Molecular determinants of nonaqueous biocatalysis

Dissertation presented to obtain the Ph.D degree in BiochemistryOver the last thirty years, the tremendous biotechnological potential of nonaqueous biocatalysis has boosted research efforts in this area. Numerous studies have tried to elucidate how enzymes work in these nonconventional media and many properties are now well understood. However, when this thesis was initiated, some aspects of this field were poorly characterized at the molecular level. In particular, the molecular determinants of protein-ion interactions, enzyme stability, and molecular memory, are important issues which were lacking a thorough molecular analysis. These three subjects are herein investigated using molecular simulation methodologies.(...

New insights into the molecular mechanism of methanol-induced inactivation of Thermomyces lanuginosus lipase: A molecular dynamics simulation study

Methanol intolerance of lipase is a major limitation in lipase-catalyzed methanolysis reactions. In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solvent-exposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak. Additionally, the catalytically relevant hydrogen bond between Asp201 OD2 and His258 ND1 in the active site was broken when the enzyme was solvated in methanol. This may affect the stability of the tetrahedral intermediates in the catalytic cycle of TLL. Furthermore, compared to those in water, some enzyme surface residues displayed enhanced movement in methanol with higher Cα root-mean-square atomic positional fluctuation values. One of such methanol-affecting surface residues (Ile241) was chosen for mutation, and MD simulation of the I241E mutant in methanol was conducted. The structural analysis of the mutant showed that replacing a non-polar surface residue with an acidic one at position 241 contributed to the stabilization of enzyme structure in methanol. Ultimately, these results, while providing molecular-level insights into the destabilizing effect of methanol on TLL, highlight the importance of surface residue redesign to improve the stability of lipases in methanol environments

Biomass processing using ionic liquids: Effects of 3-methylimidazolium cations and carboxylate anions

As the global transportation and industrial sectors continue to grow, fuels, chemicals, and products derived from lignocellulosic biomass have become a key alternative to petroleum-based products. Lignocellulosic biomass is composed of lignin, cellulose, and hemicellulose linked together in a rigid structure. This spatial arrangement contributes to its resistance to degradation and requires pretreatment and/or separation before being processed to produce valuable chemicals and fuels. Biomass pretreatment has mainly been optimized to convert carbohydrates into monosugars. However, better sustainability is attained when the entire feedstock is utilized to produce fuel and value-added chemicals and products. To achieve this goal, an integrated biorefinery will require a highly selective and economically viable fractionation process. Although traditionally used for pretreatment, recent studies have found ionic liquids to be ideal solvents for biomass dissolution, “activation”, and fractionation to produce various end products for biorefinery and industrial applications.Previous works have demonstrated that the IL 1-ethyl-3-methylimidazolium acetate ([EMIM]Acetate) is ideal for the above processes to produce sugars as well as lignin-based products. However, our study shows that three other ILs with 3-methylimidazolium cations and carboxylate anions (1-ethyl-3-methylimidazolium formate ([EMIM]Formate), 1-allyl-3-methylimidazolium formate ([AMIM]Formate), and 1-allyl-3-methylimidazolium acetate ([AMIM]Acetate)) are effective for biomass dissolution, with [AMIM]Formate having a 40% increase in biomass solubility compared to [EMIM]Acetate. Both [AMIM]Formate and [EMIM]Acetate are further evaluated for their activation and fractionation capability by studying crystallinity changes and enzymatic conversion rates of cellulose and hemicellulose into soluble sugars. Our findings show that although [AMIM]Formate is better at biomass dissolution, [EMIM]Acetate is better for biomass activation and fractionation. Following activation using [AMIM]Formate, biomass retains its most of its crystallinity and acetyl groups, whereas activation using [EMIM]Acetate significantly reduces crystallinity and acetyl groups, leading to higher enzymatic conversion of cellulose and hemicellulose. Future studies should investigate the potential for in situ saccharification in ILs using commercial cellulases and hemicellulases, as our preliminary data show that enzymes remain active in these two ILs. Ultimately, this research will provide technological breakthroughs needed to develop a robust means of biomass fractionation and subsequent conversion into high value organics and biofuels

Structure-function studies of GH7 cellulases, key enzymes in the global carbon cycle

Enzyme mixtures used for lignocellulosic ethanol production are most commonly derived from filamentous fungi, and enzymes from the glycoside hydrolase family 7 (GH7) constitute the most abundant components in these cocktails. In this thesis I have aimed to increase our understanding of this enzyme family, with focus on the interrelation between their structure and function.In a study of the two model enzymes Trichoderma reesei Cel7A (TreCel7A) and Phanerochaete chrysosporium Cel7D (Pch7D), we determined factors governing the idiosyncratic behavior of these enzymes on commonly used model compounds, and by using fluorescence titration, enzyme kinetics, structure determination and molecular dynamics simulations found specific structural features connected to nonproductive binding, playing a major role in enzyme activity on these compounds.We also determined the molecular structure of a GH7 enzyme RsSymEG1, belonging to a group of smaller GH7 endoglucanases with previously unknown structure architecture, and originating from symbiotic protozoa of wood eating lower termites. The X-ray crystal structure revealed a configuration with several keydifferences to previously known GH7 structures, and will aid in modelling and engineering of enzymes in this so far little-known group of enzymes. A further look into this group of sequences, as well as other GH7 enzymes found in the termite symbiont protists, also revealed previously unknown details about the evolution ofthis ancient enzyme family.Furthermore, we explored single molecule imaging of the model enzymeTreCel7A with novel imaging methods, providing a first proof-of-concept of usingfluorescence resonance energy transfer (FRET) for the study of inter-domaindynamics of this enzyme, as well as total internal reflection dark-field microscopy(TIRDFM) for imaging enzyme movement on cellulose surface at ultra-hightemporal resolutions

Processing of Cellulose for the Advancement of Biofuels

The enzymatic degradation of cellulose polymers is currently a rate-limiting step in the bioconversion of biomass to biofuels. Cellulose polymers self assemble to form crystalline structures stabilized by a complex network of intermolecular interactions such as hydrogen bonding. The network of interactions in crystalline cellulose (cellulose nanostructure) poses an energy barrier that limits enzymatic degradation as apparent from the activity of Cel5H. To improve the degradability of cellulose the intermolecular interactions must be disrupted. The interactions of the cellulose nanostructure prevent solubilization by water and most other common solvents, but some organic solvents aid degradation of cellulose suggesting they influence cellulose nanostructure. The objective of this work is to understand the influence of solvents on cellulose nanostructure with the goal of improving the degradability of cellulose nanostructure using solvents. To understand solvent interaction with cellulose, phosphoric acid was used to first solubilize cellulose (PAS cellulose) followed by adding an organic liquid or water to wash the phosphate from the system. The Flory Huggins theory was used to predict wash liquids that could favorably interact with cellulose. A favorable wash liquid was predicted to prevent the reformation of crystalline domains to yield a disrupted cellulose nanostructure, which should be more degradable. Low molecular weight alcohols and glycols were calculated to be favorable wash liquids. Washing PAS cellulose with the predicted favorable liquids yielded semi-transparent gel-like materials compared to the opaque white precipitate formed when water or unfavorable solvents were used in the wash. Fractal analysis of small angle neutron scattering (SANS) of these apparent gels indicated cellulose polymers likely have the properties of clustered rods. This partial disruption increased degradability relative to the water washed PAS cellulose. The apparent rod-like cellulose nanostructures suggested the presence of intra and interpolymer hydrogen bonding. Characterization of the hydrogen bonding network by Fourier transform infrared resonance (FTIR) indicated the gel-like material formed by ethanol washes was the result of heterogeneous interpolymer hydrogen bond cross-links. The interactions leading to gel-like materials were evaluated using Hansen solubility parameters, which predicted mixtures of ethanol and water may be most effective for disrupting cellulose nanostructure. Fractal analysis by SANS indicated 40 % ethanol/water was most effective. Similar results were obtained when 40 % ethanol was used to disrupt the cellulose nanostructure in municipal office waste (MOW). Ethanol washes increased the degradability of MOW by at least 30 % relative to conventional water washing. This is significant because increased degradability of MOW could further the development of cellulosic biofuels by reducing the amount of enzyme required to digest the material

Measurement and correlation of liquid - Liquid equilibria of three imidazolium ionic liquids with acetone and cyclohexane

Ionic liquids (ILs) can be recycled as extractants for their low vapor pressure and volatility. More and more applications are applied to the separation of industrial organic matter. The industrial production of ILs has gradually been realized, which also widens the way for the application of ILs. In this work, the liquid-liquid extraction of cyclohexane-acetone azeotropic mixture with different ILs {1-butyl-3-methylimidazolium bis(trifluormethylsulfonyl), 1-butyl-3-methylimidazolium trifluoromethansulfonate and 1-butyl-3-methylimidazolium dicyanamide} is studied. The extraction mechanism is discussed based on the molecular scale. The relationship between hydrogen bond donor and acceptor between ILs and acetone is analyzed by COSMO-SAC. The interaction between molecules is optimized and calculated by Materials Studio 7.0. The extraction ability of ILs is analyzed by radial distribution function, and the experimental results are verified. The liquid-liquid equilibrium test is carried out at 298.15 K. Distribution and selectivity are indices used to judge the extraction efficiency of ILs. The NRTL model and UNIQUAC model are adopted to correlate the liquid-liquid equilibrium data. The results show that all of the two models can well correlate the experimental.This work is supported by the National Natural Science Foundation of China (No. 21776145), National Natural Science Foundation of China (No. 21676152)

Catalysis and biocatalysis program

The annual report presents the fiscal year (FY) 1990 research activities and accomplishments for the Catalysis and Biocatalysis Program of the Advanced Industrial Concepts Division (AICD), Office of Industrial Technologies of the Department of Energy (DOE). The mission of the AICD is to create a balanced program of high risk, long term, directed interdisciplinary research and development that will improve energy efficiency and enhance fuel flexibility in the industrial sector. The Catalysis and Biocatalysis Program's technical activities were organized into five work elements: the Molecular Modeling and Catalysis by Design element; the Applied Microbiology and Genetics element; the Bioprocess Engineering element; the Separations and Novel Chemical Processes element; and the Process Design and Analysis element

Time-resolved and steady-state studies of biologically and chemically relevant systems using laser, absorption, and fluorescence spectroscopy

In Chapter 2 several experimental and data analysis methods used in this thesis are described. In Chapter 3 steady-state fluorescence spectroscopy was used to determine the concentration of the efflux pump inhibitors (EPIs), pheophorbide a and pyropheophorbide a, in the feces of animals and it was found that their levels far exceed those reported to be inhibitory to efflux pumps. In Chapter 4 the solvation dynamics of 6-Propionyl-2-(N,N-dimethyl) aminonaphthalene (PRODAN) was studied in reverse micelles. The two fluorescent states of PRODAN solvate on different time scales and as such care must be exercised in solvation dynamic studies involving it and its analogs. In Chapter 5 we studied the experimental and theoretical solvation dynamics of coumarin 153 (C153) in wild-type (WT) and modified myoglobins. Based on the nuclear magnetic resonance (NMR) spectroscopy and time-resolved fluorescence studies, we have concluded that it is important to thoroughly characterize the structure of a protein and probe system before comparing the theoretical and experimental results. In Chapter 6 the photophysical and spectral properties of a derivative of the medically relevant compound curcumin called cyclocurcumin was studied. Based on NMR, fluorescence, and absorption studies, the ground- and excited-states of cyclocurcumin are complicated by the existence of multiple structural isomers. In Chapter 7 the hydrolysis of cellulose by a pure form of cellulase in an ionic liquid, HEMA, and its aqueous mixtures at various temperatures were studied with the goal of increasing the cellulose to glucose conversion for biofuel production. It was found that HEMA imparts an additional stability to cellulase and can allow for faster conversion of cellulose to glucose using a pre-treatment step in comparison to only buffer