Kenneth L. Sale

Understanding the interactions of cellulose with ionic liquids: a molecular dynamics study.

Ionic liquids (ILs) have recently been demonstrated to be highly effective solvents for the dissolution of cellulose and lignocellulosic biomass. To date, there is no definitive rationale for selecting ionic liquids that are capable of dissolving these biopolymers. In this work, an all-atom force field for the IL 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] was developed and the behavior of cellulose in this IL was examined using molecular dynamics simulations of a series of (1-4) linked beta-d-glucose oligomers with a degree of polymerization n = 5, 6, 10, and 20. Molecular dynamics simulations were also carried out on cellulose oligomers in two common solvents, water and methanol, which are known to precipitate cellulose from IL solutions, to determine the extent and energetics of the interactions between these solvents and the cellulosic oligomers. Thermodynamic properties, such as density and solubility, as well as the two-body solute-solvent interaction energy terms, were calculated. The structural and dynamic behavior of solutions was analyzed and the conformations of cellulose oligomers were compared in ionic liquid and water mixtures. It was found that the interaction energy between the polysaccharide chain and the IL was stronger than that for either water or methanol. In addition to the anion acetate forming strong hydrogen bonds with hydroxyl groups of the cellulose, some of the cations were found to be in close contact with the polysaccharides through hydrophobic interactions. These results support the concept that the cation may play a significant role in the dissolution of cellulose by [C2mim][OAc]. It is also observed that the preferred beta-(1,4)-glycosidic linkage conformation of the cellulose was altered when dissolved in [C2mim][OAc] as compared to that found in crystalline cellulose dispersed in water. To our knowledge, this report is the first theoretical study that addresses the key factors in cellulose dissolution using an ionic liquid.

A Thermophilic Ionic Liquid-Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

Molecular dynamics study of polysaccharides in binary solvent mixtures of an ionic liquid and water.

Some ionic liquids (ILs) have great promise as effective solvents for biomass pretreatment, and there are several that have been reported that can dissolve large amounts of cellulose. The solubilized cellulose can then be recovered by addition of antisolvents, such as water or ethanol, and this regeneration process plays an important role in the subsequent enzymatic saccharification reactions and in the recovery of the ionic liquid. To date, little is known about the fundamental intermolecular interactions that drive the dissolution and subsequent regeneration of cellulose in complex mixtures of ionic liquids, water, and cellulose. To investigate these interactions, in this work, molecular dynamics (MD) simulations were carried out to study binary and ternary mixtures of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) with water and a cellulose oligomer. Simulations of a cellulose oligomer dissolved in three concentrations of binary mixtures of [C2mim][OAc] and water were used to represent the ternary system in the dissolution phase (high [C2mim][OAc] concentration) and present during the initial phase of the regeneration step (intermediate and low [C2mim][OAc] concentrations). The MD analysis of the structure and dynamics that exist in these binary and ternary mixtures provides information on the key intermolecular interactions between cellulose and [C2mim][OAc] that lead to dissolution of cellulose and the key intermolecular interactions in the intermediate states of cellulose precipitation as a function of water content in the cellulose/IL/water system. The analysis of this intermediate state provides new insight into the molecular driving forces present in this ternary system.

Understanding pretreatment efficacy of four cholinium and imidazolium ionic liquids by chemistry and computation

Certain ionic liquids (ILs) offer a potentially more sustainable and environmentally responsible alternative to organic solvents for many industrial applications, including biorefineries, where they are used to pretreat lignocellulose. To gain a more robust understanding of the roles of cations and anions in the process, we monitored the impact of the respective ILs on Panicum virgatum (switchgrass) in terms of lignin content, cellulose crystallinity, and enzymatic digestibility. The behaviors of four ILs, based on one of two cations, 1-ethyl-3-methylimidazolium ([C2mim]+) and cholinium ([Ch]+), and one of two anions, acetate ([OAc]−) and lysinate ([Lys]−), were compared. While all four ILs were effective in pretreating switchgrass, ILs containing [Lys]− anions provided greater delignification (70–80% vs. 16–50%) after addition of water as an anti-solvent and higher glucose yields (78–96% vs. 56–90%) compared to those obtained by the use of ILs containing [OAc]− anions. Measurements of the Kamlet–Taft parameters using a series of dyes indicated a greater hydrogen bond basicity for the ILs with [Lys]− anions as compared to acetate ILs. To understand the effective delignification ability of lysinate-based ILs, interaction energies of individual ions and ion pairs with a model dilignol substrate were determined by quantum chemical calculations. The results show that the addition of antisolvent significantly influenced the interaction energies governing lignin removal during the process.

Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry

Recent work using chemical cross‐linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G‐protein‐coupled receptor bovine rhodopsin in its native membrane using lysine‐ and cysteine‐targeted bifunctional cross‐linking reagents. Cross‐linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT‐ICR mass spectrometry with automated data‐reduction and assignment software. Tandem mass spectrometry was used to verify cross‐link assignments and locate the exact sites of cross‐link attachment. Cross‐links were observed to form between 10 pairs of residues in dark‐state rhodopsin. For each pair, cross‐linkers with a range of linker lengths were tested to determine an experimental distance‐of‐closest‐approach (DCA) between reactive side‐chain atoms. In all, 28 cross‐links were identified using seven different cross‐linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross‐link between C316 and K325 in the C‐terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross‐linking reactions. Biochemical and spectroscopic data from other studies have found that the C‐terminal region is highly mobile in solution and not fully represented by X‐ray crystallography data. Our results show that chemical cross‐linking can provide reliable three‐dimensional structural information and insight into local conformational dynamics in a membrane protein.

Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima

Tm_Cel5A, which belongs to family 5 of the glycoside hydrolases, is an extremely stable enzyme among the endo-acting glycosidases present in the hyperthermophilic organism Thermotoga maritima. Members of GH5 family shows a common (β/α)(8) TIM-barrel fold in which the catalytic acid/base and nucleophile are located on strands β-4 and β-7 of the barrel fold. Thermally resistant cellulases are desirable for lignocellulosic biofuels production and the Tm_Cel5A is an excellent candidate for use in the degradation of polysaccharides present on biomass. This paper describes two Tm_Cel5A structures (crystal forms I and II) solved at 2.20 and 1.85Å resolution, respectively. Our analyses of the Tm_Cel5A structure and comparison to a mesophilic GH5 provides a basis for the thermostability associated with Tm_Cel5A. Furthermore, both crystal forms of Tm_Cel5A possess a cadmium (Cd(2+)) ion bound between the two catalytic residues. Activity assays of Tm_Cel5A confirmed a strong inhibition effect in the presence of Cd(2+) metal ions demonstrating competition with the natural substrate for the active site. Based on the structural information we have obtained for Tm_Cel5A, protein bioengineering can be used to potentially increase the thermostability of mesophilic cellulase enzymes.

Discovery and characterization of ionic liquid-tolerant thermophilic cellulases from a switchgrass-adapted microbial community

BackgroundThe development of advanced biofuels from lignocellulosic biomass will require the use of both efficient pretreatment methods and new biomass-deconstructing enzyme cocktails to generate sugars from lignocellulosic substrates. Certain ionic liquids (ILs) have emerged as a promising class of compounds for biomass pretreatment and have been demonstrated to reduce the recalcitrance of biomass for enzymatic hydrolysis. However, current commercial cellulase cocktails are strongly inhibited by most of the ILs that are effective biomass pretreatment solvents. Fortunately, recent research has shown that IL-tolerant cocktails can be formulated and are functional on lignocellulosic biomass. This study sought to expand the list of known IL-tolerant cellulases to further enable IL-tolerant cocktail development by developing a combined in vitro/in vivo screening pipeline for metagenome-derived genes.ResultsThirty-seven predicted cellulases derived from a thermophilic switchgrass-adapted microbial community were screened in this study. Eighteen of the twenty-one enzymes that expressed well in E. coli were active in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) concentrations of at least 10% (v/v), with several retaining activity in the presence of 40% (v/v), which is currently the highest reported tolerance to [C2mim][OAc] for any cellulase. In addition, the optimum temperatures of the enzymes ranged from 45 to 95°C and the pH optimum ranged from 5.5 to 7.5, indicating these enzymes can be used to construct cellulase cocktails that function under a broad range of temperature, pH and IL concentrations.ConclusionsThis study characterized in detail twenty-one cellulose-degrading enzymes derived from a thermophilic microbial community and found that 70% of them were [C2mim][OAc]-tolerant. A comparison of optimum temperature and [C2mim][OAc]-tolerance demonstrates that a positive correlation exists between these properties for those enzymes with a optimum temperature >70°C, further strengthening the link between thermotolerance and IL-tolerance for lignocelluolytic glycoside hydrolases.

Production and extraction of sugars from switchgrass hydrolyzed in ionic liquids

BackgroundThe use of Ionic liquids (ILs) as biomass solvents is considered to be an attractive alternative for the pretreatment of lignocellulosic biomass. Acid catalysts have been used previously to hydrolyze polysaccharides into fermentable sugars during IL pretreatment. This could potentially provide a means of liberating fermentable sugars from biomass without the use of costly enzymes. However, the separation of the sugars from the aqueous IL and recovery of IL is challenging and imperative to make this process viable.ResultsAqueous alkaline solutions are used to induce the formation of a biphasic system to recover sugars produced from the acid catalyzed hydrolysis of switchgrass in imidazolium-based ILs. The amount of sugar produced from this process was proportional to the extent of biomass solubilized. Pretreatment at high temperatures (e.g., 160°C, 1.5 h) was more effective in producing glucose. Sugar extraction into the alkali phase was dependent on both the amount of sugar produced by acidolysis and the alkali concentration in the aqueous extractant phase. Maximum yields of 53% glucose and 88% xylose are recovered in the alkali phase, based on the amounts present in the initial biomass. The partition coefficients of glucose and xylose between the IL and alkali phases can be accurately predicted using molecular dynamics simulations.ConclusionsThis biphasic system may enable the facile recycling of IL and rapid recovery of the sugars, and provides an alternative route to the production of monomeric sugars from biomass that eliminates the need for enzymatic saccharification and also reduces the amount of water required.

Neutron Reflectometry and QCM-D Study of the Interaction of Cellulases with Films of Amorphous Cellulose

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) of the interaction of a fungal enzyme extract ( T. viride ) and an endoglucanse from A. niger with amorphous cellulose films. The use of amorphous cellulose is motivated by that the fact that several biomass pretreatments currently under investigation disrupt the native crystalline structure of cellulose and increase the amorphous content. NR reveals the profile of water through the film at nanometer resolution and is highly sensitive to interfacial roughness, whereas QCM-D provides changes in mass and film stiffness. NR can be performed using either H(2)O- or D(2)O-based aqueous reservoirs. NR measurement of swelling of a cellulose film in D(2)O and in H(2)O revealed that D/H exchange on the cellulose chains must be taken into account when a D(2)O-based reservoir is used. The results also show that cellulose films swell slightly more in D(2)O than in H(2)O. Regarding enzymatic digestion, at 20 °C in H(2)O buffer the T. viride cocktail rapidly digested the entire film, initially roughening the surface, followed by penetration and activity throughout the bulk of the film. In contrast, over the same time period, the endoglucanase was active mainly at the surface of the film and did not increase the surface roughness.

Simulations Reveal Conformational Changes of Methylhydroxyl Groups during Dissolution of Cellulose Iβ in Ionic Liquid 1-Ethyl-3-methylimidazolium Acetate

In this work, we use molecular dynamics (MD) simulations to study the dissolution of microcrystalline cellulose in the ionic liquid 1-ethyl-3-methylimidazolium acetate (abbreviated as [C2mim][OAc]) at 20 wt % loading. The interactions of [C2mim][OAc] with the Iβ cellulose structure at 120 °C were studied. The results show that both the cation and the anion of [C2mim][OAc] penetrate into the cellulose Iβ crystal structure but that the anion in particular forms strong hydrogen bonds with cellulose. Our results also show that the methylhydroxyl groups of cellulose solvated in [C2mim][OAc] are predominantly in the gauche-trans (gt) conformation, in contrast to the dominant trans-gauche (tg) conformation of cellulose Iβ in air or the gauche-gauche (gg) conformation for cellulose chains in water or after pretreatment with ammonia. Because the gt conformation is found mainly in cellulose II, these simulations suggest that regenerated cellulose under similar pretreatment conditions is composed mainly of cellulose II, and this result was confirmed by X-ray diffraction of samples processed under similar pretreatment conditions. These simulations provide new insight into the efficacy of [C2mim][OAc] pretreatment, suggesting that [C2mim][OAc] interacts with and biases the methylhydroxyl groups of cellulose toward orientations that are consistent with the experimentally observed more easily hydrolyzed cellulose II.