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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Spray Drying Enzyme-Treated Cellulose Nanofibrils

Enzyme-treated cellulose nanofibrils (CNFs) were produced via a lab-scale mass colloider using bleached kraft pulp (BKP) to evaluate their processability and power requirements during refining and spray-drying operations. To evaluate the energy efficiency in the CNF refining process, the net energy consumption, degree of polymerization (DP), and viscosity were determined. Less energy was consumed to attain a given fines level by using the endoglucanase enzymes. The DP and viscosity were also decreased using the enzymes. The morphological properties of the enzyme-pretreated spray-dried CNF powders (SDCNFs) were measured. Subsequently, the enzyme-pretreated SDCNFs were added to a PP matrix with MAPP as a coupling agent. The mixture was then compounded through a co-rotating twin-screw extruder to determine whether the enzyme treatment of the CNFs affects the mechanical properties of the composites. Compared to earlier studies on enhancing PMCs with SDCNF powders, this research investigates the use of enzyme-pretreated SDCNF powders. It was confirmed that the strength properties of PP increased by adding SDCNFs, and the strength properties were maintained after adding enzyme-pretreated SDCNFs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Neural network based analysis of multimodal bond distributions using extended x-ray absorption fine structure spectra

Knowledge of the local coordination environment around atomic species in functional materials is critical for understanding their mechanisms of operation. Heterogeneous mixtures of metal complexes are ubiquitous in catalysts, ionic liquids, molten salts, biological enzymes, and geochemical systems, among many others. Extracting information from ensemble-average measurements about the structural and compositional descriptors of each type of coordination complex comprising the mixture is not generally possible, especially when they possess multimodal bond-length distributions. Here, we developed a method that enables the mapping of an x-ray absorption spectrum on the radial distribution function describing the average environment of the metal ions. The supervised neural network based method utilizes an objective training set, for which the choice of the local structural motifs is completely agnostic to the theoretically expected structure and dynamics of the modeled system. The method was validated using first-principles modeling of structural dynamics of nickel complexation in molten salts, and it applies to a large class of heterogeneous systems, including those studied under in situ and operando conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Engineering an aldoxime dehydratase with high activity and isomer tolerance for biosynthesis of an O -protected primary cyanohydrin

O-protected primary cyanohydrins (glycolonitriles) are important building blocks for many difunctionalized compounds and precursors to known bioactive molecules. Their synthesis, however, utilizes toxic cyanide, which raises significant safety concerns for industrial synthesis. Here, in this study, we present a cyanide-free enzymatic synthesis of an o-benzyl protected primary cyanohydrin from an (E)- or (Z)-α-oxygen protected aldoxime using an engineered aldoxime dehydratase enzyme from Bacillus sp. OxB-1 (OxdB). In contrast to many evolved enzymes that tend to “specialize” as their activity increases, we used directed evolution to engineer OxdB for efficient dehydration of both isomers in a mixture of (E)- or (Z)-α-oxygen aldoximes with high activity and substrate loading to achieve near quantitative yield. Using this enzyme, we further demonstrate a cyanide-free chemoenzymatic pathway to an o-protected primary cyanohydrin starting from a readily available aldehyde, where the aldehyde is first condensed with hydroxylamine, followed by dehydration using our evolved enzyme. This pathway was readily scaled up to 1 g scale with high substrate loading, demonstrating its utility in industrial synthesis of these important building block functional groups.

Aldoxime dehydratase↗
DOE OSTI2021

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Hagen, Live↗
DOE OSTI2026

Catalytic Triad-Inspired Nanozyme Catalysts for Ester Hydrolysis in Organic Solvent Mixtures

We synthesized a series of molecular nanozyme catalysts containing functional groups (imidazole, carboxylic acid, and hydroxyl groups) inspired by the catalytic triad found in natural serine hydrolases. Different structural features were incorporated using two distinct synthesis routes to investigate the influence of interactions beyond the active site on catalytic activity in designing molecular nanozymes. Molecular dynamics simulations suggest that nanozyme activity is affected by structural features that influence nanozyme hydrophilicity and the organization of the local solvent environment. The most active nanozyme showed activity comparable to the enzyme α-chymotrypsin for the hydrolysis of a model ester (4-nitrophenyl 4- hydroxybenzoate) in a 95:5 (v/v%) water/acetonitrile mixture at ambient temperature. Increasing the temperature and organic solvent content decreases the activity of α-chymotrypsin while enhancing the activity of the nanozymes. The nanozymes can be immobilized on supported metal nanoparticles using a dithiol self-assembled monolayer, which facilitates their removal from the postreaction solution. These results demonstrate the potential in creating solvent-tolerant bioinspired catalysts, thereby combining the advantages of biocatalysts and chemical catalysts as next-generation industrial catalysts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Cryo-EM structures of DNA-free and DNA-bound BsaXI: architecture of a Type IIB restriction–modification enzyme

Abstract We have determined multiple cryogenic electron microscopy (cryo-EM) structures of the Type IIB restriction–modification enzyme BsaXI. Such enzymes cleave DNA on both sides of their recognition sequence and share features of Types I, II, and III restriction systems. BsaXI forms a heterotrimeric (RM)2S assemblage in the presence and absence of bound DNA. Two unique structural motifs—a multi-helical “knob” and a long antiparallel double-helical “paddle”—are involved in DNA binding and cleavage. Binding of the DNA target triggers a large conformational change from an ‘open’ to ‘closed’ configuration, resulting in a mixture of two different conformations with respect to the positioning of the S subunit and its target recognition domains on the enzyme’s bipartite DNA target site. Structure-guided mutagenesis studies implicated two clusters of residues in the RM subunit as being critical for DNA cleavage, both are located proximal to a DNA cleavage site. One corresponds to a canonical PD-(D/E)xK endonuclease site in the N-terminal endonuclease domain, while the other corresponds to residues clustered within the paddle motif (near to the C-terminal end of the RM subunit). This analysis facilitates a comparison of three potential mechanisms by which such enzymes cleave DNA on each side of the bound target.

Shen, Betty W.↗
DOE OSTI2024

Lignin molecular weights of Populus trichocarpa residues after CELF pretreatment

Here we present a dataset of molecular weights of lignin from a woody energy crop (Populus trichocarpa) residues after a series of co-solvent enhanced lignocellulosic fractionation (CELF) pretreatment. The natural poplar variant GW-9947 from the Center for Bioenergy Innovation (CBI) was used. The poplar was knife milled and passed through a 1 mm sieve and CELF pretreatment was performed in a Parr autoclave reactor with 7.5 wt % solids loading, 0.5 wt% H2SO4 as catalyst at 150°C with various time. Tetrahydrofuran was added in a 1:1 mass ratio with water as the pretreatment solvent. Lignin was isolated from the pretreated samples after ball-milling in a porcelain jar with ceramic balls via Retsch PM 200 at 580 rpm for 2.5 h followed by enzymatic hydrolysis in acetate buffer (pH 4.8, 50 °C) for 48 h. The solid residue was isolated by centrifugation and hydrolyzed again with freshly added buffer and enzymes for another 48 h. After filtration, the solid residue was extracted twice with 96% (v/v) 1,4-dioxane/water mixture at room temperature overnight. The extracts were combined, rotary evaporated, and freeze-dried to recover lignin. The lignin samples were then derivatized in an acetic anhydride/pyridine (1:1, v/v) mixture and stirred at room temperature for 24 h. Ethanol was added to the reaction mixture, left for 30 min and then removed with a rotary evaporator. The addition and removal of ethanol was repeated at least 3 times until all traces of acetic acid were removed. Acetylated lignin samples were then dissolved in tetrahydrofuran (THF) at a concentration of 1.0 mg/mL. The molecular weight of acetylated lignin was measured by a gel permeation chromatography (GPC) on a PSS-Polymer Standards Service (Warwick, RI, USA) GPC SECurity 1200 system featuring Agilent HPLC 1200 components equipped with four Waters Styragel columns (HR1, HR2, HR4 and HR6) and an UV detector (270 nm). Tetrahydrofuran was used as the mobile phase and flow rate was 0.3 mL/min. The Polymer Standards Service WinGPC Unity software (Build 6807) was used for data processing for all the samples. The data provides information about the effects of CELF pretreatment time at 150 ºC on lignin molecular weights.

09 BIOMASS FUELS↗
DOE OSTI2023

Factors that influence the activity of biomass-degrading enzymes in the presence of ionic liquids—a review

Ionic liquids (ILs) are seen as a more sustainable alternative to volatile organic solvents that are accelerating innovations in many industries such as energy storage, separations, and bioprocessing. The ability to effectively deconstruct lignocellulosic biomass is a significant hurdle in the biorefining/bioprocessing industry and presents limitations towards the commercial production of bioproducts (such as biofuels, biomaterials, etc. ). Certain ILs have been shown to promote effective lignin removal, cellulose recovery, and sugar yields from various biomass feedstocks such as corn stover, wheat straw, sugarcane bagasse, sorghum, switchgrass, miscanthus, poplar, pine, eucalyptus, and certain mixtures of municipal solid waste. However, these improvements are often counteracted by the limited biocompatibility of ILs, which results in an IL-induced reduction in enzyme activity and stability—an important downstream step in the conversion of biomass to biofuels/bioproducts. As a result, significant efforts have been made to discover and engineer compatible enzyme-IL systems and to improve our understanding on the effect that these ILs have on these systems. This review seeks to examine the impact of ionic liquids on enzymes involved in lignocellulosic biomass deconstruction, with a specific focus on their relevance in the context of pretreatment. Beyond presenting an overview of the ionic liquid pretreatment landscape, we outline the main factors that influence enzyme activity and stability in the presence of ILs This data is consolidated and analyzed to apply this body of knowledge towards new innovations that could lead to improvements in the processing of biomass to biofuels and bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l - and d -amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC 50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

42 ENGINEERING↗
DOE OSTI2021

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accu- mulation of by-products due to overflow metabolism of glucose hampered efficient production of 2' -FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engi- neered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to pro- duce 2' -FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. In conclusion, as yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumu- lating yeast cells.

2′ -fucosyllactose↗
DOE OSTI2020

Data from Enhanced 2′-Fucosyllactose Production by Engineered Saccharomyces cerevisiae using Xylose as a Co-Substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from E. coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Conversion↗
DOE OSTI2024

Enhanced Interfacial Electron Transfer in Photocatalyst-Natural Enzyme Coupled Artificial Photosynthesis System: Tuning Strategies and Molecular Simulations

Laccase is capable of catalyzing a vast array of reactions, but its low redox potential limits its potential applications. The use of photocatalytic materials offers a solution to this problem by converting absorbed visible light into electrons to facilitate enzyme catalysis. Herein, MIL-53(Fe) and NH 2 -MIL-53(Fe) serve as both light absorbers and enzyme immobilization carriers, and laccase is employed for solar-driven chemical conversion. Electron spin resonance spectroscopy results confirm that visible light irradiation causes rapid transfer of photogenerated electrons from MOF excitation to T1 Cu(II) of laccase, significantly increasing the degradation rate constant of tetracycline (TC) from 0.0062 to 0.0127 min -1 . Conversely, there is only minimal or no electron transfer between MOF and laccase in the physical mixture state. Theoretical calculations demonstrate that the immobilization of laccase's active site and its covalent binding to the metal-organic framework surface augment the coupled system's activity, reducing the active site accessible from 27.8 to 18.1 Å. In conclusion, the constructed photo-enzyme coupled system successfully combines enzyme catalysis’ selectivity with photocatalysis's high reactivity, providing a promising solution for solar energy use.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Pseudomonas sp. Strain 273 Degrades Fluorinated Alkanes

Fluorinated organic compounds have emerged as environmental constituents of concern. We demonstrate that the alkane degrader Pseudomonas sp. strain 273 utilizes terminally monofluorinated C 7 –C 10 alkanes and 1,10-difluorodecane (DFD) as the sole carbon and energy sources in the presence of oxygen. Strain 273 degraded 1-fluorodecane (FD) (5.97 ± 0.22 mM, nominal) and DFD (5.62 ± 0.13 mM, nominal) within 7 days of incubation, and 92.7 ± 3.8 and 90.1 ± 1.9% of the theoretical maximum amounts of fluorine were recovered as inorganic fluoride, respectively. With n-decane, strain 273 attained (3.24 ± 0.14) × 10 7 cells per μmol of carbon consumed, while lower biomass yields of (2.48 ± 0.15) × 10 7 and (1.62 ± 0.23) × 10 7 cells were measured with FD or DFD as electron donors, respectively. The organism coupled decanol and decanoate oxidation to denitrification, but the utilization of (fluoro)alkanes was strictly oxygen-dependent, presumably because the initial attack on the terminal carbon requires oxygen. Fluorohexanoate was detected as an intermediate in cultures grown with FD or DFD, suggesting that the initial attack on the fluoroalkanes can occur on the terminal methyl or fluoromethyl groups. Overall, the findings indicate that specialized bacteria such as Pseudomonas sp. strain 273 can break carbon–fluorine bonds most likely with oxygenolytic enzyme systems and that terminally monofluorinated alkanes are susceptible to microbial degradation. The findings have implications for the fate of components associated with aqueous film-forming foam (AFFF) mixtures.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Determination of Thiol Protonation States by Sulfur X-ray Spectroscopy in Biological Systems

Cysteine is one of the most functionally diverse of the proteinogenic amino acids, owing to its reactive thiol side chain that can undergo deprotonation to form a strongly nucleophilic thiolate. However, few techniques can directly interrogate sulfur charge and covalency in cysteine, particularly in proteins. X-ray spectroscopies provide an element specific probe of sulfur. We demonstrate the sensitivity of S Kβ and Kα X-ray emission spectroscopy (XES) to cysteine ionization and compare it to S K-edge X-ray absorption spectroscopy (XAS) in the physiologically relevant biomolecules l-cysteine and N-acetyl-l-cysteine at room temperature in solution phase. Kβ XES and K-edge XAS are most sensitive to chemical changes at the cysteine thiol and can be used to evaluate the composition of thiol/thiolate mixtures. Furthermore, these results provide a foundation for assessing the pK a of functionally significant cysteine residues in proteins and open the door to time-resolved studies of cysteine-dependent enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Superior performance biodiesel from biomass-derived fusel alcohols and low grade oils: Fatty acid fusel esters (FAFE)

We demonstrated production of a superior performance biodiesel referred to here as fatty acid fusel alcohol esters (FAFE) – by reacting fusel alcohols (isobutanol, 3-methyl-1-butanol, and (S)-(-)-2-methyl-1-butanol) with oil (glyceryl trioleate) using lipase from Aspergillus oryzae. Reaction conditions corresponding to a molar ratio of 5:1 (fusel alcohols to oil), enzyme loading of 2% w/w, reaction temperature of 35 °C, shaking speed of 250 rpm, and reaction time of 24 h achieved >97% conversion to FAFE. Further, FAFE obtained from reacting a fusel alcohol mixture with corn oil were evaluated for use as a fuel for diesel engines. FAFE mixtures showed superior combustion and cold-flow properties, with the derived cetane numbers up to 4.8 points higher, cloud points up to -6 °C lower, and the heat of combustion up to 2.1% higher than the corresponding FAME samples, depending on the fusel mixture used. This represents a significant improvement for all three metrics, which are typically anti-correlated. Finally, FAFE provides a new opportunity for expanded usage of biodiesel by addressing feedstock limitations, fuel performance, and low temperature tolerance.

09 BIOMASS FUELS↗