylglycerol-β-guaiacyl ether (GGE), tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF), chromium (III) acetylacetonate (Cr(acac)3), and ethyl acetate were purchased from Tokyo Chemical

Industry Co. (Portland, OR, USA). The ILs, cholinium lysinate ([Ch][Lys]), and

53 diethylamine hydrogensulfate ([DEA][HSO4]) were synthesized using the anion and cation precursors according to previously developed protocols 167.

2.3.2 Biocompatibility screening

The biocompatibility of three ILs, [C2C1Im][OAc], [Ch][Lys], and [DEA][HSO4] in their aqueous solution (approximate 1-10% w/v), was screened with T. versicolor laccase. To delineate the effect of pH, the IL solutions were adjusted to pH 5.0 using 1 M sodium hydroxide or 1 M hydrochloric acid prior to testing. Activity was screened with

20 mM sodium citrate buffer, AIL (0, 1, 2, 3, 4, or 10% w/v) and 2 mM ABTS in clear, flat bottom 96-well Costar assay plates (Corning Inc., Kennebunk, ME). Laccase was loaded at 3 concentrations: low (0.6 μg mL-1), medium (1.2 μg mL-1), and high (3.0 μg mL-1). The plate was incubated at 40oC in a SpectraMax M2 plate reader (Molecular Devices,

Sunnyvale, CA). Absorbance readings were taken every 30 seconds for 5 min at a wavelength of 420 nm. Plates were shaken for 5 seconds prior to initial reading and 3 seconds before each reading to ensure well mixing. Oxidation of ABTS in buffer and AILs without laccase were measured as blanks.

2.3.3 Inhibition kinetics Michaelis-Menten curves were generated for different IL concentrations by measuring initial velocities for 11 different ABTS concentrations at a medium loading (1.2

μg mL-1) of laccase. ABTS oxidation was measured using the same method as described in the biocompatibility screening in 96-well plates. The oxidized ABTS was quantified using an absorbance coefficient ε = 36,000 M-1 cm-1 258. A unit of activity was defined as the amount of laccase that oxidizes 1 μmol of ABTS per minute. Laccase initial activity

54 as a function of ABTS concentration ([S]) was fit to the Michaelis-Menten curve, shown in equation 2-1.

V ×[S] V = max (2-1) Km+[S]

These curves were linearized using the Hanes-Woolf method, resulting in equation 2-2.

[S] 1 K = × [S] + m (2-2) V Vmax Vmax

Equation 2 was used to calculate the kinetic coefficients, Km and Vmax of laccase in different

AILs.

2.3.4 Molecular docking simulations In order to determine likely interactions between IL molecules and laccase, docking simulations were performed using the YASARA Structure software package (YASARA

Biosciences GmbH, Vienna, Austria) with the AutoDock Vina plugin 259. Ion structures and parameters were generated in YASARA from SMILES representations and energy minimized. The crystal structure of T. versicolor laccase (PDB ID: 1GYC) was used as a target in the docking simulations 86. Cations and anions were treated as separate ligands, with 25 simulations run for each, and the target laccase was held rigid throughout all simulations. Results of docking were visualized with PyMol (Schrodinger LLC, New

York, NY).

2.3.5 Lignin depolymerization in aqueous IL GGE dimer: Prior to experiments with the model compound, all solutions were adjusted to pH 5.0 with 1 M sodium hydroxide or 1 M hydrochloric acid. Laccase was loaded to a final concentration of 1.2 μg mL-1 and ABTS was added for a final concentration of 2 mM. The GGE model compound was added for a final concentration

55 of 0.5 mg mL-1. AILs were added for final concentrations of 2% (w/v) [C2C1Im][OAc],

1% [Ch][Lys], and 10% [DEA][HSO4]. Reactions were carried out in 1 mL glass tubes under 30 psi O2 pressure at 40oC inside a 100 mL Parr reactor (4593 bench top reactor,

Alloy C276, Parr Instruments, IL). The reactor was purged with pure O2 for 1 minute and then the release valve was closed to maintain O2 pressure. The reaction was allowed to proceed for an additional 2 h.

After reaction, product profiles were analyzed by isocratic HPLC using an 80% water – 20% acetonitrile mobile phase at a flow of 0.5 mL min-1 for 30 minutes through an

Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) equipped with ultraviolet (UV) detector. Separation was achieved through use of a Zorbax Eclipse XDB-

C18 column (5 μm particle size, 150 x 4.6 mm i.d., Agilent Technologies, Santa Clara,

CA) at 25oC. Elution profiles were measured by UV absorbance at 280 nm. The molecule weight distribution of the products was characterized using gel permeation chromatography (GPC) while liquid chromatography–mass spectrometry (LC-MS) was used to identify the major products in GGE experiment as described in the “Lignin

Characterization” section.

Alkaline lignin: The same reaction protocol was used for alkaline lignin samples as for the GGE dimer except that 500 mg of kraft lignin was loaded with the reactants (1.2 μg mL-1 laccase, 2 mM ABTS, AILS) in the Parr reactor with a final reaction volume of 10 mL. After the reaction, residual lignin was precipitated by addition of 20 mL water and centrifugation at 7,000 rpm for 15 minutes. The aqueous fraction was subjected to liquid- liquid extraction using a 1:1 volume of ethyl acetate: liquid fraction, with the extraction

56 repeated 3 times. The ethyl acetate was evaporated in a vacuum oven set at room temperature for 24 h to leave a thick oil. Oil yield was calculated using equation 2-3.

weight of oil Oil Yield (%yield) = × 100 (2-3) weight of initial lignin

The precipitated solids (residual lignin) were washed with 50 mL water three times to remove any excess IL and dried in a convection oven at 45oC for 48 h. Lignin conversion was calculated using equation 2-4.

weight of initial lignin−weight of remaining lignin Conversion (%) = × 100 (2-4) weight of initial lignin

The oil was dissolved in 750 μL MeTHF and analyzed by GC-MS for lignin decomposition products. Identification and quantification of the monomeric products from the depolymerization reaction were performed on an Agilent 7890B GC coupled to a

5977B MS with an HP-5ms (60 m × 0.32 mm) capillary column 260. The temperature program started at 50°C and increased to 120°C at 10°C min−1 with a holding time of 5 min; then it was raised to 280°C at 10°C min−1 with a holding time of 8 min and to 300°C at 10°C min−1 with holding time of 2 min 261. The inlet was held in splitless mode at 250oC using helium as a carrier gas at a flow rate of 1.2 mL min−1. Calibration curves were created using commercially available pure compounds: guaiacol, syringol, vanillin, acetovanillone, and homovanillic acid (Sigma Aldrich, St. Louis, MO). Monomer yields were calculated on basis of mg/g starting lignin.

2.3.6 Lignin characterization Gel Permeation Chromatography (GPC): The weight-average molecular weight

(Mw) and number-average molecular weight (Mn) of both precipitated lignin and lignin products in the liquid phase following reactions were determined by GPC. The precipitated

57 lignin was acetylated via acetobromination 262. Briefly, 10 mg of the lignin was dissolved in 2.5 mL of 92:8 (v/v) acetic acid and acetyl bromide and mixed at 50oC for 2 h. Excess acetic acid and acetyl bromide was evaporated by purging with N2. The acetylated lignin was dissolved in 750 uL THF and analyzed by direct injection into an HPLC system equipped with a Mixed-D PLgel column (5 μm particle size, 300 x 7.5 mm i.d., molecular weight range of 200 – 400,000 μm, Polymer Laboratories, Amherst, MA, USA) at 50°C.

The THF flow rate was 0.5 mL min-1. Eluted molecular species were detected by absorbance at 300 nm, which was calibrated using a polystyrene standards kit (Sigma-

Aldrich, St. Louis, MO). The molecular weight distribution of model compound products was analyzed by direct injection into the HPLC system using the aforementioned method.

Fourier Transform Infrared Spectroscopy (FTIR): Chemical structures of alkaline lignin and treated lignin were determined using a Thermo Nicolet 870 FTIR-ATR spectrometer. Lignin sample spectra were obtained with 64 scans at wavelengths from 700 to 4000 cm-1 with a spectral resolution of 2 cm-1. The raw FTIR spectra were baseline corrected and normalized using Omnic 6.1a software and compared in the range 750-4000 cm−1.

Differential Scanning Calorimetry (DSC): DSC measurements of alkaline and treated lignin samples were performed using a DSC Q20 (TA Instruments, New Castle,

DE) equipped with an autosampler. Analysis was carried out in a temperature range of 40-

500°C at a rate of 10°C min-1 and N2 flow of 50 mL min-1.

1H-13C HSQC NMR: Approximately 100 mg lignin sample along with ca. 1 mg

Cr(acac)3 were dissolved in DMSO-d6/pyridine-d5 (4:1) under mild heat and sonication in a NMR tube until a homogeneous mixture was obtained. NMR spectra were acquired on

58 a 500 MHz JEOL ECZr (Peabody, MA, USA) spectrometer equipped with a 5-mm Royal

Probe. The central DMSO solvent peak was used as an internal reference (C 39.5, H 2.5 ppm). The 1H–13C correlation experiment was an HSQC experiment (JEOL standard pulse sequence ‘hsqc_dec_club_pn’). HSQC experiments were carried out using the following parameters: acquired from 12 to 0 ppm in f2 (1H) with 1024 data points (acquisition time

136 ms), 220 to 0 ppm in f1 (13C) with 256 increments and 150 scans with a 1 s interscan delay. In all cases, processing used typical sine bell (90o) in f2 and squared sine-bell (90o) in f1 (first point 0.5). Volume integration of contours in HSQC plots used Maetrelabs

MestReNova 12.0 (Mac version) software. Integrals were from volume-integration of C/H pairs with similar properties, the α-C/H correlations of A (β-O-4'), B (β-β'), and C (β-5) units. Spectra are displayed in absolute value mode and color coded (in Adobe Illustrator

CC 2018) using literature reference standards 263.

Model Compound Mass Spectrometry: Mass spectrometry, to identify large molecular weight products from GGE experiments, was performed on a Q-Exactive

(ThermoScientific, Waltham, MA, USA) orbitrap spectrometer. The instrument parameters were as follows; a 3.8 kV spray voltage, 225oC inlet temperature, sheath auxiliary gas flow of 2 (arbitrary units), a mass resolution of 140,000, with nitrogen as the

HCD collision gas. A 10 L aliquot of each sample was reduced to dryness under nitrogen.

The dry residue was dissolved in 100 L of a solution containing 50% acetonitrile and 50%

10 mM aqueous sodium hydroxide. The final solution was infused directly into the mass spectrometer by syringe pump at a flow rate of 3 mL per minute. For the HCD tandem mass spectrometry experiments, precursor ions were isolated using a window of 3 m/z, and

59 a normalized collision energy of 25 NCE was applied. Data were processed using Xcalibur software (ThermoScientific, Waltham, MA, USA).

2.4 Results and Discussion

2.4.1 Biocompatibility screening In order to test whether laccase is still active in AIL solution, three ILs were screened for their biocompatibility with T. versicolor laccase (TvL) at concentrations ranging from 1-10% IL in water. Results indicate that [Ch][Lys] and [C2C1Im][OAc] are more inhibitory to TvL with 2% [Ch][Lys] or 5% [C2C1Im][OAc] causing significant loss

(>70%) of TvL activity (Fig. 2-1). In contrast, [DEA][HSO4] is more compatible with TvL, as indicated by the modest loss (<20%) of TvL activity even at 10% concentration. The tested IL concentrations represented a reported range of AIL biomass pretreatment conditions. For example, Liu et al. showed that 10% [Ch][Lys] was highly effective in removing lignin from switchgrass varieties during pretreatment 172, and [C2C1Im][OAc] at

20-50% in water can remove a large portion of the hemicellulose and lignin when compared to pure IL 171, 264. Gschwend et al. have demonstrated that 80 wt% triethylammonium hydrogen sulfate ([TEA][HSO4]) is capable of improving Miscanthus saccharification yields and lignin removal after pretreatment for 15 minutes at 180oC 243.

0.15-0.00 0.32-0.16 0.49-0.33 0.65-0.50 0.82-0.66 1.00-0.83

Both the cations and anions of the AILs could impact their activity. It has been shown that the toxicity of imidazolium-based ILs increases with increasing cation size 265.

[C2C1Im][OAc] at 2% concentration strongly inhibited cellulase activities and growth of common biofuel fermentation microbes such as yeast and E. coli 266 while [Ch][Lys] exhibited low toxicity to yeast and E. coli 170. A more recent study further suggested that cellulase enzymes and yeast can tolerate 5% [Ch][Lys] 267. However, it appeared that

[Ch][Lys] was more inhibitory to the TvL than [C2C1Im][OAc] and [DEA][HSO4]. This case agrees with previous reports showing that ILs with sulfate anions are generally more biocompatible with laccases 149, 268. However, we did not control for the cation between each AIL so the biocompatibility cannot be solely attributed to the anion in this case.

2.4.2 Inhibition mechanisms To better understand the different effects of the three ILs on TvL, we determined their inhibition kinetics using ABTS as a substrate. [C2C1Im][OAc] and [Ch][Lys] are mixed inhibitors of TvL, decreasing Vmax and increasing Km (Table 2-1). This type of inhibition suggests that the ILs do not directly compete with the substrate for the active site, but rather affect the residues surrounding the active site to interfere with substrate binding. [DEA][HSO4] in contrast is a non-competitive inhibitor, not affecting substrate binding but decreasing the catalytic rate of the enzyme. To the best of our knowledge, there have not been any previous studies assessing the inhibitory mechanisms of [Ch][Lys] and [DEA][HSO4] on laccases. The effects of alkylimidazolium ILs on TvL kinetics have been reported, though no studies have been performed with [C2C1Im][OAc]. Tavares et al. found that 3 ILs with a [C2C1Im]+ cation reduced both the Vmax and Km of TvL with

ABTS as a substrate, supporting our finding with [C2C1Im][OAc] 191.

Table 2-1. Kinetic constants of ABTS oxidtion by T. versicolor laccase in AILs

Ionic Liquid Concentration

Ionic Liquid 0% (w/w) 2% (w/w) 5% (w/w)

Vmax (U  mg-1 TvL)

[C2C1Im][OAc] 23.1 ± 1.0a 16.2 ± 2.4b 9.0 ± 0.4c

[Ch][Lys] 23.1 ± 1.0a 7.2 ± 0.3b 5.8 ± 0.3b

[DEA][HSO4] 23.1 ± 1.0a 17.9 ± 0.3b 21.6 ± 0.3a

Km (mM ABTS)

[C2C1Im][OAc] 0.065 ± 0.023a 0.531 ± 0.090b 1.469 ± 0.012c

[Ch][Lys] 0.065 ± 0.023a 1.154 ± 0.124b 2.847 ± 0.300c

[DEA][HSO4] 0.065 ± 0.023a 0.104 ± 0.013a 0.111 ± 0.023a

2.4.3 Docking simulations To gain a deeper insight into the inhibition mechanisms, docking simulations were carried out with the IL cations and anions (Fig. 2-2). A high probability binding site for

[C2C1Im][OAc] and [Ch][Lys] was found near the Type I copper, thus possibly decreasing the catalytic rate by obstructing removal of electrons during substrate oxidation at the Type

I copper. Binding of these ILs at this site may also interfere with ABTS interaction, accounting for the observed increase in Km. In contrast, a binding site for [DEA][HSO4] was not identified, with any significant probability near the active site, consistent with the observed weak noncompetitive inhibition.

(a) (b) (c) (d)

Docking simulation of [C2C1Im][OAc] in this study corroborates a previous study on alkylimidazolium ILs and laccases. MD simulations with alkylimidazolium cations found that cations of varying chain length interacted with the active site via hydrophobic interactions 269. The shorter chain length cations (C2-C6) were capable of diffusing into the active site; longer chain length cations (C8-C10) were limited to binding to Leu residues around the active site entrance. Several studies have shown that enzyme-IL interactions,

63 particularly interactions with the anion, lead to disruption of protein stability as predicted by the Hofmeister series 133, 270, 271. The more chaotropic anions lead to disruption of the water shell around a protein, causing destabilization of the protein. The specific anion effect can only be assessed when the cation is consistent, but [HSO4]- has been observed to be among the most stabilizing of the anions 33.

2.4.4 GGE dimer transformation The activity screening results support the possibility of using TvL in dilute or more concentrated AIL solutions, depending on the IL introduced, to process lignin. The phenolic lignin dimer (GGE) was first tested because the dimer is linked by β-O-4' bond which is the most common interunit linkage in lignin. In absence of ABTS, TvL in 1%

[Ch][Lys] or 2% [C2C1Im][OAc] was inhibited from processing GGE, whereas TvL in 10%

[DEA][HSO4] experiences no inhibition to processing GGE (Fig. 2-3a). Some studies have found that acidic ILs are capable of cleaving the β-O-4' linkage on their own at elevated temperatures 272. The temperature used in this study (40oC) was much lower than previous studies (150oC), so it is unlikely the [DEA][HSO4] itself was responsible for any major changes made to GGE during the reaction. Upon addition of ABTS, TvL is able to process

GGE in the presence of all ILs (Fig. 2-3b).

(a)

(b)

To better assess changes made to GGE during the reaction, the products were further analyzed with GPC and LC-MS. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and the polydispersity index (PDI) of all samples increased during the reaction with GGE (Table 2-2). The samples with an IL present had

65 a lower Mw and higher PDI after 120 minutes compared to the control without AIL. The

Mw of the control after 120 minutes was 1,315 g mol-1, whereas the highest Mw of samples with IL was 1,087 g mol-1 in 1% [Ch][Lys]. The PDI of the No IL control was 1.18 after

120 minutes, whereas the PDI of samples with ILs were 1.57 (1% [Ch][Lys]), 1.69 (2%

[C2C1Im][OAc]), and 1.90 (10% [DEA][HSO4]). The Mn of IL samples decreased between

30 and 120 minutes, which led to the substantial increase in PDI at the end of the reaction.

Molecular weight distribution plots (Fig. 2-4) suggest this is a result of low-molecular weight species appearing in the 120 min samples. To identify any higher-order structures, e.g. dimers, trimers, or oligomers, extracted products were identified with LC-MS.

Table 2-2. Molecular weight distribution of GGE before (0 min), during (30 min), and after (120 min) treatment with TvL, ABTS, and ILs1

Mw (g mol-1) IL 0 min 30 min 120 min No IL 447 941 1315 2% [C2C1Im][OAc] 447 877 975 1% [Ch][Lys] 447 881 1087 10% [DEA][HSO4] 447 993 984 Mn (g mol-1) IL 0 min 30 min 120 min No IL 402 817 1118 2% [C2C1Im][OAc] 402 742 576 1% [Ch][Lys] 402 741 694 10% [DEA][HSO4] 402 762 519 PDI IL 0 min 30 min 120 min No IL 1.11 1.15 1.18 2% [C2C1Im][OAc] 1.11 1.18 1.69 1% [Ch][Lys] 1.11 1.19 1.57 10% [DEA][HSO4] 1.11 1.30 1.90 1Mw: weight-average molecular weight, Mn: number-average molecular weight, PDI: polydispersity index

1.2 1.2 0 min 0 min 30 min 30 min 1.0 120 min 1.0

120 min

s n

0.8 n 0.8

0.6 d 0.6

a a

0.4 0.4

N N 0.2 0.2

0.0 0.0 104 103 102 101 104 103 102 101 Molecular Weight (g mol-1) Molecular Weight (g mol-1)

1.0 120 min 1.0 120 min

s n

0.8 n 0.8

0.6 d 0.6

a a

0.4 0.4

N N 0.2 0.2

0.0 0.0 104 103 102 101 104 103 102 101 Molecular Weight (g mol-1) Molecular Weight (g mol-1)

Figure 2-4. Molecular weight distributions of products after GGE reaction with TvL, ABTS, and a) buffer without IL, b) 2% [C2C1Im][OAc], c) 1% [Ch][Lys], d) 10% [DEA][HSO4]

Direct infusion mass spectrometry experiments using a high-resolution accurate- mass orbitrap mass spectrometer (Q-Exactive) (ThermoScientific, Waltham, MA, USA) in the negative ion mode with sodium hydroxide as a pH modifier were performed. The infusion mass spectra were dominated by three ion peaks with m/z values much greater than the starting GGE dimer. High-resolution, accurate mass spectra and tandem mass spectrometry allowed the probable identity of these species to be defined. The ion peak at m/z 637.1919 was consistent with the coupling of two GGE dimers (GGE dimer MW 320 g mol-1) with the proposed structure shown in Figure S2-2a (calculated m/z 637.1927, observed m/z 637.1918, error 1.3037 ppm). The ion peak at m/z 953.3242 was consistent

67 with the coupling of three GGE dimers with the proposed structure shown in Figure S2-2b

(calculated m/z 953.3237, observed m/z 953.3241, error 0.3765 ppm). Finally, the ion peak at m/z 1073.3454 was consistent with the addition of a G monomer unit to the three

GGE oligomer (m/z 953.3242) with the proposed structure shown in Figure S2c (calculated m/z 1073.3449, observed m/z 1073.3448, error -0.0172 ppm).

Previous studies have shown that laccases facilitate both the depolymerization and polymerization of lignin model compounds. LC-MS analysis of extracted products formed during guaiacylic and syringilic lignin model compound oxidation by Melanocarpus albomyces and Trametes hirsuta laccases showed that GGE homodimerized to form a single product via a 5-5' coupling mechanism, identical to that shown in Figure S2-2a 240.

ABTS was also found to participate in the polymerization of a β-O-4' lignin model dimer and guaiacol when treated with T. hirsuta laccase, although the structures of the products formed during the reaction were not identified 273. Neither of these studies investigated the role of ILs during polymerization of lignin model compounds. We have shown that the presence of ABTS and, possibly the ILs, facilitates the polymerization of GGE into structures larger than the single GGE homodimer reported previously 240. Additionally, the molecular weight distributions of the reaction products suggest that the presence of ILs promotes the formation of low-molecular weight compounds during model compound polymerization/depolymerization.

2.4.5 Depolymerization of alkaline lignin To further investigate the utility of TvL-AIL systems, we tested their ability to depolymerize commercial alkaline lignin with the same reactors used for other studies. In comparison to a control lacking IL (2.12% conversion based on the mass of residual solids),

68 higher lignin conversions were achieved for TvL catalysis of alkaline lignin in AIL systems

(Table 2-3). The highest conversion was obtained with TvL in 10% [DEA][HSO4]

(21.66%), followed by TvL in 1% [Ch][Lys] (17.24%) and TvL in 2% [C2C1Im][OAc]

(11.72%). However, 1% [Ch][Lys] resulted in the highest yield of oil (1.28%) as compared to 2% [C2C1Im][OAc] (0.84%) and 10% [DEA][HSO4] (0.74%). Analysis of extracted products with GC-MS revealed vanillin to be the primary degradation product (consisting

55-70% of the quantifiable monomers), followed by syringaldehyde, acetovanillone, and guaiacol (Fig. 2-5). The discrepancy between lignin conversion and oil yield, which was particularly marked for [DEA][HSO4] (21.66% conversion vs. 0.74% oil yield), could be attributed to less extractable oligomeric compounds formed by the laccase, similar to the condensation of the GGE model compound, that cannot be detected by GC-MS.

Table 2-3. Converstion and yield of alkaline lignin degradation products

Identifiable monomer yield (mg∙g-1) Conversion

Sample (%) Lignin oil yield* (%) Guaiacol Vanillin Syringaldehyde Acetovanillone Total

No IL 2.12 0.74 0.00 1.02 0.59 0.46 2.07

2% [C2C1Im][OAc] 11.72 0.84 0.00 1.15 0.68 0.54 2.37

1% [Ch][Lys] 17.24 1.28 0.54 1.86 1.24 0.98 4.62

10% [DEA][HSO4] 21.66 0.74 0.18 1.45 0.81 0.68 3.12

* Fraction extracted using ethyl acetate

1e+6 No IL 2% [C C Im][OAc] 2 1 2 8e+5 1% [Ch][Lys] 10% [DEA][HSO4] 3 5 6 1

6e+5

t i

s 4

t n I 4e+5

2e+5

0 6 7 8 9 10 16 18 20 22 24

Time (min)

Gel permeation chromatography (GPC) results revealed all treatments led to a small increase in average molecular weight when compared to untreated alkaline lignin (Table

2-4). Additionally, the PDI decreased for all treatments when compared to untreated alkaline lignin. Treatment of alkaline lignin with laccase and 1-hydroxybenzatriazole

(HBT), another mediator, resulted in a reduced PDI and increased molecular weight 113.

GPC of supernatants revealed the presence of higher molecular weight solids in treatments with ILs compared to treatment without IL. These results indicate the laccase-AIL system could facilitate both depolymerization of lignin to a low molecular weight fraction as well as larger products resulting from subsequent polymerization. This polymerization could be limited by a capping agent like phenol, which has been used to improve monomer yields from organosolv olive tree pruning lignin 241, although the amount of phenol required is

70 quite high (2:1 phenol:lignin). The results with the model compound suggest that some

AILs could act as biocompatible capping agents, though this possibility will need to be investigated further in a separate study.

Table 2-4. Processed lignin molecular weight distribution and polydispersity of lignin streams1

Solid Phase Liquid Phase

Sample Mw (g mol-1) Mn (g mol-1) PDI Mw (g mol-1) Mn (g mol-1) PDI

Alkaline lignin 5677 1974 2.88 N/A N/A N/A

No IL 5457 2470 2.21 693 450 1.54

2% [C2C1Im][OAc] 5358 2482 2.16 709 542 1.31

1% [Ch][Lys] 5772 2454 2.35 654 535 1.22

10% [DEA][HSO4] 6170 2068 2.98 667 526 1.27

1Mw: weight average molecular weight; Mn: number average molecular weight; PDI: polydispersity; N/A: not applicable. The chemical and structural properties of the resulting lignin stream were further characterized using FTIR, DSC and 2D HSQC NMR. Analysis of FTIR spectra indicated minimal differences between untreated and treated lignin samples (Fig. S2-3a). The only difference can be seen is in the peak at 1,110 cm-1. This peak corresponds to the aromatic

C-H plane deformation in S-lignin, and is reduced for treated lignin samples when compared to untreated alkaline lignin, which is consistent with the identification of derivatives of S-lignin with GC-MS. The bands at 1,220 cm-1 corresponding to C-C, C-O, and C=C stretching in G-lignin were unchanged for treated lignin when compared to untreated alkaline lignin. Preferential breakdown of S-lignin has been observed before via oxidation of alkaline lignin using transition metal catalysts, so it’s possible that the

71 biocatalyst follows the same trend 254, 274. Thermal profiles as shown by differential scanning calorimetry are similar for all samples of untreated and treated alkaline lignin

(Fig. S2-3b). Exothermic and endothermic regions are indicated by positive and negative values of Hf, respectively. An exothermic region between 50-100oC was observed for all samples. Similarly, an endothermic region between 400-450oC was observed for all samples. The mild reaction conditions, relatively low temperature and low IL concentrations did not appear to result in any significant changes to the lignin structure.

2D HSQC NMR spectra show a small amount of S-lignin in the starting material, however there does not appear to be a substantial reduction in these peaks after processing suggesting only a small portion of the starting material was solubilized during processing

(Fig. 2-6). There appears to be shifts in the ratios of β-O-4' and β-β' linkages in alkaline lignin after treatment, but these changes are not above experimental variation. The most substantial changes were the relative ratios of β-O-4' and β-β' linkages in the alkaline lignin treated with laccase and ABTS (KLA), while alkaline lignin treated with laccase and ABTS in [C2C1Im][OAc] (EKLA) saw relatively minor changes in the HSQC. β-O-4' linkages in alkaline lignin treated without any AILs present were reduced from 54% to 41%. Alkaline lignin treated with aqueous [C2C1Im][OAc] in addition to laccase and ABTS resulted in increased β-O-4' (54% → 56%) and reduced β-β' (29% → 26%) linkages. Previously, Li et al., reported the water-insoluble fraction of alkaline lignin after treatment with laccase and the mediator HBT had increased β-O-4' (61.5% → 68.5%) and reduced β-β’ (5.8% →

5.7%) content compared to the starting material, suggesting the presence of less branched lignin structure when compared to the starting material 113.

It is possible that there are IL-specific effects that altered the changes in linkage ratios seen after treatment with laccase and ABTS. The relative content of ether bond was based on NMR analysis of the residual solids rather than the liquid products. Results suggest that the relative ether bond content stayed unchanged or slightly increased in the

73 residuals solids. The relatively small increase in ether bonds from KLA to EKLA is likely negligible, however, it is possible that dehydrogenation reactions of the β-β linkages rendered a small amount of these linkages without C-H groups at the Cα-H and Cβ-H position causing a small increase in the β-O-4 content. A similar phenomenon has been observed when kraft lignin was treated with T. versicolor laccase and 1- hydroxybenzotriazole hydrate (HBT), another mediator 275. The insoluble lignin fraction in their study had a higher ether bond content than the starting material. It is also noted that these numbers should not be over-interpreted as HSQC NMR is a semi-quantitative method (due to variations in T2 relaxations and 1JC-H constants) for quantifying the linkages in their relative ratios. It is surprising that S-lignin was identified in the raw and treated kraft lignin samples. We encountered the similar problem in our previous work 255 where a small quantity of S-lignin was observed in the original Sigma-Aldrich alkaline lignin.

While the majority of the lignin is likely softwood, it is possible that the lignin streams used for the Sigma Aldrich kraft lignin are made of a small amount of other species (e.g., hardwood).

The source of the lignin itself could play a role in the depolymerization since the alkaline (kraft) lignin has been reported to be heterogeneous and condensed 276. To fully understand these effects, a less recalcitrant lignin source may be tested as the commercial alkaline/kraft lignin tends to be more condensed (more C-C bonds) than the native lignin in plants 277. Due the intrinsic complexity of real lignin (alkaline lignin in this study) and the limitations of current lignin analytical approaches, many of the proposed lignin transformation mechanisms are based lignin model compounds such as monolignols and

GGE dimmer. In this work, we were able to identify the major products in GGE experiment

74 using LC-MS and propose possible reaction pathways. The lignin transformation mechanism with GGE dimmer could be partially transferable to the alkaline lignin substrate; however, to fully reveal the alkaline lignin transformation mechanism is still very challenging based on current lignin analytical techniques.

Summarizing the results of GGE dimer and alkaline lignin depolymerization, it was found that TvL in aqueous [C2C1Im][OAc] and [Ch][Lys] require the presence of the mediator ABTS to process the β-O-4' lignin model compound. Alternatively, TvL in

[DEA][HSO4] does not require ABTS to process with the compound. GPC and LC-MS analysis of the reaction products indicates that GGE was initially polymerized into dimers, trimers, and oligomers of the model compound. As the reaction proceeded, the ILs limited this polymerization and appeared to promote the formation of small-molecular weight products. TvL in AIL promoted both the depolymerization and polymerization of the alkaline lignin, and the improved monomer yield could be attributed to the cleavage of the

β-O-4' linkages. Obtaining high lignin monomer yield is important from lignin valorization standpoint. As the main focus of our study is to understand the interactions among lignolytic enzymes, lignin and IL solvent, we were not set to optimize lignin depolymerization; indeed, we picked relatively mild reaction conditions to better probe the initial stage of the reaction and catch the product profile. Further optimizing the reaction conditions would likely improve the yield of aromatic monomers from enzymatic lignin depolymerization. Native plant lignin can be used as a substrate in future studies, in place of the highly condensed kraft lignin used in this study. Additionally, other laccases or laccase-engineering strategies can be employed to develop a biocatalyst with resistance to higher ionic liquid concentrations or operating temperatures.

2.5 Conclusions

This study provides insight into the interactions between TvL and AILs. The effects of three AILs with distinct properties on TvL activity, inhibition mechanism, and oxidative capability were investigated. Results indicate that [DEA][HSO4] did not impede the ability of TvL to oxidize ABTS, whereas [C2C1Im][OAc] and [Ch][Lys] were inhibitory at all but the lowest concentrations. Enzyme kinetics and docking simulations suggest that

[C2C1Im][OAc] and [Ch][Lys] may bind at the entrance to the active site to interfere with substrate interaction and catalysis, while [DEA][HSO4] likely does not. Both depolymerized and polymerized products were observed when the TvL enzyme was applied to a model GGE dimer and alkaline lignin in AILs. The reaction conditions still need to be optimized by using different lignins and thermophilic laccases with better resistance to

AIL inhibition and higher temperatures. Collectively, this study provides a better understanding of the interactions between a fungal laccase and AILs and serves as a foundation for developing lignin valorization via biocatalysis in AILs.

2.6 Author Contributions

Joseph Stevens, Lalitendu Das, Justin Mobley, Shardrack Asare, Bert Lynn, David

Rodgers, and Jian Shi conceptualized the work, designed the experiments, and analyzed the data. Shardrack Asare and Bert Lynn performed the LCMS experiments, generated the figures, and wrote the experimental portions. Justin Mobley performed the HSQC NMR experiments, generated the figures, and wrote the experimental portions. Joseph Stevens performed all other experiments and wrote all other portions of the manuscript.

2.7 Acknowledgements

The authors acknowledge the National Science Foundation under Cooperative

Agreement No. 1355438 and 1632854 (2016 – 2020). This is a publication of the Kentucky

Agricultural Experiment Station and is published with the approval of the Director. This work is also supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch-Multistate project under accession number 1003563 (2018 – 2023).

2.8 Supplemental Information

(a)

(b)

(c)

Figure S 2-1. Hanes-Woolf curves of T. versicolor laccase in a) [C2C1Im][OAc], b) [Ch][Lys], and c) [DEA][HSO4]

(a) (b)

(c)

(a)

(b)

(a)

(b)

Figure S 2-4. Molecular weight distribution of a) solid-phase and b) liqui-phase alkaline lignin after treatment with laccase, AILs, and ABTS

(a) (b) (c) (d) (e)

r A

CHAPTER 3. CHARACTERIZATION AND ENZYME ENGINEERING OF A HYPERTHERMOPHILIC LACCASE TOWARD IMPROVING ITS ACTIVITY IN IONIC LIQUID1

3.1 Abstract

Ionic liquids (ILs) are organic salts molten at room temperature that can be used for a wide variety of applications. Many ILs, such as 1-ethyl-3-methylimidazolium acetate

([C2C1Im][OAc]), have been shown to remove a significant fraction of the complex biopolymer lignin from biomass during pretreatment. Valorizing lignin via biological pathways (e.g. enzymes) holds promise but is limited by the low biocompatibility of many

ILs used for pretreatment. The discovery of thermostable enzymes and the application of enzyme engineering techniques have yielded biocatalysts capable of withstanding high concentrations of ILs. Converting lignin from a waste product to value-added chemicals is vital to the success of future cellulosic biorefineries. To that end, we screened the activity of the lignolytic enzyme laccase from a hyperthermophilic bacterium (Thermus thermophilus) in aqueous [C2C1Im][OAc]. Despite the thermophilicity (Topt > 90 ºC) of this laccase, significant activity loss (>50%) was observed in only 2% (w/v)

[C2C1Im][OAc]. Kinetics studies show that the IL can bind to the free enzyme and the enzyme-substrate complex. Docking simulations suggest that the cation favors binding to a region close to the active site. We then used a rational design strategy to improve the activity of the laccase in [C2C1Im][OAc]. A total of 8 single amino acid mutations were made; however, there were no significant improvements in the activity of the mutants in

[C2C1Im][OAc] compared to the wild type. The results of this study shed light on the

1This chapter in its whole has been accepted for a publication in Frontiers in Energy Research, 2020. DOI: 10.3389/fenrg.2020.00158.

83 complex nature of enzyme-IL interactions and the challenges faced when designing a biological lignin valorization strategy.

3.2 Introduction

Lignin is a complex biopolymer that makes up ~30% of the terrestrial plant biomass on earth 40, 244. It is made primarily of 3 phenylpropanoid subunits, syringyl (S), guaiacyl

(G) and p-hydroxyphenyl (H) 41, 43. These subunits are joined via complex networks of ether and carbon-carbon linkages with the β-aryl ether (β-O-4), phenylcoumaran (β-5) and pinoresinol (β-β) linkages being the most common 45, 278. Additionally, the ratio of subunits is not homogeneous across all sources of biomass 44. Due to this monomeric and structural heterogeneity, lignin is recalcitrant to biological and chemical depolymerization processes for production of aromatic compounds at high selectivity and yield. Lignin is currently generated as waste in large volumes in the paper and pulping industry and in cellulosic biofuel production 1, 247. Converting lignin to value-added products will not only reduce waste production, but will also add value to the paper and pulping industries and future cellulosic biorefineries 3, 4.

Although applications of lignin are limited in its polymeric form, the phenolic products derived from lignin can be used for production of fuels or as building blocks for chemicals used in the plastics, food, or pharmaceutical industries 248, 249. Broadly speaking current methods used for converting lignin to value-added products can be divided into thermochemical, including pyrolysis, hydrogenolysis, and catalytic oxidation, and biological, including the use of lignolytic enzymes or microbes to break down lignin 6, 279,

280. Biological methods utilize milder reaction conditions, with associated cost and safety

84 advantages, and can potentially improve the yield and selectivity of lignin breakdown products due to the inherent efficiency of biocatalysts 6, 12. Several lignolytic pathways have been identified in biomass-degrading fungi and bacteria. These include the heme peroxidases (versatile, lignin, and manganese), laccases, and the recently discovered NAD or glutathione-dependent enzymes from the soil bacterium Sphingobium SYK-6 13, 15, 281.

The application of these enzymes in an industrial setting has been limited by several factors, including the high cost of enzyme production, the poor solubility of lignin in a biocompatible solvent, and low selectivity of lignin-derived monomers by the biocatalyst

282, 283.

Many of the known lignin solvents, e.g. dimethyl sulfoxide (DMSO) or alkaline solutions, reduce or eliminate enzyme activity 18, 19. Furthermore, these solvents require the use of high temperatures for enhanced lignin solubilization 284, which also eliminates enzyme activity. Therefore, a solvent that is both capable of solubilizing lignin and supporting enzyme activity is required for developing biological lignin valorization strategies. Ionic liquids are molten, organic salts (Tm < 100 ºC) that can adopt a variety of properties by selecting the appropriate cation and anion 127, 128, 131, 133. Many alkylimidazolium ILs, particularly 1-ethyl-3-methylimidazolium acetate

([C2C1Im][OAc]), have been extensively studied for use in biomass pretreatment at relatively low temperatures 251-253. Due to the high cost of alkylimidazolium ILs, recent efforts have focused on driving down the cost of ILs by using inexpensive, bio-derived cations (e.g. choline and ammonium) and anions (e.g. amino acids and carboxylic acids) and increasing the fraction of water during pretreatment 167, 285. ILs are favored over other biomass pretreatment solvents due to their ability to reduce biomass recalcitrance, increase

85 yield of fermentable sugars from enzymatic hydrolysis, and solubilize high fractions (5 –

20%) of cellulose 171, 286, 287.

Laccases are a member of the superfamily of multi-copper oxidases (E.C. 1.10.3.2).

First discovered in the extract from the Japanese lacquer tree Toxicodendron vernicifluum, laccases have since been identified in fungi, bacteria, and archaea 84, 85, 90, 91, 93. Laccases differ from the other major lignolytic enzymes found in nature, the heme peroxidases, as they are copper containing enzymes that utilize molecular oxygen, as opposed to stronger oxidants, in their catalytic mechanism 71, 80, 86. The mild reaction conditions required for laccase activity have made them attractive targets for use in a variety of biotechnological applications, including lignin valorization 288. Additionally, laccases can oxidize nonphenolic lignin compounds when coupled with small molecule mediator compounds, like 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 1- hydroxybenzotriazole (HBT) 97, 256, 289. Recently, some ILs, including those investigated for use in biomass pretreatment have been found to be biocompatible with the activity of laccases. Several alkylimidazolium ILs were found to be biocompatible with the activity of a mesophilic fungal laccase from Trametes versicolor (TvL) at different concentrations in water; over 80% of laccase activity remained in 50% 1-ethyl-3-methylimidazolium ethylsulfate ([C2C1Im][EtSO4]) 193, for example. However, this IL was shown to be the most biocompatible of the 4 aklylimidazolium ILs screened in the study. TvL activity was reduced by 50% in less than 40% 1-butyl-3-methylimidazolium chloride ([C4C1Im][Cl]),

1-hexyl-3-methylimidazolium bromide ([C6C1Im][Cl]), and 1-decyl-3-methylimidazolium chloride ([C10C1][Cl]) 290. Additionally, we showed in a previous study that the same laccase loses >50% activity in as little as 10% (w/v) [C2C1Im][OAc] and 1% (w/v)

[Ch][Lys], far below the IL concentrations required for effective biomass pretreatment 291.

If laccases are to be used as biocatalysts for lignin valorization, they must be able to withstand higher concentrations of ILs.

Recent strategies to improve enzyme activity and stability in ILs can be classified into 3 categories: chemical, physical, and biotechnological modifications. By reducing the ratio of positively charged to negatively charged surface residues via acetylation or succinylation, the activity and stability of enzymes can be improved in ILs 202, 292.

Immobilization of laccase from Myceliophthora thermophila (MtL) on glyoxyl-agarose beads improved enzyme stability in aqueous concentrations (0 – 75%) of [C2C1Im][EtSO4]

198. Single and multiple amino acid mutations have been used to improve the activity of laccases in alkylimidazolium ILs 265, 293, 294. Recently, several thermotolerant organisms have been found to produce laccases or laccase-like enzymes. Due to the ability of these organisms to survive in extreme conditions of extreme temperature, pH, and salinity they could prove to be useful sources of biocatalysts for industrial applications such as lignin valorization in aqueous ILs 199. Bacillus subtilis produces a laccase-like enzyme (Topt > 70

ºC) that is capable of oxidizing canonical laccase substrates such as syringaldazine and

ABTS in the presence of several alkylimidazolium chloride ILs 91, 265. The laccase produced by Thermus thermophilus (Topt > 90 ºC), is the most thermotolerant laccase identified to date 93. However, there has been no work to characterize the behavior of the

T. thermophilus laccase in ILs.

In this study we sought to characterize the interactions between the IL

[C2C1Im][OAc] and the laccase produced by T. thermophilus (TtL). To do this, we first screened the biocompatibility of [C2C1Im][OAc] with recombinantly produced TtL. We

87 then used enzyme kinetics and docking simulations to better understand how

[C2C1Im][OAc] interacts with TtL. Finally, we used a rational design approach to make a series of single amino acid mutations in an effort to improve the activity of TtL in

[C2C1Im][OAc]. This study expands our understanding of the effects ILs can have on laccases and highlights some of the challenges faced when designing a biological lignin valorization strategy.

3.3 Materials and Methods

3.3.1 Materials

ABTS tablets were purchased from Thermo Fisher and the IL [C2C1Im][OAc] was purchased from MilliporeSigma. The TtL sequence was codon optimized for E. coli expression and inserted into pET32a(+) vector at NcoI and BamHI sites by GenScript

(Piscataway, NJ) to make the pET32aTtL expression plasmid. Mutant primers were purchased from Eurogentec (Seraing, Belgium). The PfuUltra High-Fidelity DNA

Polymerase, buffer, and dNTP mixture were purchased from Agilent.

3.3.2 Expression of T. thermophilus Laccase

E. coli Rosetta (DE3) cells transformed with pET32aTtL were cultured in 1 L LB broth containing ampicillin (50 µg/mL) and chloramphenicol (34 µg/ mL) with shaking at

37 ºC until the optical density at 600 nm reached 0.5. The laccase expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was transferred to a 16 ºC shaker incubator overnight. Cells were collected by centrifugation and resuspended in 40 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and stored at -20 ºC until purification.

3.3.3 Purification and copper-induced laccase folding

The frozen cells were thawed and subsequently sonicated on ice. The cell debris was removed by centrifugation at 15,000 rpm for 50 min at 8 ºC. The supernatant was heated for 1 h at 60 ºC to precipitate any heat sensitive protein, centrifuged for 30 min at

15,000 rpm, then filtered and loaded onto a column with 2.5 mL cobalt resin equilibrated with lysis buffer. The bound protein was washed with 15 mL lysis buffer followed by 5 mL wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove any protein bound to the column by non-specific binding. Finally, the protein was eluted with 5 mL elution buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0).

After purification, the native, copper-containing enzyme was produced according to a previously used protocol 295. The eluted protein was dialyzed twice against 1 L of a copper containing buffer (20 mM sodium acetate, 0.1 mM CuSO4, pH 6.0) for 12 h at 4 ºC.

The protein was then dialyzed twice against 1 L of a copper-free buffer (20 mM sodium acetate, pH 4.5) for 12 h at 4 ºC to remove any remaining copper from the first dialysis step. Precipitate was removed by centrifugation at 15,000 rpm for 20 min at 4 ºC. The final protein concentration was determined by measuring the absorbance at 280 nm and using the molar absorption coefficient Ɛ280 = 46,065 M-1 cm-1.

3.3.4 Biocompatibility screening

The biocompatibility of [C2C1Im][OAc] in aqueous solution (approximately 1-10% w/v), was screened with recombinant TtL. To reduce the effect of pH, the IL solution was adjusted to pH 4.5 using 1 M hydrochloric acid prior to testing. Activity was screened with

20 mM sodium acetate buffer (pH 4.5), 0.1 mM CuSO4, IL (0, 1, 2, 3, 4, or 10% w/v) and

2 mM ABTS in quartz cuvettes with a 1 cm path length. Absorbance readings were taken continuously for 10 min at 61.5 ºC. Oxidation of ABTS in buffer and AILs without laccase were measured as blanks. The relative activity of TtL in ILs relative to buffer was calculated using equation 3-1.

퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푖푛 퐼퐿 푅푒푙푎푡푖푣푒 푎푐푡푖푣푖푡푦 = (3-1) 퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푖푛 푏푢푓푓푒푟

3.3.5 Inhibition kinetics

Michaelis-Menten curves were generated for IL concentrations (0, 1, 2, 3, and 4% w/v) by varying the concentration of ABTS (0.75 – 2.00 mM) and measuring initial velocities. ABTS oxidation was measured using the same method as described in the biocompatibility screening. The oxidized ABTS was quantified by measuring the absorbance at 420 nm and using the absorbance extinction coefficient Ɛ420 = 36,000 M-1 cm-1 295. An activity unit was defined as 1 µmol ABTS oxidized in 1 min (µmol

ABTS/min). Initial velocity (V) as a function of ABTS concentration ([S]) was fit to the

Michaelis-Menten curve, shown in equation 3-2.

푉 ∗[푆] 푉 = 푚푎푥 (3-2) 퐾푚+[푆]

The kinetic coefficients were calculated by plotting equation 3-2 in Sigma Plot and calculating Km and Vmax using the ligand binding curve fitting algorithm.

3.3.6 Docking Simulations

The 3D structures and PDB files of the ligands were prepared in YASARA

Structure (YASARA Biosciences GmbH, Vienna, Austria) using the SMILES strings obtained from PubChem. The PDBQT files of the ligands and TtL (PDBID: 2XU9) were

90 prepared with AutoDock (version 4.2.6, MGLTools, La Jolla, CA). Charges for the coppers were added by manually editing the TtL PDBQT file. AutoGrid parameters were as follows: space value of 0.375Å, (x, y, z) grid centered at (-28.148, -27.636, 14.016), and grid size of 126 in all directions. AutoDock parameters were as follows: Lamarckian GA,

1,000 genetic algorithm runs, and 25,000,000 max eval size. [C2C1Im]+ and [OAc]- were docked separately to both unbound TtL and TtL with ABTS bound to the active site. The structure of TtL with ABTS in the active site was prepared in PyMol by aligning TtL with

B. subtilis CotA containing ABTS in the active site (PDBID: 1OF0).

3.3.7 Site-directed mutagenesis of TtL

Primer design and site-directed mutagenesis of TtL was carried out using the

QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA, USA). Table

S1 lists the primers used in this study. The WT TtL plasmid (pET32aTtL) was used as the template for all PCR reactions. The PCR reaction mixture (50 uL) contained 5 ng of DNA template, 125 ng each of forward and reverse primers, 1 uL dNTP mix, and 2.5 U of polymerase. Reactions were conducted using the following sequence heated for 30 seconds at 95 ºC followed by 16 cycles of heating (95 ºC, 30 sec), annealing (55 ºC, 1 min), and elongation (68 ºC, 7.3 min). Following PCR, DpnI restriction enzyme was added to digest the methylated template plasmid. Plasmid sequences were verified by Eurofins Genomics

(Ebersberg, Germany). Expression, purification, and biocompatibility screening of TtL mutants were carried out using the previously described protocols in sections 3.3.3 and

3.3.4.

3.4 Results

3.4.1 WT TtL Biocompatibility Screening

To determine the biocompatibility of [C2C1Im][OAc], we screened the activity of

TtL in a range of IL concentrations in water. Figure 3-1A shows the activity of TtL in different concentrations of [C2C1Im][OAc] relative to the activity in buffer. A 50% reduction of TtL initial velocity in buffer was seen in 2% [C2C1Im][OAc], in contrast with recent studies of thermophilic enzymes in [C2C1Im][OAc]. A thermophilic cellulase (Topt

= 80 ºC) retained 40% initial activity in 20% [C2C1Im][OAc], while a hyperthermophilic cellulase (Topt > 95 ºC) retained 90% initial activity in 20% [C2C1Im][OAc] 199. The range of IL concentrations tested is relevant to previous studies investigating the effect of water on biomass pretreatment with ILs. When studying the role of water during pretreatment with [C2C1Im][OAc], it was found that pretreatment efficacy is reduced when more than

50% water is present 171.

(A) 1.0 (B) 0,4 0.9 0% [C2C1Im][OAc] 1% [C2C1Im][OAc]

0.8 )

) 2% [C2C1Im][OAc]

1 -

- 0,3

g g y 0.7 3% [C C Im][OAc]

t 2 1

v i

U 4% [C C Im][OAc]

U t ( 2 1

0.6 (

v c

0.5 i e

t 0,2

0.4 V

a i

R t

0.3 i

p I

S 0,1 0.2

0.1

0.0 0,0 0 2 4 6 8 10 0,0 0,5 1,0 1,5 2,0 [C2C1Im][OAc] (%w/v) [ABTS] (mM)

There are several factors that mediate the IL’s effect on enzyme activity. In the case of alkylimidazolium ILs, the ability of the anion to disrupt the water shell around the

92 protein (chaotropicity) and the alkyl chain length of the cation are thought to be largely responsible for the IL biocompatibility. The activity of TvL was relatively unaffected by

50% 1-ethyl-3-methylimidazolium ethyl sulfate ([C2C1Im][EtSO4]), but was severely inhibited in ILs with longer alkyl chain length cations ([C4C1Im]+, [C6C1Im]+, and

[C10C1Im]+) and more chaotropic anions (Cl- and Br-) 193. An increase in the alkyl chain length of the alkylimidazolium chloride ([CnC1Im][Cl]) ILs (C2 -> C4 -> C6) decreased the activity of MtL, followed by a greater decrease in activity when the alkyl chain length was further increased (C6 -> C10) 269. Therefore, the short alkyl chain length [C2C1Im]+ cation does not play a significant role in TtL inhibition. Rather, it is the chaotropic [OAc]- anion that causes the inhibition in low IL concentrations.

3.4.2 Enzyme Kinetics

To further understand the interaction between [C2C1Im][OAc] and TtL, we carried out a more in depth investigated of enzyme kinetics using ABTS as a substrate (Figure 3-

1B). With increasing concentrations of [C2C1Im][OAc], Km increased while the kcat decreased (Table 3-1). This suggests that [C2C1Im][OAc] is a mixed competitive and noncompetitive inhibitor of TtL. Such behavior could be the result of [C2C1Im][OAc] interaction remote from the active site that decreases both substrate binding and catalysis, or the IL could be binding both at the active site and a remote location. As indicated in

Table 3-1, kcat was lowered from 0.42 s-1 in 0% [C2C1Im][OAc] to 0.29 s-1 in 1%

[C2C1Im][OAc]. However, a smaller reduction in kcat was observed between 1%

[C2C1Im][OAc] (0.29 s-1) and 4% [C2C1Im][OAc] (0.20 s-1). In contrast, Km steadily increased between 0% [C2C1Im][OAc] (0.52 mM ABTS) and 4% [C2C1Im][OAc] (1.90 mM ABTS). The different Km and kcat variations with IL concentration support the

93 presence of 2 major binding sites. The first is a high affinity noncompetitive binding site that lowers the enzyme activity and is almost fully occupied in 1% [C2C1Im][OAc]. The second is a low affinity competitive binding site that likely overlaps with the substrate binding site, which lowers the substrate affinity of the enzyme.

Table 3-1. Kinetic constants of the WT TtL in different concentrations of [C2C1Im][OAc]. Values shown are mean ± standard error (SE) of 3 experiments.

[C2C1Im][OAc] Km Vmax kcat kcat/Km (%w/v) (mM ABTS) (U mg TtL-1) (s-1) (s-1 mM-1)

0% [C2C1Im][OAc] 0.52 ± 0.05 0.34 ± 0.01 0.41 ± 0.01 0.78

1% [C2C1Im][OAc] 0.76 ± 0.08 0.24 ± 0.01 0.29 ± 0.01 0.38

2% [C2C1Im][OAc] 0.96 ± 0.10 0.22 ± 0.01 0.27 ± 0.01 0.28

3% [C2C1Im][OAc] 1.34 ± 0.30 0.20 ± 0.02 0.24 ± 0.03 0.17

4% [C2C1Im][OAc] 1.90 ± 0.33 0.17 ± 0.02 0.20 ± 0.02 0.11

Previous kinetics work with laccases and ILs have shown that ILs can affect both

Km and kcat of several laccases. Several [C2C1Im]+ ILs lowered the activity and increased the substrate affinity of MtL by uncompetitive inhibition 191. In contrast, the IL

[C2C1Im][Cl] was found to be a competitive inhibitor of the same laccase 269. The activity of TvL and MtL are increased by the ILs choline dihydrogen phosphate ([Ch][H2PO4]) and

[C2C1Im][EtSO4], respectively; enzyme kinetics suggest these ILs decrease substrate affinity (increase Km) but increase enzyme activity (increase Vmax) via an uncompetitive inhibition mechanism 150, 198.

3.4.3 Docking simulations

To gain insight into the possible binding locations of [C2C1Im][OAc] on the surface of TtL, we performed docking simulations with AutoDock. Figures 3-2A and 3-2B show

94 the most populated IL docking poses to the surfaces of [E] and [E][S], respectively, after

1,000 docking simulations with AutoDock. Neither the cation nor the anion binds close enough to the coppers to suggest that the IL disrupts the residues that coordinate and stabilize the coppers. The binding locations of the cation and the anion to the surface of

TtL is affected by the presence of ABTS in the active site. The binding location of the cation to [E] (Fig. 3-2A) appears to overlap with ABTS bound in the active site, leading to the displacement of the cation when docked to [E][S] (Fig. 3-2B). This suggests that the cation directly competes with the ABTS for the active site which leads to the increased Km seen in the enzyme kinetics. Similarly, there is a shift in the anion binding site when docked to [E] vs [E][S]; however, the anion binding location does not overlap with the

ABTS. This suggests that the anion plays a role decreasing kcat but may also contribute to the change in Km, since substrate affinity would be decreased by the cost of displacing the anion.

(A) (B)

Docking results from this work are consistent with previous simulations examining docking locations of several ILs to TvL and docking of alkylimidazolium cations to MtL.

Highly populated binding sites were found close to the T1 copper when [C2C1Im][OAc],

[Ch][Lys], and diethylamine hydrogen sulfate ([DEA][HSO4]) were docked to TvL 291.

When docked to MtL, short alkyl chain length cations (C2 – C6) diffused into the active site whereas the long chain length cations (C8 – C10) interacted with leucine residues around the active site 269. However, the activity of MtL is increased in the presence of

[C2C1Im][EtSO4] 198, therefore IL docking close to the T1 copper does not imply the IL will inhibit laccase activity.

3.4.4 Rational Design of TtL

The mutations were chosen based on several approaches aimed at improving enzyme activity and stability in ILs. Recent directed evolution and semi-rational design studies have identified the L1 loop in TvL as a key target for laccase engineering. A triple mutant targeting the alanines on the L1 loop in TvL, A310D/A312P/A318R, retained 21% initial activity in 40% [C2C1Im][EtSO4], compared to 0% remaining activity for the WT laccase 294. The L1 loop in TtL presents 3 alanines (Ala320, Ala332, and Ala341) (Fig. S3-

1A). The mutations of these alanines were chosen based on recent chemical modifications of enzymes in ILs. Reducing the ratio of positively charged amines to negatively charged acids on the enzyme surface by acetylation or succinylation improved their activity and stability in ILs 202, 292. For this reason, the Ala320, Ala332, and Ala341 from TtL L1 loop were mutated to: A320D/E, A332D/E, and A341D/E. Although we could have selected additional mutations based on previous studies with TvL (e.g. A320P or A332R), we chose the mutations to aspartate and glutamate in order to limit our design space. Additionally, this strategy of engineering the L1 loop is based on studies with a fungal laccase; however, we felt it appropriate to apply this strategy to the bacterial laccase used in this study. We

96 also made mutations to Glu170 based on previous work improving the activity of a laccase- like enzyme from B. subtilis in ILs. The catalytic efficiency (kcat/Km) of B. subtilis HR03 mutants E188F and E188Y was increased compared to the WT in several [CnC1Im][Cl] ILs

265. Through sequential alignment, we identified a homologous glutamate residue in TtL,

Glu170 (Fig. S3-1B), which was mutated to both tyrosine (E170Y) and phenylalanine

(E170F).

Aiming to improve the activity of TtL in [C2C1Im][OAc], we made 8 single amino acid mutations to TtL. With the exception of the E170Y mutant at 0% IL, none of the mutations significantly improved the activity of TtL in buffer or [C2C1Im][OAc] when compared to WT (Table 3-2). The A320 mutations significantly decreased the activity in buffer and some IL concentrations, as did the E170F mutation at 2% [C2C1Im][OAc]. There were no significant differences from WT when normalizing each variant to its activity in

0% IL, except for the lower activity of the E170Y mutant in 2% [C2C1Im][OAc], indicative of its generally greater sensitivity to low [C2C1Im][OAc] concentrations.

Table 3-2. Activity of WT and mutants relative to activity in buffer in different concentrations of [C2C1Im][OAc]. Values shown are mean ± standard deviation (SD) of 3 experiments1.

Relative to Activity in Buffer

IL (%w/v) WT A320D A320E A332D A332E A341D A341E E170F E170Y

0 1.00a 1.00a 1.00a 1.00a 1.00a 1.00a 1.00a 1.00a 1.00a

1 0.70 ± 0.04a 0.72 ± 0.07a 0.75 ± 0.05a 0.70 ± 0.03a 0.64 ± 0.01a 0.72 ± 0.04a 0.70 ± 0.03a 0.71 ± 0.01a 0.58 ± 0.04a

2 0.49 ± 0.03a 0.42 ± 0.05a 0.44 ± 0.05a 0.45 ± 0.02a 0.42 ± 0.04a 0.45 ± 0.05a 0.43 ± 0.03a 0.39 ± 0.02a 0.33 ± 0.03b

3 0.38 ± 0.01a 0.41 ± 0.05a 0.42 ± 0.04a 0.40 ± 0.03a 0.40 ± 0.04a 0.38 ± 0.03a 0.35 ± 0.04a 0.37 ± 0.04a 0.29 ± 0.01a

4 0.28 ± 0.06a 0.26 ± 0.04a 0.31 ± 0.06a 0.32 ± 0.01a 0.31 ± 0.05a 0.28 ± 0.04a 0.28 ± 0.04a 0.32 ± 0.05a 0.22 ± 0.02a

10 0.09 ± 0.02a 0.09 ± 0.02a 0.09 ± 0.02a 0.09 ± 0.00a 0.09 ± 0.03a 0.10 ± 0.01a 0.09 ± 0.01a 0.09 ± 0.02a 0.11 ± 0.00a

Relative to WT Activity in Buffer

0 1.00a 0.85 ± 0.00b 0.73 ± 0.03b 0.96 ± 0.01a 0.94 ± 0.06a 1.00 ± 0.05a 1.07 ± 0.04a 0.94 ± 0.04a 1.23 ± 0.04b

1 0.70 ± 0.04a 0.58 ± 0.05a 0.52 ± 0.06b 0.67 ± 0.02a 0.63 ± 0.04a 0.72 ± 0.04a 0.75 ± 0.03a 0.68 ± 0.05a 0.71 ± 0.04a

2 0.49 ± 0.03a 0.33 ± 0.04b 0.32 ± 0.03b 0.43 ± 0.02a 0.40 ± 0.04a 0.45 ± 0.05a 0.46 ± 0.03a 0.37 ± 0.02b 0.41 ± 0.04a

3 0.38 ± 0.01a 0.33 ± 0.04a 0.30 ± 0.03a 0.38 ± 0.03a 0.37 ± 0.04a 0.38 ± 0.03a 0.38 ± 0.04a 0.35 ± 0.04a 0.36 ± 0.01a

4 0.28 ± 0.06a 0.20 ± 0.03a 0.22 ± 0.04a 0.28 ± 0.04a 0.29 ± 0.04a 0.29 ± 0.04a 0.30 ± 0.04a 0.30 ± 0.05a 0.27 ± 0.02a

10 0.09 ± 0.02a 0.06 ± 0.00a 0.07 ± 0.02a 0.10 ± 0.02a 0.09 ± 0.03a 0.10 ± 0.01a 0.10 ± 0.01a 0.09 ± 0.02a 0.12 ± 0.01a

1The superscript letters “ab” indicate the significance levels (α = 0.05) of the means compared to the WT means Despite our results showing that engineering the L1 loop of TtL with single amino acid mutations does not increase activity in ILs, it is important to note that single mutations can be deleterious as it was observed for the A318V mutant of TvL, which decreased activity in ILs relative to WT 296. It is only when multiple amino acid mutations are made to the L1 loop that the laccase activity increased in ILs 294. Additionally, the increase in the catalytic efficiency of the HR03 mutants was attributed to increased π – π and π – anion interactions between the mutants and several aspartate and tyrosine residues present on the enzyme surface 265. Figure S3-2 shows there are relatively fewer aromatic and charged residues surrounding Glu170 in TtL that could help stabilize the E170Y/F mutants.

3.5 Discussion

In this study we showed that unlike most thermophilic enzymes the activity of the hyperthermophilic, bacterial laccase from Thermus thermophilus is sensitive to the presence of the IL [C2C1Im][OAc]. Through in vitro and in silico techniques, we provide insightful information about 1) the inhibition of TtL by [C2C1Im][OAc] and 2) rational design of TtL to improve activity in [C2C1Im][OAc]. The biocompatibility screening showed that >50% initial enzyme velocity is lost in just 2% [C2C1Im][OAc]. The docking simulations, along with the enzyme kinetics results, suggest that [C2C1Im][OAc] inhibits the activity of TtL by interfering with ABTS binding to the active site (competitive inhibition) and interfering with the ABTS oxidation once it is bound (uncompetitive inhibition). We made several single amino acid mutations to TtL with the aim of improving the activity in [C2C1Im][OAc]. These mutations were made based on previous studies that focused on engineering the L1 loop in fungal laccases as well as previous work that 98 improved the activity of a bacterial laccase in ILs. While the E170Y mutant displayed significantly higher activity in buffer, none of the mutants had significantly higher levels of activity in [C2C1Im][OAc] when compared to the WT enzyme. We have shown that thermophilicity alone does not indicate whether or not an enzyme will have high activity in ILs. Future studies might aim to better understand the mechanism underlying inhibition of TtL by [C2C1Im][OAc] or other ILs using molecular dynamics (MD) simulations or co- crystallizing TtL with the IL, along with employing other techniques (e.g. multiple amino acid mutations, chemical modifications, immobilization, computational design) to improve

TtL activity in ILs.

3.6 Author Contributions

Joseph Stevens, David Rodgers, Claire Dumon, and Jian Shi conceptualized the work, designed the experiments, and analyzed the data. Joseph Stevens conducted the experiments and wrote the manuscript.

3.7 Acknowledgements

The authors acknowledge the support from the 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 number 1018315 (2018 – 20203). 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).

3.8 Supplemental Information

Table S 3-1. List of primer names and sequences for making the mutants in this study. Mutated nucleotides are underlined.

Oligoname Sequence (5’ -› 3’) Position

A320D_Fo gaa acc ctg ctg tat ctg att gat ccg aaa aac ccg aaa cc 320

A320D_Rev gg ttt cgg gtt ttt cgg atc aat cag ata cag cag ggt ttc 320

A320E_Fo gaa acc ctg ctg tat ctg att gaa ccg aaa aac ccg aaa cc 320

A320E_Rev gg ttt cgg gtt ttt cgg ttc aat cag ata cag cag ggt ttc 320

A332D_Fo ctg ccg ctg ccg aag gat ctg agc ccg ttc cc 332

A332D_Rev gg aaa cgg gct cag atc ttt cgg cag cgg cag 332

A332E_Fo ctg ccg ctg ccg aaa gaa ctg agc ccg ttt cc 332

A332E_Rev gg aaa cgg gct cag ttc ttt cgg cag cgg cag 332

A341D_Fo g ttc ccg acc ctg ccg gat ccg gtg gtg ac 341

A341D_Rev gt cac cac cgg atc cgg cag ggt cgg aaa c 341

A341E_Fo g ttt ccg acc ctg ccg gaa ccg gtg gtg ac 341

A341E_Rev gt cac cac cgg ttc cgg cag ggt cgg aaa c 341

E170F_Fo gaa ctg cgc gaa gcg ttt gaa cat ctg ctg gtg ctg aag 170

E170F_Rev ctt cag cac cag cag atg ttc aaa cgc ttc gcg cag ctc 170

E170Y_Fo gag ctg cgt gaa gcg tat gaa cac ctg ctg gtg c 170

E170Y_Rev g cac cag cag gtg ttc ata cgc ttc acg cag ctc 170

100

(A)

(B)

101

(A) GLU171 (B) ASP190

TYR250

GLU168 ASP187 TYR189 GLU246 HIS172 GLU188 GLU170

102

Figure S 3-3. Codon optimized sequence of TtL (NCBI Accession Number: I7AL37)

103

CHAPTER 4. MODIFYING SURFACE CHARGES OF A THERMOPHILIC LACCASE TOWARDS IMPROVING ACTIVITY AND STABILITY IN AQUEOUS IONIC LIQUID

4.1 Abstract

The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid. However, the ionic liquid concentrations required for biomass pretreatment severely inhibit laccase activity.

Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in [C2C1Im][EtSO4], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C2C1Im][EtSO4], the biocompatibility of [C2C1Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C2C1Im][OAc]. Based on previous studies, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. These modifications did not improve laccase activity or stability in [C2C1Im][OAc] in contrast with the previous studies, although it is unknown if all modifications were made to the enzyme. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc]- are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C2C1Im][OAc] towards M. thermophila laccase is not dependent upon the IL interacting with the enzyme surface.

104

4.2 Introduction

Lignin is among the most abundant terrestrial biopolymers that makes up ~30% of the weight of biomass 40, 244. It is primarily made of 3 phenylpropanoid subunits: p- hydroxyphenyl (H), guaiacyl (G), and syringyl (S) that can be linked together via several condensed (C-C) or ether (C-O) bonds, with the phenylcoumaran (β – 5), β – aryl ether (β

– O – 4), and pinoresinol (β – β) being the most common 39, 41, 43, 45. Additionally, the ratio of subunits present in the lignin network is dependent upon the source of the biomass 44.

As a result of lignin’s structural and monomeric heterogeneity, thermochemical and biological methods achieve poor selectivity and yield of breakdown products. Lignin is currently generated in high volumes as a waste product by the paper and pulping industry and during production of cellulosic biofuels 1, 297. Converting lignin from a waste product to value added chemicals via new technologies will not only reduce waste lignin accumulation but also add value to the paper & pulping and future cellulosic biorefineries

3, 4. While the applications of polymeric lignin are limited, the phenolic products obtained from lignin deconstruction can be used as fuels or precursors for chemicals in the food, pharmaceutical, or plastic industries; many of these fuels and chemicals are currently derived from petroleum 248, 249.

Current methods for lignin deconstruction can be divided into thermochemical (e.g. high temperature pyrolysis, catalytic oxidation, and hydrogenolysis) and biological (e.g. lignin degrading enzymes [LDEs] or microbes) 6, 279, 280. Much of the research into the thermochemical methods has focused on improving the catalyst performance at high temperatures and improving the selectivity of lignin-derived products 255, 298, 299. The use of LDEs in biological lignin deconstruction strategies could enable lignin deconstruction

105 at lower temperatures and improve product selectivity due to the inherent selectivity and efficiency of biocatalysts 6. Many LDEs such as the peroxidases (lignin, manganese, and versatile) and laccases have been discovered in bacteria, plant, and fungi 85, 91, 93.

Additionally, catabolic lignin pathways in soil bacteria, such as Sphingobium sp. SYK-6 that catabolizes lignin using NAD or glutathione dependent enzymes have recently been identified 281. The application of LDEs for lignin deconstruction has been limited by a number of factors, chief among these being the high cost of enzyme production and the low yield of products due to the poor solubility of lignin in solvents biocompatible with

LDE activity 282, 283.

Many of the known lignin solvents (e.g. dimethyl sulfoxide [DMSO] and alkaline solutions) reduce or altogether eliminate enzyme activity 18, 19. Additionally, these solvents require high temperatures to facilitate the complete dissolution of plant derived lignin, leading to a further loss in enzyme activity 284. Therefore, a solvent system that is capable of solubilizing lignin at low temperatures and mild pH is desirable. Ionic liquids (ILs) are molten, organic salts liquid at temperatures <100oC, the properties of which can be tuned by selecting the appropriate cation and anion 127, 128, 131, 144. The alkylimidazolium ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3- methylimidazolium chloride ([C4C1Im][Cl]), have been the focus of numerous studies on biomass pretreatment at relatively low temperatures 251-253. Due to the high cost associated with using these ILs, recent efforts have focused on developing low cost ILs from bioderived cations (e.g. choline and ammonium) and anions (e.g. carboxylic acids and amino acids) that can be used in low concentrations during pretreatment 167, 285, 300. One

106 example is cholinium lysinate ([Ch][Lys]) which, at only 10% (w/v) in water, was shown to remove 80% of lignin from genetically engineered switchgrass during pretreatment 254.

Laccases are a member of the multicopper oxidase superfamily of enzymes (E.C.

1.10.3.2). They were first discovered in extract from the Japanese lacquer tree

(Toxicodendron vernicifluum) in 1883 84. Laccases differ from the other group of lignolytic enzymes, the heme peroxidases, in that they are copper-containing enzymes that do not require the presence of a strong oxidant, such as hydrogen peroxide, for the reaction mechanism 71, 86. The substrate is oxidized at the T1 catalytic copper via 4-electron removal, after which the electrons are shuttled 13Å to the trinuclear copper cluster where molecular oxygen is reduced to water 301. The T1 catalytic copper and the trinuclear coppers (a T2 and 2 T3) are coordinated by 4 conserved HXH motifs; X = cysteine in the case of the T1 copper 86. The T1 copper is also coordinated by a methionine or leucine/phenylalanine in bacterial/plant and fungal laccases, respectively 302. Figure 4-1 shows the conserved copper coordinating motifs in the sequences of laccases from different organisms.

107

Laccases are also capable of oxidizing nonphenolic lignin compounds when used alongside a small molecule mediator, such as 1-hydroxybenzotriazole (HBT) or 2,2’-azino- bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) 97, 256, 289. The relatively mild reaction conditions required for laccase activity along with the oxidation of mediator compounds make laccases a desirable biocatalyst for use in many biotechnological applications, including biological lignin deconstruction strategies 222, 288. Many ILs have been shown to be biocompatible with the activity of laccases at low concentrations in water. When Galai et al. investigated the effect of 56 ILs on the activity of T. versicolor laccase for dye decoloring applications, they found that 13 of the ILs increased the activity of the laccase;

10 mM choline dihydrogen phosphate ([Ch][H2PO4]) increased activity by 451% 150. The

108 initial activity of laccase from the white rot fungus Trametes versicolor was only reduced by 20% in a reaction mixture containing 50% (v/v) 1-ethyl-3-methylimidazolium ethylsulfate ([C2C1Im][EtSO4]) 290. However, other alkylimidazolium ILs have been found to be less biocompatible with laccase activity. The initial activity of T. versicolor laccase was reduced by 50% in reaction mixtures containing just 3, 20, 40, and 5% (v/v) of [C2C1Im][OAc], [C4C1Im][Cl], 1-hexyl-3-methylimidazolium bromide ([C6C1Im][Br]), and 1-decyl-3-methylimidazolium chloride ([C10C1Im][Cl]), respectively 290, 291.

Recent studies have shown that ILs are more biocompatible with thermophilic enzymes compared to mesophilic enzymes. Hydrolysis of carboxymethylcellulose (CMC) by hyperthermophilic (Topt > 95oC) and thermophilic (Topt = 80oC) cellulases was minimally impacted in 0 – 20% (v/v) [C2C1Im][OAc] while a mesophilic cellulase (Topt =

37oC) was unable to hydrolyze CMC in only 10% (v/v) [C2C1Im][OAc] 199. The activity of laccase from the thermophilic fungi Myceliophthora thermophila (MtL) is increased 3- fold in 25% (v/v) [C2C1Im][EtSO4] 198. The bacteria Bacillus subtilis produces a laccase- like spore coat protein (CotA) that can oxidize canonical laccase substrates ABTS and syringaldazine (SGZ) in the presence of several alkylimidazolium chloride ([CnC1Im][Cl])

ILs; [CnC1Im][Cl] ILs have been shown to severely inhibit the activity of a mesophilic laccase at low concentrations 91, 265. However, we showed in a previous study that the most thermophilic laccase identified to date, produced by the hyperthermophilic bacterium

Thermus thermophilus, is highly sensitive to low concentrations (<10% v/v) of

[C2C1Im][OAc], suggesting that enzyme thermophilicity alone does not guarantee high activity in ILs 93.

109

In this study we sought to determine the biocompatibility of aqueous

[C2C1Im][OAc] with MtL. To do this we first screened the activity of MtL in different concentrations of [C2C1Im][OAc] up to 50% in water. We also measured the effect of low concentrations of [C2C1Im][OAc] on the thermostability of MtL. Following the initial biocompatibility and stability screenings, we made several surface charge modifications with the aim of improving MtL activity and stability in [C2C1Im][OAc]. We also used molecular docking simulations to understand how [C2C1Im][OAc] docks to the surface of

MtL and the 4 charge variants we produced. The results of this study expand our understanding of laccase-IL interactions and the difficulties faced when trying to improve enzyme activity in ILs.

4.3 Methods and Materials

4.3.1 Materials

Laccase from M. thermophila expressed in Aspergillus oryzae (Batch #

OMN07029) was kindly provided by Novozymes (Bagsværd, Denmark). The 1-Step™

ABTS Substrate Solution was purchased from ThermoFisher Scientific (Waltham, MA).

All other reagents, including the IL [C2C1Im][OAc], were purchased from MilliporeSigma

(St. Louis, MO).

4.3.2 Surface Charge Modifications

MtL was purified by sequentially spin filtering with 100 kDa and 30 kDa MWCO filters at 8oC prior to surface charge modifications. Surface charges were modified according to previously reported protocols 202, 292. Cationized MtL was produced by modification with 0.5 M ethylenediamine hydrochloride in 200 mM MES buffer (pH 4.5)

110 containing a 20:1 molar ratio of the crosslinking reagent N-(3-dimethylaminopropyl)-N’- ethylcarbodiimide (EDC)-to-acid sites for 2.5 h at room temperature; neutralized MtL was similarly produced using diethylamine in place of ethylenediamine hydrochloride.

Succinylated MtL was produced by addition of 4 aliquots of succinic anhydride to the enzyme in 1 M sodium carbonate buffer (pH 8.5) over 3 h at room temperature. Acetylated

MtL was produced by addition of 3 aliquots of acetic anhydride to the enzyme in 100 mM sodium phosphate buffer (pH 7.0) over 1.5 h at room temperature. The final molar ratio of acetic or succinic anhydride to enzyme containing primary amines was approximately 30:1.

Excess reagent was removed with 30 kDa MWCO spin filters at 4oC and the enzymes were stored in 100 mM sodium phosphate buffer (pH 7.0) prior to experiments. Protein content was measured using the Bradford assay.

4.3.3 Biocompatibility Screening

The biocompatibility of [C2C1Im][OAc] in aqueous solution (approximately 1-50% w/v), was screened with all MtL variants. To reduce the effect of pH, the IL solution was adjusted to pH 4.5 using 1 M hydrochloric acid prior to testing. Activity was screened with

50 mM citrate/100 mM phosphate buffer (pH 4.5), IL (0, 1, 2, 3, 4, or 10% w/v) and 50 μL

ABTS solution in clear, flat bottom, 96-well Costar assay plates (Corning Inc., Kennebunk,

ME). Absorbance readings were taken every 15 seconds for 10 min at 40 ºC in a

SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). Plates were shaken for

3 seconds prior to each reading to ensure a homogeneous well solution. Oxidation of ABTS in buffer and AIL without laccase were measured as blanks. The activity of MtL in ILs relative to buffer was calculated using equation 4-1.

111

퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푖푛 퐼퐿 푅푒푙푎푡푖푣푒 푎푐푡푖푣푖푡푦 = (4-1) 퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푖푛 푏푢푓푓푒푟

4.3.4 MtL Thermostability

To measure the thermostability of MtL, all charge variants were incubated at 40oC in 0, 2.5, or 5% [C2C1Im][OAc] and 50 mM citrate/100 mM phosphate buffer, pH 4.5. At each time interval (0, 30, 60, 120, 180, and 240 min) aliquots were removed and placed on ice prior to measuring residual enzyme activity. Activity was screened using the same method in 4.3.3. Biocompatibility Screening. The residual activity of MtL and the charge variants after incubation was calculated using equation 4-2.

퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푎푓푡푒푟 푖푛푐푢푏푎푡푖표푛 푅푒푠푖푑푢푎푙 푎푐푡푖푣푖푡푦 = (4-2) 퐼푛푖푡푖푎푙 푉푒푙표푐푖푡푦 푎푓푡푒푟 0 min 푖푛푐푢푏푎푡푖표푛

4.3.5 Docking Simulations

The 3D structures and PDB files of the ligands were prepared in YASARA

Structure (YASARA Biosciences GmbH, Vienna, Austria) using the SMILES strings obtained from PubChem. The PDBQT files of the ligands and MtL (PDBID: 6F5K) were prepared with AutoDock (version 4.2.6, MGLTools, La Jolla, CA). Charges for the coppers were added by manually editing the MtL PDBQT file. AutoGrid parameters were as follows: space value of 0.375Å, (x, y, z) grid centered at (4.840, 28.290, -18.567), and grid size of 126 in all directions. AutoDock parameters were as follows: Lamarckian GA,

100 genetic algorithm runs, and 25,000,000 max eval size. Docking results were visualized using PyMol (Schrodinger LLC, New York, NY). To simulate the surface charge modifications, target surface residues were mutated in PyMol. These mutations are shown in Table 4-2.

112

Table 4-1. Summary of mutations to used mimic surface charge modifications in PyMol

Charge Modification Target PyMol Variant Chemistry Residues Mutations

Unmodified N/A N/A N/A

Primary Lys -> Acetylated Amines Ala (Lys)

Asp -> Carboxylic Ala Neutralized Acid (Asp or Glu -> Glu) Ala

Primary Lys -> Succinylated Amines Asp (Lys) Asp -> Carboxylic Lys Cationized Acid (Asp or Glu -> Glu) Lys

4.4 Results and Discussion

4.4.1 Biocompatibility Screening

To understand the biocompatibility of [C2C1Im][OAc], we measured the activity of

MtL and all of the charged variants in aqueous solutions (0 – 50% w/v) of [C2C1Im][OAc]

113 in water. None of the surface charge modifications improved the activity of MtL in aqueous

[C2C1Im][OAc] relative to the unmodified MtL, however we were unable to confirm whether or not the modifications were successfully introduced to the enzyme surface

(Figure 4-2). Minimal inhibition of MtL activity is observed in 1 – 10% [C2C1Im][OAc], however severe inhibition of MtL activity is observed in 15 – 50% [C2C1Im][OAc]. Since the coppers are not coordinated by any modifiable residues, it is unlikely that the inhibition is due to the loss of the coppers. The range of [C2C1Im][OAc] concentrations tested in this study are based on recent reports that reduced lignin removal, lower fermentable sugar yields, and lower cellulose solubilization are observed when less than 50% IL is present in aqueous solutions of [C2C1Im][OAc] and water 171.

Strong Inhibition

) v / 0%

w 0.15 – 0.00

% (

1% n

o 0.32 – 0.16 i

t 2%

r t

n 3%

e 0.49 – 0.33 c

n 4% o

C 0.65 – 0.50

] 10%

c A

O 15%

[ 0.82 – 0.66 ]

m 25%

1 C

2 50% 1.00 – 0.83

C [ d d d e e ed e ed e d No Inhibition fi t t z z u i la la i li r d y y n a C o t n io tr m e i t u c cc a e n A u C U S N

Figure 4-2. Heatmap showing the activity of MtL and the charge variants in [C2C1Im][OAc] relative to MtL activity in buffer.

The cation and anion of the IL play a role in determining the biocompatibility of the IL with enzyme activity. Previous studies have shown that as the alkyl chain length of

114 the cation increases, alkylimidazolium ILs become more inhibitory 265, 290. The inhibitory effect of the anion has been shown to follow the Hofmeister series 133, 303. ILs with chaotropic anions (e.g. NTf2- and Br-) more strongly destabilize proteins when compared to kosmotropic anions (e.g. SO42- and PO43-). For example, the activity of T. versicolor laccase is increased 451% in 10 mM choline dihydrogen phosphate ([Ch][H2PO4]), an IL with a kosmotropic anion, but is not affected by the same concentration of choline bis([trifluoromethyl]sulfonyl)imide ([Ch][NTf2]), an IL with a chaotropic anion 150.

Nordwald et al. previously showed that acetylation and succinylation improves enzyme activity and stability in 1-butyl-3-methylimidazolium chloride ([C4C1Im][Cl]) by reducing the interaction of the chaotropic Cl- anion with the enzyme surface. Additionally, low enzyme activity in higher concentrations of [C2C1Im][OAc] can be attributed to the buffering capacity of [C2C1Im][OAc]. Optimal ABTS oxidation occurs at pH < 5 for most fungal and baterial laccases, but [C2C1Im][OAc] buffers at a pH well above this range thereby lowering MtL activity in high concentrations of [C2C1Im][OAc] 304.

4.4.2 Stability [C2C1Im][OAc]

Although the surface charge modifications did not improve the activity of MtL, we sought to understand if the modifications improved the stability of MtL in [C2C1Im][OAc].

All charge variants of MtL were incubated in 50 mM citrate/100 mM phosphate buffer (pH

4.5) with 0%, 2.5%, and 5% [C2C1Im][OAc] at 40oC and the activity was measured at different time intervals up to 4 hours. The surface charge modifications, assuming they were successfully introduced to the enzyme surface, did not improve the stability of MtL in low concentrations of [C2C1Im][OAc] (Figure 4-3). Minimal activity loss was observed after 4 h in buffer for all charge variants. When incubated in 2.5% [C2C1Im][OAc] >50%

115 initial activity is lost after 2 h, while >50% initial activity is lost after only 1 h in 5%

[C2C1Im][OAc]. The IL concentrations tested are based on the results of the biocompatibility screening: 2.5 and 5% [C2C1Im][OAc] represent ranges of IL concentrations in which no or minimal activity loss is observed, respectively.

0% [C2C1Im][OAc] 0 min 30 min 60 min Low Residual Activity 120 min 180 min 0.15 – 0.00

e 240 min 2.5% [C C Im][OAc] m 2 1 0.32 – 0.16 i 0 min

T 30 min 0.49 – 0.33

n 60 min

o 120 min

i 0.65 – 0.50

t 180 min

a 240 min

b 0.82 – 0.66 5% [C2C1Im][OAc]

u 0 min c

30 min 0.83 – 1.00 n

I 60 min 120 min High Residual Activity 180 min 240 min d ed ed ed ed e fi at at iz iz i l l n l d y y o a o et n i tr m c i at u n cc e A u C N U S

Figure 4-3. Heatmaps showing the residual activity of MtL charge variants after incubation in 50 mM citrate/100 mM phosphate buffer and 2.5 or 5% [C2C1Im][OAc] at pH 4.5 and 40oC.

Previously, enzymes with surface charge modifications in ILs were shown to have a higher or lower half-life relative to the wild type in the IL. Both succinylation and

116 acetylation improved the half-life of lipase and α - chymotrypsin in 40% [C4C1Im][Cl] and

55% [C2C1Im][EtSO4], respectively, while acetylation improved the half-life of papain in

30% [C4C1Im][Cl]; however, neutralization and cationization reduced the half-life of lipase and α - chymotrypsin in IL, respectively 292. Succinylated and acetylated enzymes are thought to be more stable in ILs due to 2 factors: their similarity to halophilic enzymes and the formation of salt bridges. Halophilic enzymes have a large number of acidic surface residues which attract water molecules, increasing protein hydration and preventing the enzyme from aggregating, while the salt bridges further increase enzyme stability in saline environments 94, 305, 306.

4.4.3 Docking Simulations

Due to the unknown concentration of ABTS in the 1-Step solution used in the biocompatibility screening, we were unable to use enzyme kinetics analyses to understand the inhibition of MtL in [C2C1Im][OAc]. Therefore, docking simulations were used to better understand the effect of the surface charge modifications on the major binding locations of [C2C1Im][OAc] to the enzyme surface. The results of the docking simulations show that the modification chemistry affects the major docking locations of the IL to the surface of MtL (Figure 4-4). The major docking location of the IL to the surface of the unmodified MtL was limited to a region close to the T1 copper (Figure 4-4a). When MtL was acetylated or succinylated the IL docked close to the T1 copper in addition to a new

[OAc]- binding location away from the T1 copper (Figure 4-4b). When MtL was cationized or neutralized [C2C1Im]+ docked close to the T1 copper while [OAc]- docked further from the T1 copper but still close to the active site entrance (cationized) or away from the active site (cationized and neutralized) (Figure 4-4c).

117

a) b) c)

A closer inspection of the [C2C1Im]+ docking location reveals the presence of

several aromatic residues that would not be affected by any of the surface charge

modifications. These aromatic residues would be able to stabilize [C2C1Im]+ via π – π or

π – cation interactions in all charge variants. While these results are consistent with a

previous report that [C2C1Im]+ docks to MtL in the presence of aromatic residues, we did

not observe [C2C1Im]+ entering the active site 269. This study did not restrict the possible

[C2C1Im]+ binding locations, whereas the previous study focused the docking around the

MtL active site which could affect the degree to which the cation entered into the active

site. Arg456 and His457 stabilize [OAc]- close to the active site of unmodified,

succinylated, and acetylated MtL. When docking to succinylated and acetylated MtL,

[OAc]- is stabilized by Arg226 and Arg228 at the 2nd [OAc]- binding site. The major

[OAc]- docking location close to the active site of cationized MtL contains 2 modified

residues (Asp360 and Glu420), in addition to several lysines. The 2nd [OAc]- binding site

to cationized and neutralized MtL contains several arginines, lysines, and modified

residues.

118

The shift in [OAc]- binding is determined by the modification chemistry.

Acetylation and succinylation reduce the positive surface charges that stabilize [OAc]-, driving the anion to the arginine-rich binding pocket. Neutralization and cationization further increase the favorability of anion binding to an arginine-rich pocket containing modified glutamate and aspartate. The previous work on modifying surface charges suggest that acetylation and succinylation discourage the chaotropic anion (e.g. Cl- or

[OAc]-) from interacting with the enzyme surface, thereby improving enzyme activity and stability in IL 202, 292. Our docking results show that all surface charge modifications encourage [OAc]- binding away from the active site, however neither MtL activity nor stability in [C2C1Im][OAc] are affected by the modifications. Laccase activity is dependent upon the presence of a T1 copper in the active site, a trinuclear copper cluster, and a Cys-

His pathway between the T1 copper and the trinuclear cluster. If the surface charge modifications are confirmed, then the following mechanism can be proposed: the loss of

MtL activity and stability in [C2C1Im][OAc] is likely due to destabilization of the coppers and the Cys-His pathway by the IL via a mechanism that cannot be influenced by modifying the enzyme surface charges. Without confirming the presence of the enzyme surface modifications this mechanism cannot be supported by the current data.

4.5 Conclusions and Future Work

In this study we examined the biocompatibility of aqueous [C2C1Im][OAc] with the activity of the thermophilic fungal laccase from Myceliophthora thermophila. Results show severe inhibition of MtL activity in only 15% (w/v) [C2C1Im][OAc], far below the IL concentration necessary for effective biomass pretreatment. In an effort to reduce this inhibition of MtL activity, we made several surface charge modifications to increase the

119 acid or amine surface residues. Although previous studies have shown that surface charge modifications can increase enzyme activity and stability in aqueous ILs, the stability and activity of all MtL charge variants in [C2C1Im][OAc] were unchanged compared to the unmodified enzyme. However, it is not known at this time if the modifications were successfully introduced to the enzyme surface. Docking simulations show that the IL docks close to the T1 copper on all charge variants, with some additional [OAc]- docking locations on the surface of modified enzymes. Further work might seek to 1) confirm the presence of the surface charge modifications, 2) better understand the mechanism by which the IL inhibits MtL activity through the use of enzyme kinetics and molecular dynamics

(MD) simulations and 3) employ additional techniques (e.g. rational design or immobilization) to improve MtL activity in [C2C1Im][OAc].

Methods used to assess the effects of ILs on enzymes can be divided into in vitro

(e.g. activity assays, stability assays, and enzyme kinetics) and in silico (e.g. docking simulations and molecular dynamics [MD] simulations) techniques. In vitro techniques can be used to provide a broad overview of how the ILs are affecting the enzyme activity, stability, and substrate affinity. By measuring the activity of TvL in ILs, past studies have shown that [C2C1Im][EtSO4] is the most biocompatible alkylimidazolium IL, suggesting that this laccase-IL combination would be best suited for lignin deconstruction applications

198, 290. However, these activity assays are done on a short time scale which does not provide any information about the stability of the laccase in the IL. Although the initial activity of MtL immobilized on glyoxyl-agarose beads is lower in 25% [C2C1Im][EtSO4] than the free laccase initial activity, the immobilized laccase is far more stable in 75% IL than the free laccase over a 7 day period 198. Enzyme kinetics studies, performed by

120 measuring the initial velocity of enzymes in different substrate concentrations, can further indicate if the loss in activity is due to the IL affecting the enzyme activity or substrate binding. Kinetic studies of MtL in [C2C1Im][Cl] showed that the IL is a competitive inhibitor that competes with the substrate for binding in the active site 269.

[C2C1Im][EtSO4] was found to interact with MtL as an uncompetitive inhibitor that does not affect substrate binding, but rather affects substrate oxidation once it has bound 198.

Some ILs are also able to affect both substrate binding and oxidation, for example

[C2C1Im][OAc] and [Ch][Lys], which were found to be mixed inhibitors that decreased substrate affinity and activity of TvL 291 .

To further understand the laccase-IL interactions seen with kinetic studies, recent studies have used docking simulations with ILs and static protein models. Docking simulations showed that the diffusion of [CnC1Im]+ cations (n = 2, 4, 6, 8, 10) into the active site of MtL is determined by the alkyl chain length 269. Similarly, [C2C1Im][OAc] and [Ch][Lys] were found to dock close to the T1 copper of TvL 291. In both of these cases the docking simulations supported the previous kinetics results showing the ILs can affect substrate binding and oxidation. MD simulations can also be used to better capture the dynamic nature of enzyme-IL interactions. After in vitro experiments showed

[C2C1Im][OAc] is more biocompatible with thermophilic cellulases than a mesophilic cellulase, MD simulations revealed the IL effect is limited to local disturbances of thermophilic cellulases or secondary structure unfolding of a mesophilic cellulase 199, 307.

MD simulations also provided further insight into enzyme-IL interactions that increase the thermostability of a Fusarium solani serine protease cutinase in 1-butyl-3- methylimidazolium hexafluorophosphate ([C4C1Im][PF6]) 308. Further insight into the

121 effect of the IL on enzymes can be gained by applying different methods to understand the binding free energy of the IL or substrate to the enzyme surface or active site 309.

In this study we applied a single enzyme engineering approach to try and improve

MtL activity and stability in [C2C1Im][OAc]. However, previous studies have applied other enzyme engineering strategies to achieve the same goal (Table 4-1). The enzyme used in this study was not produced recombinantly, therefore we were unable to apply and biotechnological (e.g. directed evolution or rational design) methods to improve MtL activity and stability. Future studies with MtL in [C2C1Im][OAc] might apply immobilization techniques similar to those previously used to improve stability in

[C2C1Im][EtSO4] 198. Additionally, recombinant expression of MtL would enable future researchers to apply other enzyme engineering techniques. The L1 loop can be targeted for mutations with a directed evolution or computationally assisted strategy, similar to previous studies with TvL 293, 294. Additionally, novel targets for mutations can be identified using a computationally assisted approach like that used to identify the Glu170 target in B. subtilis CotA 265. The application of more robust methods for analyzing laccase-IL interactions alongside targeted enzyme engineering strategies will facilitate the identification of laccase-IL combinations suited for use in future lignin valorization systems.

122

Table 4-2. Previous studies using various methods to improve enzyme activity in aqueous ILs

Method Enzyme Outcome Citation

Computationally assisted T. versicolor laccase Triple and quadruple mutants with 294

protein engineering increased activity in [C2C1Im][EtSO4]

Directed evolution T. versicolor laccase Double mutant with increased activity 293

in [C2C1Im][EtSO4]

Computationally assisted B. subtilis laccase Single mutants with increased catalytic 265

protein engineering efficiency (kcat/Km) in 3 [CnC1Im][Cl]

ILs

Surface charge Bovine pancreas α- Succinylation and acetylation 292

modification chymotrypsin, Carica improved activity and stability in

papaya papain, Candida [C4C1Im][Cl] and [C2C1Im][EtSO4]

rugosa lipase

Surface charge Trichoderma reesei Succinylation improved cellulose 202

modification cellulase hydrolysis in [C4C1Im][Cl]

Immobilization M. thermophila laccase Immobilization on glyoxyl-agarose 198

beads improved stability in

[C2C1Im][EtSO4]

4.6 Author Contributions

Joseph Stevens and Jian Shi conceptualized the work, designed the experiments, and analyzed the data. Joseph Stevens conducted the experiments and wrote the manuscript.

4.7 Acknowledgements

The authors acknowledge the support from the National Science Foundation under

Cooperative Agreement No. 1355438 and 1632854 (2016 – 2020) and the National 123

Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch-Multistate project under accession number 1018315 (2018 – 2023).

124

CHAPTER 5. A MIXED TECHNIQUE APPROACH TOWARDS UNDERSTANDING IONIC LIQUID INHIBITION OF A PLANT LACCASE

5.1 Abstract

High concentrations of alkylimidazolium ionic liquids are required for biomass pretreatment, increasing their cost of use and lowering their biocompatibility with the lignolytic enzyme laccase. Due to the low concentration of [Ch][Lys] required for biomass pretreatment, it is an attractive target to be used alongside laccases in a low-cost biological lignin valorization system. We investigated the biocompatibility of [Ch][Lys] with a plant laccase from Toxicodendron vernicifluum. Severe inhibition of laccase activity was seen in only 3% [Ch][Lys]. Enzyme kinetics and docking simulations suggest the [Ch][Lys] inhibits via a mixed competitive and noncompetitive mechanism. [Ch]+ acts as the competitive inhibitor, directly interfering with DMP binding the active site, while [Lys]+ acts as the noncompetitive inhibitor that interferes with DMP catalysis once it is bound.

Subsequent molecular dynamics simulations were used to better capture the dynamic nature of the laccase-IL interactions, the first time these simulations have been used with a laccase. These simulations revealed that [Ch][Lys] does not perturb the enzyme structure, but rather lowers the stability of DMP in the active site. These results show for the first time that IL inhibition of laccase activity could be due to substrate-IL interactions in addition to the laccase-IL interactions seen in previous studies.

125

5.2 Introduction

Laccases are a member of the multicopper oxidase superfamily of enzymes (E.C.

1.10.3.2). First identified in extract from the Japanese lacquer tree Toxicodendron vernicifluum, several laccases and laccase-like enzymes have been discovered in bacteria, fungi, and archaea 84, 85, 90, 91, 93. Laccases differ from the other lignolytic enzymes, the heme peroxidases, in that they are copper containing enzymes that require molecular oxygen, as opposed to hydrogen peroxide, for their oxidation mechanisms 71, 80, 310.

Laccases are also capable of oxidizing non-phenolic lignin compounds when coupled with a small molecule mediator such as 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

(ABTS) or 1-hydroxybenzotriazole (HBT) 97, 256, 289. As a result laccases have been the target of numerous biotechnological applications, including lignin valorization 288.

Lignin is among the most abundant biopolymers on earth, making up about 30% of the weight of terrestrial biomass 40, 244. It is primarily made of 3 phenylpropanoid subunits: p-hydroxyphenyl (H-lignin), guaiacyl (G-lignin), and syringyl (S-lignin) 41, 43. These subunits are linked by a complex network of condensed (e.g. β – β or β – 5) and ether (e.g.

β – O – 4 or β – O – 5) bonds 39, 45. The ratio of subunits present in the lignin network is also dependent upon the biomass source 44. As a result of its monomeric and structural heterogeneity, lignin is very recalcitrant towards thermochemical and biological depolymerization methods for production of chemicals at high selectivity. Lignin is generated in large volumes as a waste product by the paper and pulping industry and during the production of cellulosic biofuels 1, 247 . Developing technologies that convert lignin from waste to value-added products will not only reduce waste but add value to the paper and pulping industry and cellulosic biorefineries 3, 311.

126

Numerous lignolytic enzymes have been identified in bacteria and fungi, most notably the heme peroxidases (manganese, versatile, and lignin) and laccase; lignin breakdown pathways have also been identified in the soil bacterium Sphingobium sp. SYK-

6 which metabolizes aromatic compounds via NADP and glutathione dependent enzymes

13, 15, 281. The use of enzymes in biological lignin valorization can be beneficial from both an operating cost and safety standpoint as a result of the mild operating conditions (low temperature, low pressure, mild pH) and improved yield of lignin breakdown products due to the efficiency of biocatalysts 6, 12. These monomeric lignin breakdown products can be used in place of petroleum-derived precursors for chemicals used in the pharmaceutical, food, and plastic industries 248, 249. However, many of the known lignin solvents (e.g. dimethylsulfoxide [DMSO], acetone, and alkaline solutions) reduce or altogether eliminate enzyme activity 18, 19. These solvents also require the use of high temperatures for complete dissolution of native plant lignin, further eliminating enzyme activity in the process 284.

The potential for biological lignin valorization strategies is great, however many challenges

(e.g. high cost of enzyme production and poor monomer yield due to low lignin solubility) must be overcome by using a low-cost biocompatible solvent that is capable of dissolving lignin at low temperatures 16, 117.

Ionic liquids (ILs) are molten salts that are liquid at temperatures below 100oC.

Considered to be “green solvents” due to their relatively low toxicity and vapor pressure, the properties of ILs can be tuned by selecting the appropriate cation and anion 127, 128, 131,

133. Alkylimidazolium ILs, for example 1-ethyl-3-methylimidazolium acetate

([C2C1Im][OAc]), have been studied for their ability to reduce cellulose crystallinity and extract lignin during relatively low temperature biomass pretreatment 251-253. Many

127 alkylimidazolium ILs have been shown to be biocompatible with the activity of laccase from the white rot fungi Trametes versicolor at different concentrations in water; >80% initial activity remained in 50% (v/v) 1-ethyl-3-methylimidazolium ethylsulfate

([C2C1Im][EtSO4]) 290. Due to the high cost associated with using [C2C1Im][OAc], recent efforts have focused on developing ILs for biomass pretreatment using low-cost, bio- derived cations (e.g. choline and ammonium) and anions (e.g. amino acids and carboxylic acids) that can be used at low concentrations in water 164, 167, 243. For example, when switchgrass was pretreated with 10% [Ch][Lys] in water, greater than 80% lignin removal was observed 254. The low concentration of [Ch][Lys] required for effective biomass pretreatment could improve its biocompatibility since enzyme activity in ILs is strongly dependent upon IL concentration 290, 293. However, we previously found that T. versicolor laccase is severely inhibited in just 1% [Ch][Lys], far below the concentration required for effective biomass pretreatment 291. The tunability and biocompatibility of ILs makes them attractive solvents to be used with laccases in biological lignin valorization strategies, however relatively little is known of laccase-IL interactions beyond in vitro experiments

Molecular dynamics (MD) simulations can be used to understand the interactions between enzymes and ILs at an atomistic resolution. After initial in vitro experiments determined that [C2C1Im][OAc] is more biocompatible with thermophilic cellulases than a mesophilic cellulase, subsequent MD simulations revealed that the main effect of the IL was limited to local disturbances, in the case of the thermophilic cellulases, or complete unfolding of the enzyme secondary structure, in the case of the mesophilic cellulase 199, 307.

MD simulations of a serine protease cutinase from Fusarium solani in 1-butyl-3- methylimidazolium hexafluorophosphate ([C4C1Im][PF6]) found that the IL stabilizes the

128 protein structure at high temperatures, a result already obtained from in vitro lab experiments 308. Previous studies on laccase-IL interactions have used docking simulations to identify docking locations of the IL on the enzyme surface, for example T. versicolor laccase with [C2C1Im][OAc], [Ch][Lys], and [DEA][HSO4] or M. thermophila laccase with several alkylimidazolium cations 269, 291. In both of these cases the docking results supported the kinetics data, however the ILs were docked to a static model of the laccases which fails to capture the dynamic nature of the laccase-IL interactions. Additionally, previous kinetics data suggest that ILs not only interfere with substrate binding but also affect oxidation once the substrate is bound 150, 198, 291; an in-depth investigation of laccase-

IL interactions would greatly benefit from the atomistic resolution of MD simulations.

5.3 Methods and Materials

5.3.1 Materials

Laccase from T. vernicifluum (RvL, ≥50 units/mg solid), 2,6-dimethoxyphenol

(DMP), choline hydroxide, L-lysine, and all other reagents were purchased from

MilliporeSigma (St. Louis, MO, USA). Cholinium lysinate ([Ch][Lys]) was synthesized using the anion and cation precursors according to previously developed protocols 167.

5.3.2 Plant laccase purification

RvL was purified from the crude acetone extract using the method previously published 312. First, 35 mg of the crude extract was mixed with 10 mL buffer (0.1 M potassium phosphate, pH 6.0) at room temperature for 16-20 h. After overnight mixing, any undissolved material was filtered using 0.45 and 0.2 um syringe filters. The filtered material (10 mL) was then mixed with 3g of (NH4)2SO4 then centrifuged for 20 min at

129

15000g to remove any precipitated material. Finally, the supernatant was applied at a rate of 0.5 mL min-1 to a column (11 mm diameter x 10.4 cm height) of phenyl-Sepharose

(Tosoh Bioscience, Tokyo, Japan) equilibrated with 2.67 M (NH4)2SO4 in 0.01 M potassium phosphate buffer (pH 6.0). The protein was eluted off the column by reducing the concentration of (NH4)2SO4 at a gradient from 2.67 M to a final concentration of 0.27

M. Protein content was determined using the Bradford method.

5.3.3 Biocompatibility screening

The biocompatibility of [Ch][Lys] in aqueous solution (approximately 1-10% w/v), was screened with purified RvL. To reduce the effect of pH, the IL solution was adjusted to pH 8.0 using 1 M hydrochloric acid prior to testing. Activity was screened with 25 mM

MES, HEPES, glycine, and acetic acid buffer (pH 8.0); IL (0, 1, 2, 3, 4, or 10% w/v); and

25 mM DMP into a clear, flat-bottom, 96-well Costar assay plate. Absorbance readings were taken continuously for 10 min at 25 ºC using a SpectraMax M3 Microplate reader

(Molecular Devices, Sunnyvale, CA). Oxidation of DMP in buffer and AILs without laccase were measured as blanks. The relative activity of RvL in ILs relative to buffer was calculated using equation 5-1.

퐴푐푡푖푣푖푡푦 푖푛 퐼퐿 푅푒푙푎푡푖푣푒 푎푐푡푖푣푖푡푦 = (5-1) 퐴푐푡푖푣푖푡푦 푖푛 푏푢푓푓푒푟

5.3.4 Enzyme Kinetics

Michaelis-Menten curves were generated for IL concentrations (0, 2, and 4% w/v) by varying the concentration of DMP (2 – 25 mM) and measuring initial velocities. DMP oxidation was measured using the same method as described in the biocompatibility screening. The oxidized DMP was quantified by measuring the absorbance at 469 nm and 130 using the absorbance extinction coefficient Ɛ469 = 35,645 M-1 cm-1. An activity unit was defined as 1 µmol DMP oxidized in 1 min (µmol DMP/min). Initial velocity (V) as a function of DMP concentration ([S]) was fit to the Michaelis-Menten curve, shown in equation 5-2.

V ×[S] V = max (5-2) Km+[S]

The kinetic coefficients were calculated by plotting equation 5-2 in Sigma Plot and calculating Km and Vmax using the ligand binding curve fitting algorithm.

5.3.5 Plant laccase modelling and copper parameterization

The 3D model of plant laccase was built using the Modeller Software, version 9.23

313. The sequences of Zea mays laccase 3 (PDBID: 6KLG, sequence id: 48.91%) and

Cucurbita pepo ascorbate oxidase (PDBID: 1AOZ, sequence id: 31.80%) were used as templates in Modeller. Multiple sequence alignment was performed with MAFFT

Software, version 7.310 314. The 100 models generated by Modeller were ranked based on their Discrete Optimized Protein Energy (DOPE) score 315. The model with the lowest

DOPE score was selected as the best model. The parameters (force constants, geometry, and charges) of the coppers and their coordinating residues were calculated using

MCPB.py, a python based metal center parameter builder 316. All quantum calculations were computed using Gaussian 09 D.01 with B3LYP-631G* functionality 317.

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5.3.6 Docking Simulations

The 3D structures and PDB files of the ligands were prepared in YASARA

Structure (YASARA Biosciences GmbH, Vienna, Austria) using the SMILES strings obtained from PubChem. The PDBQT files of the ligands and the RvL model were prepared with AutoDock (version 4.2.6, MGLTools, La Jolla, CA). Charges for the coppers were added by manually editing the RvL PDBQT file. AutoGrid parameters were as follows: space value of 0.375Å, (x, y, z) grid centered at (80.297, 66.064, 8.193), and grid size of 126 in all directions. AutoDock parameters were as follows: Lamarckian GA;

100 genetic algorithm runs; 0.5Å RMS tolerance; and 25,000,000 max eval size. [Ch]+ and

[Lys]+ were docked separately to both unbound RvL and RvL with DMP bound to the active site. The structure of RvL with DMP in the active site was prepared in PyMol by aligning the RvL model with Melanocarpus albomyces laccase containing DMP in the active site

(PDBID: 3FU8).

5.4 Results and Discussion

5.4.1 Biocompatibility of AILs with laccase activity

To determine the biocompatibility of [Ch][Lys] with RvL, the activity of RvL was measured in aqueous concentrations of [Ch][Lys] (1-10%) in water. The activity of RvL in different concentrations of [Ch][Lys] relative to the activity in buffer is shown in Figure

5-1. RvL is strongly inhibited by [Ch][Lys], only 50% initial activity is remaining in 3%

[Ch][Lys]. As the concentration of [Ch][Lys] continues to increase, the decrease in RvL activity is not as severe although only 20% initial activity remains in 10% [Ch][Lys]. The range of [Ch][Lys] tested is relevant to recent work investigating the effect of water during

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IL pretreatment of biomass. The pretreatment efficacy of [C2C1Im][OAc] is severely reduced at a concentration of 50% in water 171. High sugar yields were obtained after pretreating switchgrass at 180oC for 15 min using triethylammonium hydrogen sulfate

([TEA][HSO4]) in 20% water 285. In contrast, 10% [Ch][Lys] in water removes 80% of lignin from genetically engineered switchgrass during pretreatment 254.

1.0

0.8

0.6

0.4

Relative Activity Relative

0.2

0.0 0 2 4 6 8 10 [Ch][Lys] (%w/v)

Figure 5-1. Effect of aqueous [Ch][Lys] on the activity of RvL. Values shown are the mean ± SD of 3 experiments

Both the cation and the anion play a role in enzyme inhibition. As the alkyl chain length increased (C2 -> C10) the activity of Myceliophthora thermophila laccase decreased in several alkylimidazolium chloride ([CnC1Im][Cl]) ILs 269. A decrease in the activity of

Trametes versicolor laccase was seen as the alkyl chain length increased (C2 -> C10) and the chaotropicity of the anion increased 290. Chaotropic anions, e.g. bis(trifluoromethylsolfonyl) imide ([NTf2]-) and bromide (Br-), are more inhibitory than less chaotropic anions, e.g. chloride (Cl-) and phosphate (PO43-), closely following the

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Hofmesiter series 148. Previous experiments have shown that the activity of T. versicolor laccase was increased 451% by 10 mM choline dihydrogenphosphate ([Ch][H2PO4]), not affected by 10 mM ([Ch][NTf2]), and severely inhibited by 1% [Ch][Lys] 150, 291. Our results, along with the results of the previous studies indicate that lysine is not biocompatible with laccase activity.

5.4.2 Inhibition Mechanisms

We used enzyme kinetics to elucidate the inhibition mechanisms of [Ch][Lys] towards RvL activity using DMP as a substrate. We chose to investigate the kinetics of

DMP oxidation at 0, 2, and 4% [Ch][Lys] based on the results of the biocompatibility screening. The changes in kcat and Km with increasing [Ch][Lys] concentration suggest the presence of 2 binding sites (Table 5-1). A high affinity competitive binding site that is completely occupied in 2% [Ch][Lys] increases Km while a low affinity non-competitive binding site lowers kcat in all concentrations of [Ch][Lys].

Table 5-1. Kinetic constants of RvL in different concentrations of [Ch][Lys]. Values shown are mean ± standard error of 3 experiments

[Ch][Lys] Km kcat kcat/Km

(%w/v) (mM DMP) (s-1) (s-1 mM-1)

0% [Ch][Lys] 28.88 ± 3.61 3.15 ± 0.15 0.11

2% [Ch][Lys] 39.74 ± 2.88 2.75 ± 0.05 0.07

4% [Ch][Lys] 40.58 ± 3.14 2.10 ± 0.03 0.05

Previous kinetics work has shown that several ILs are capable of acting as competitive, noncompetitive, or uncompetitive inhibitors. Aqueous solutions of

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[C2C1Im][OAc] and 1-ethyl-3-methylimidazolium sulfate ([C2C1Im][EtSO4]) acted as competitive (increased Km) and noncompetitive (decreased Vmax) inhibitors of T. versicolor laccase 183. [Ch][H2PO4] and [C2C1Im][EtSO4] increased Km and kcat of T. versicolor and

M. thermophila laccase, respectively, by binding to [E][S] as uncompetitive inhibitors 150,

290.

5.4.3 Copper parameterization and stabilization during simulations

The calculated charges of the coppers and hydroxyl groups bounded to the coppers are shown in Figure S5-2. The distance between the coppers and their coordinating residues were recorded during a 100 ns simulation to ensure the stability of the coppers.

As seen in Figure 5-2, the distances between the coppers and their coordinating residues are around 2Å, consistent with the distances seen in laccase crystal structures 86, 318.

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Figure 5-2. Distances between (top) T1 copper, (middle) T2 copper, and (bottom) T3 coppers and coordinating residues during 100 ns simulations

5.4.4 IL Docking

To better understand where [Ch][Lys] binds to the surface of RvL, we performed docking simulations using AutoDock. Major docking poses of [Ch][Lys] are shown in

Figure 5-3. Three major docking locations were identified on both [E] and [E][S]. [Ch]+ docks close to the T1 copper and the trinuclear copper cluster in a pocket close to the C-

136

terminus. [Lys]+ binds to the same C-terminus pocket and a 3rd location close to the

trinuclear copper cluster. These results suggest that [Ch]+ acts as both a competitive and

noncompetitive inhibitor, while [Lys]+ only acts as a noncompetitive inhibitor.

(a) (b)

Previous results docking ILs to laccases have shown that ILs are capable of docking

close to or within the active site. When alkylimidazolium cations were docked to M.

thermophila laccase, it was found that short chain length cations (C2 – C6) were able to

diffuse into the active site, whereas the longer chain length cations (C8 – C10) were limited

to interacting with leucines surrounding the active site 269. We showed in a previous study

that [C2C1Im][OAc] and [Ch][Lys] dock with high probability to a region close to the

active site of T. versicolor laccase 291.

5.4.5 Steered Molecular Dynamics of DMP in [Ch][Lys]

To better understand the effect of [Ch][Lys] on the stability of DMP in the active

site of RvL, we analyzed the free energy profile of DMP using steered molecular dynamics

137

(SMD) analysis. The free energy of DMP was calculated at a range of distances between the hydroxyl group of DMP and the T1 copper of RvL. Figure 5-4 below shows the free energy profile of DMP in 0, 2, and 4% [Ch][Lys] at different distances from the T1 copper.

The lower free energy 6 – 8 Å away from the T1 copper is consistent with the location of

DMP when docked into the active site of RvL from the M. albomyces crystal structure.

Additionally, the free energy within this range becomes less negative as the concentration of [Ch][Lys] increases, suggesting that [Ch][Lys] lowers the stability of DMP in the active site. These results are consistent with the kinetics, which showed that [Ch][Lys] disrupts substrate binding and oxidation, and the docking simulations, which showed that [Ch][Lys] can dock close to the T1 copper in close proximity to DMP.

6.8Å

5.5 Author Contributions

Joseph Stevens, Jérémy Esque, Isabelle André, David Rodgers, and Jian Shi conceptualized the work, designed the experiments, and analyzed the data. Jérémy Esque performed the RvL modelling, copper parameterization, and MD simulations. Joseph

Stevens conducted the all other experiments and wrote the manuscript.

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5.6 Acknowledgements

The authors acknowledge the support from the 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 number 1018315 (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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5.7 Supplemental Information

0.8 0% [Ch][Lys]

) 2% [Ch][Lys]

-1 4% [Ch][Lys] 0.6

0.4

0.2

Specific Activity (U * mg * (U Activity Specific

0.0 0 5 10 15 20 25

[DMP] (mM)

Figure S 5-1. Michaelis-Menten curves of RvL in aqueous [Ch][Lys]. Data were plotted and models were fit in Sigma-Plot. Values plotted are mean ± SD of 3 experiments.

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Figure S 5-2. Final charges and geometry of coppers and coordinating residues

141

CHAPTER 6. CONCLUSIONS AND FUTURE WORK

6.1 Conclusions

The aim of this body of work was to create a biological lignin valorization system using a biocompatible IL and a laccase. Initial studies showed that the selected ILs, 1- ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]), cholinium lysinate ([Ch][Lys]), and diethylamine hydrogen sulfate ([DEA][HSO4]), were biocompatible with a mesophilic fungal laccase from Trametes versicolor in low concentrations. When Kraft lignin was treated with the laccase and ILs, few overall changes were made to the lignin network and product yields were low. These results prompted the search for a more robust biocatalyst to be used alongside the ILs.

Prior studies showing improved biocompatibility of [C2C1Im][OAc] with thermophilic cellulases lead to the investigation of thermophilic laccases as biocatalysts for lignin valorization. The first of these laccases was from the hyperthermophilic bacterium Thermus thermophilus. Despite this being the most thermotolerant laccase identified to date (Topt = 92oC), severe loss of initial activity was observed in only 2%

[C2C1Im][OAC]. A rational design approach to improve activity in [C2C1Im][OAc] subsequently failed to produce any mutants with improved activity in the IL, highlighting the challenging nature of designing a lignin valorization system with laccases and ILs.

To continue the search for a suitable biocatalyst, the biocompatibility of

[C2C1Im][OAc] was screened with a thermophilic fungal laccase from Myceliophthora thermophila. Although this laccase was not as thermotolerant as the bacterial laccase, previous studies have shown it to be active and stable in high concentrations of 1-ethyl-3- methylimidazolium ethylsulfate ([C2C1Im][EtSO4]). Minimal activity loss was observed

142 in 10% [C2C1Im][OAc], however severe inhibition was observed in concentrations greater than 15%. Previous studies have shown that modifying the surface charges of enzymes to increase the number of acid residues improves enzyme activity and stability in ILs. Due to the ubiquity of this approach, the surface charges of M. thermophila laccase were modified to improve activity and stability in [C2C1Im][OAc]. These modifications did not improve the activity or stability of M. thermophila laccase in the IL, however docking simulations revealed that they do affect the binding of [OAc]- to the enzyme surface. These results suggest that the laccase-IL interactions leading to M. thermophila laccase inhibition cannot be mediated by changing the interaction of the IL with the enzyme surface.

The previous studies failed to produce a laccase with improved activity and stability in [C2C1Im][OAc] by applying methods previously shown to do just that. These studies had also used enzyme kinetics and molecular docking simulations to understand where the

IL is docking to the enzyme surface, however these simulations fail to accurately capture the dynamic nature of enzyme-IL interactions at high resolution. Therefore, we used molecular dynamics (MD) simulations to gain insight into the interactions between the IL cholinium lysinate ([Ch][Lys]) and a plant laccase from Toxicodendrom vernicifluum.

These simulations showed that the IL does not affect the laccase structure, but rather decreases the stability of the substrate in the active site. The initial goal of designing a biological lignin valorization system was unmet, however we gained great insight into laccase-IL interactions and the challenges of using enzyme engineering methods to improve the performance of a biocatalyst for an industrial application. The content of these studies can provide a good foundation for future work investigating and engineering laccase-IL interactions for lignin valorization.

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6.2 Future Work

This body of work lays the foundation for many possible avenues of investigating laccase-IL interactions in the future. Perhaps most obvious are future enzyme engineering efforts to improve the activity and stability of T. thermophilus laccase in [C2C1Im][OAc].

Our approach was limited in scope and would have benefited from a directed evolution or computationally guided approach, rather than relying on previous research on fungal laccases to produce many of the mutants screened in that study. The MD simulations in

Chapter 5 provided useful insight into the nature of laccase-IL, or rather substrate-IL, interactions. This approach could be used with other laccase-IL combinations to understand these interactions with atomistic resolution. These results can then be used to guide enzyme engineering efforts by identifying key residues that mediate the IL interactions on the enzyme surface. Similar to T. thermophilus laccase, a more thorough analysis of the interactions between [C2C1Im][OAc] and M. thermophila laccase could guide enzyme engineering efforts to further improve activity and stability in the IL.

This groundwork could also be applied to systems other than laccases and ILs. The next generation of designer solvents is arriving with the advent of deep eutectic solvents

(DES). Many studies have already been published that show DESs to be cheaper and easier to synthesize than ILs. These same DESs are comparable to ILs when studied for their ability to fractionate biomass during pretreatment. Future studies may investigate laccase-

DES interactions with many of the same methods used in this study. These results may guide enzyme engineering efforts to improve laccase activity in DES for a cost-effective and sustainable biological lignin valorization system.

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VITA

Joseph Stevens

EDUCATION Ph.D., Biosystems and Agricultural Engineering, University of Kentucky Expected August 2020

B.S., Biological Systems Engineering, Virginia Tech Graduated May 2016

PROFESSIONAL EXPERIENCE

STEM Chateaubriand Fellow, Toulouse Biotechnology Institute January to October 2019

OTC Graduate Fellow, UK Office of Technology Commercialization January to December 2018

Graduate Research Assistant, UK Biosystems and Agricultural Engineering May 2016 to Present

Undergraduate Researcher, Virginia Tech Biomolecular Engineering Lab January 2015 to May 2016

Undergraduate Researcher, Virginia Tech Bioenergy and Biotechnology Laboratory June to December 2014

RESEARCH SUMMARY Journal Articles 1. Stevens J, Rodgers D, Dumon C, Shi J. “Characterization and Enzyme Engineering of a Hyperthermophilic Laccase toward Improving Its Activity in Ionic Liquid” Front Energy Res. 2020 2. Das L, Li W, Dodge L, Stevens J, Williams D, Hu H, Li C, Ray A, Shi J. “Comparative Evaluation of Industrial Hemp Cultivars: Agronomical Practices, Feedstock Characterization and Potential for Biofuels and Bioproducts” ACS Sus Chem. 2020 3. Stevens J, Das L, Mobley J, Asare S, Lynn B, Rodgers D, Shi J. “Towards biocatalytic lignin valorization in aqueous ionic liquids: inhibition mechanisms and product profiling” ACS Sus Chem. 2019 4. Stevens J, Shi J. “Biocatalysis in Ionic Liquids for Lignin Valorization: Opportunities and Recent Developments” Biotech Adv. 2019 5. Muley P, Mobley J, Tong X, Novak B, Stevens J, Moldovan D, Shi J, and Boldor D. “Rapid microwave-assisted biomass delignification and lignin depolymerization in deep eutectic solvents” En Conv Manag. 2019

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6. Das L, Li M, Stevens J, Li W, Pu Y, Ragauskas A, Shi J. “Characterization and catalytic transfer hydrogenolysis of deep eutectic solvent extracted sorghum lignin to phenolic compounds” ACS Sus Chem. 2018 7. Sheng J, Stevens J, Feng X. “Pathway compartmentalization in peroxisome of Saccharomyces cerevisiae to produce versatile medium chain fatty alcohols” Sci Rep. 2016 8. Chen Y, Sheng J, Jiang T, Stevens J, Feng X, Wei N. “Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae” Biotechnol Biofuels. 2016

Conference Presentations and Posters 1. Stevens, J., Hunter, J., Dumon, C., Rodgers, D., and Shi, J. (2019, November). “Improving resistance of hyperthermophilic laccase to ionic liquids for in situ lignin valorization” Paper presented at the 2019 AIChE Annual Meeting, Orlando, FL. 2. Vin-Nnajiofor, M., Shi, J., and Debolt, S. (2019, November). “Evaluating endocarp biomass as potential feedstocks for biorefinery” Paper presented for author at the 2019 AIChE Annual Meeting, Orlando, FL. 3. Stevens, J., Hunter, J., Esque, J., André, I., Dumon, C., Rodgers, D., and Shi, J. (2019, October). “A mixed technique approach to better understand interactions between laccases and ionic liquids” Poster presented at the 3rd Annual Commonwealth Computational Summit, Lexington, KY. 4. Stevens, J., Hunter, J., Dumon, C., Rodgers, D., and Shi, J. (2019, July). “Towards biocatalytic lignin valorization: Interactions between laccase and aqueous ionic liquids” Paper presented at the 15th International Conference on Renewable Resources and Biorefineries, Toulouse, France. 5. Li, W., Cheng, Y-T., and Shi, J. (2019, July). “Lignin derived carbon-silicon nanocomposite materials for energy storage applications” Paper presented for author at the 15th International Conference on Renewable Resources and Biorefineries, Toulouse, France. 6. Kalinoski, R., Stevens, J., Dodge, L., Das, L., Muley, P., Boldor, D., Nokes, S., and Shi, J. (2018, October). “Exploring Antimicrobial Properties of Lignin Derived Compounds and Materials” Paper presented at the AIChE Annual Meeting, Pittsburgh, PA. 7. Stevens, J., Mobley, J., Das, L., Rodgers, D., and Shi, J. (2018, October). “Towards Biocatalytic Lignin Valorization: Exploring Laccase-Ionic Liquid Interactions” Paper presented at the AIChE Annual Meeting, Pittsburgh, PA. 8. Stevens, J., Mobley, J., Das, L., Rodgers, D., and Shi, J. (2018, July). “Towards Biocatalytic Lignin Valorization: Exploring Laccase-Ionic Liquid Interactions” Poster presented at the Biocatalysis Gordon Research Conference, Biddeford, ME. 9. Stevens, J., Mobley, J., Das, L., Rodgers, D., and Shi, J. (2018, July). “Towards Biocatalytic Lignin Valorization: Exploring Laccase-Ionic Liquid Interactions” Poster presented at the Biocatalysis Gordon Research Seminar, Biddeford, ME. 10. Stevens, J., Mobley, J., Das, L., Rodgers, D., and Shi, J. (2018, March). “Lignin valorization via biocatalysis in aqueous ionic liquids” Poster presented at the 2018 EPSCoR Interjurisdictional Conference, Baton Rouge, LA.

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11. Stevens, J., Das, L., and Shi, J. (2017, December). “Directing product formation from lignin decomposition via biocatalysis in aqueous ionic liquids” Poster presented at the 7th Annual Sustainability Forum, Lexington, KY. 12. Stevens, J., Das, L., and Shi, J. (2017, November). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Poster presented at the 25th NSF EPSCoR National Conference, Missoula, MT. 13. Stevens, J., Liu, E., Das, L., and Shi, J. (2017, August). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Poster presented at the 2017 EPSCoR Interjurisdictional Conference, Lexington, KY. 14. Stevens, J. and Shi, J. (2017, June). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Paper presented at the 2017 KY NSF EPSCoR PechaKucha Challenge, Lexington, KY. 15. Stevens, J., Kalinoski, R., Das, L., and Shi, J. (2017, May). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Paper presented at the 2017 Renewable Energy & Energy Efficiency Workshop, Louisville, KY. 16. Stevens, J., Kalinoski, R., Das, L., and Shi, J. (2017, May). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Poster presented at the 2017 Renewable Energy & Energy Efficiency Workshop, Louisville, KY. 17. Stevens, J., Kalinoski, R., Das, L., and Shi, J. (2017, May). “Exploring Biocatalysis of Laccase in Ionic Liquids for Selective Decomposition of Lignin” Poster presented at the 2017 Symposium on Biotechnology for Fuels and Chemicals, San Francisco, CA. 18. Stevens, J., Sheng, J., and Feng, X. (2016, July). “Metabolic Engineering of Saccharomyces cerevisiae to produce long chain and medium chain fatty alcohols” Paper presented at the 2016 International ASABE Meeting, Orlando, FL.

PROFESSIONAL SERVICE • UK BAE Student Recruitment and Outreach Committee, 2019 - 2020 • UK BAE External Review Committee, 2018

AWARDS AND HONORS • University of Kentucky o 1st Place, 3rd Annual Commonwealth Computational Summit Poster Competition, 2019 o UK BAE Outstanding Ph.D. Student, 2019 o Chateaubriand STEM Fellowship, Embassy of France in the US, 2018-2019 o OTC Graduate Fellowship, Office of Technology Commercialization, 2018 o 1st Place, KY NSF EPSCoR PechaKucha Challenge, 2017 o 1st Place, BAE Graduate Student Poster Competition, 2017 • Virginia Tech o Kentuckiana Alumni Chapter Scholarship, Kentuckiana Alumni Chapter, 2012- 2013 o Distinguished University Scholar, Virginia Tech, 2012-2013 o J. Lawrence and Lucille G. Calhoun Endowment, Biosystems Engineering, 2013- 2016 o Peter H. Fitzgerald Endowment, Biosystems Engineering, 2014-2015

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