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Results for “enzymatic conversion”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Solid-State NMR Studies of Solvent-Mediated, Acid-Catalyzed Woody Biomass Pretreatment for Enzymatic Conversion of Residual Cellulose

Enzymes selectively hydrolyze the carbohydrate fractions of lignocellulosic biomass into corresponding sugars, but these processes are limited by low yields and slow catalytic turnovers. Under certain conditions, the rates and yields of enzymatic sugar production can be increased by pretreating biomass using solvents, heat, and dilute acid catalysts. However, the mechanistic details underlying this behavior are not fully elucidated, and designing effective pretreatment strategies remains an empirical challenge. Herein, using a combination of solid-state and high-resolution magic-angle-spinning NMR, infrared spectroscopy, and X-ray diffractometry, we show that the extent to which cellulase enzymes are able to hydrolyze solvent-pretreated biomass can be understood in terms of the ability of the solvent to break the chemical linkages between cellulose and noncellulosic materials in the cell wall. This finding is of general significance to enzymatic biomass conversion research, and implications for designing improved biomass conversion strategies are discussed. Furthermore, these findings demonstrate the utility of solid-state NMR as a tool to elucidate the key chemical and physical changes that occur during the liquid-phase conversion of real biomass.

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

Stereospecific Enzymatic Conversion of Boronic Acids to Amines

Boronic acids and esters are highly regarded for their safety, unique reactivity, and versatility in synthesizing a wide range of small molecules, bioconjugates, and materials. They are not exploited in biocatalytic synthesis, however, because enzymes that can make, break, or modify carbon–boron bonds are rare. We wish to combine the advantages of boronic acids and esters for molecular assembly with biocatalysis, which offers the potential for unsurpassed selectivity and efficiency. Here, we introduce an engineered protoglobin nitrene transferase that catalyzes the new-to-nature amination of boronic acids using hydroxylamine. Initially targeting aryl boronic acids, we show that the engineered enzyme can produce a wide array of anilines with high yields and total turnover numbers (up to 99% yield and >4000 TTN), with water and boric acid as the only byproducts. We also demonstrate that the enzyme is effective with bench-stable boronic esters, which hydrolyze in situ to their corresponding boronic acids. Exploring the enzyme’s capacity for enantioselective catalysis, we found that a racemic alkyl boronic ester affords an enantioenriched alkyl amine, a transformation not achieved with chemocatalysts. The formation of an exclusively unrearranged product during the amination of a boronic ester radical clock and the reaction’s stereospecificity support a two-electron process akin to a 1,2-metallate shift mechanism. Here, the developed transformation enables new biocatalytic routes for synthesizing chiral amines.

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

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

Abstract Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc‐dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl‐putrescine or ‐spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin‐labelled acetyl‐spermidine derivative to measure its PDAC activity, which is suitable for high‐throughput screening. Using this assay, we identified potent inhibitors of HDAC10‐mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10‐inhibitor structure using X‐ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

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

Data for Process Strategies for Recovery of Sugars, Lipids, and Lignin from Oilcane Bagasse Using Natural Deep Eutectic Solvents (NADES)

Sugarcane is being enhanced as a bioenergy crop by engineering it to accumulate and store lipids along with polymeric sugars in vegetative tissues. However, there is no existing process that allows for processing this new crop to recover both lipid and cellulosic sugars from the oilcane bagasse. Therefore, a comprehensive investigation of two pretreatment methods—natural deep eutectic solvents (NADES) and chemical-free hydrothermal pretreatment (HT) was conducted to judge their suitability for recovering fermentable sugars, lipids, and lignin from bagasse. Two NADES, i.e., choline chloride: lactic acid (ChCl:LA) and betaine: lactic acid (BT:LA) were prepared using a 1:2 M ratio and were evaluated for pretreatment of oilcane bagasse at 10, 20, and 50 % (w/w) solids, followed by enzymatic hydrolysis at 10 % (w/w) solids. Notably, ChCl:LA NADES treatment at 10 % (w/w) solids at 140 °C for 2 h, solubilized 78.8 % of lignin and 80.4 % of hemicellulose and allowed 82.7 % enzymatic conversion of glucans to glucose. In contrast, HT pretreatment removed approximately 87.6 % of the hemicellulose and provided an enzymatic glucose yield of 69.7 %. Furthermore, ChCl:LA operated at 50 % solids loading the enriched lipids 2.6-fold (9.2 wt%) in recovered solids compared to HT (6.4 %) and BT:LA (5.1 %) pretreatment processes. NMR-HSQC and GPC analysis showed that ChCl:LA also cleaved the most lignin β–O–4 linkages and demonstrated lower molecular weight compared to HT. This study demonstrates that NADES pretreatment is an effective green processing method for recovering lipids, sugars, and lignin from bioenergy crops at high solid loading (50 % w/w) within the context of an integrated biorefinery.

Conversion↗
DOE OSTI2024

Process strategies for recovery of sugars, lipids, and lignin from oilcane bagasse using natural deep eutectic solvents (NADES)

Sugarcane is being enhanced as a bioenergy crop by engineering it to accumulate and store lipids along with polymeric sugars in vegetative tissues. However, there is no existing process that allows for processing this new crop to recover both lipid and cellulosic sugars from the oilcane bagasse. Therefore, a comprehensive investi- gation of two pretreatment methods—natural deep eutectic solvents (NADES) and chemical-free hydrothermal pretreatment (HT) was conducted to judge their suitability for recovering fermentable sugars, lipids, and lignin from bagasse. Two NADES, i.e., choline chloride: lactic acid (ChCl:LA) and betaine: lactic acid (BT:LA) were prepared using a 1:2 M ratio and were evaluated for pretreatment of oilcane bagasse at 10, 20, and 50 % (w/w) solids, followed by enzymatic hydrolysis at 10 % (w/w) solids. Notably, ChCl:LA NADES treatment at 10 % (w/ w) solids at 140 °C for 2 h, solubilized 78.8 % of lignin and 80.4 % of hemicellulose and allowed 82.7 % enzymatic conversion of glucans to glucose. In contrast, HT pretreatment removed approximately 87.6 % of the hemicellulose and provided an enzymatic glucose yield of 69.7 %. Furthermore, ChCl:LA operated at 50 % solids loading the enriched lipids 2.6-fold (9.2 wt%) in recovered solids compared to HT (6.4 %) and BT:LA (5.1 %) pretreatment processes. NMR-HSQC and GPC analysis showed that ChCl:LA also cleaved the most lignin β–O–4 linkages and demonstrated lower molecular weight compared to HT. As a result, this study demonstrates that NADES pretreatment is an effective green processing method for recovering lipids, sugars, and lignin from bioenergy crops at high solid loading (50 % w/w) within the context of an integrated biorefinery.

09 BIOMASS FUELS↗
DOE OSTI2021

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol

Abstract Hydroxycinnamic acids such as p -coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum , Escherichia coli, and Bacillus subtilis . The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

4-hydroxystyrene↗
DOE OSTI2021

Project Liberty: Launch of an Integrated Bio-Refinery with Eco-Sustainable and Renewable Technologies. Conversion of Corn Stover Biomass to Bio-Ethanol, Final Report

POET-DSM Advanced Biofuels, in collaboration with parent companies, POET and DSM, has successfully designed, constructed, and continuously operated Project Liberty, a 770 bone-dry ton per day (tpd) nameplate capacity, 20 million gallon (cellulosic bioethanol) per year demonstration scale integrated biorefinery (IBR). The IBR produced cellulosic bio-ethanol from agriculture waste (corn stover) feedstock using next-generation pretreatment and enzymatic conversion technologies. The IBR plant was built and demonstrated operations in Emmetsburg, Iowa. The schedule for this IBR project is illustrated in Figure E1. Due to uncontrollable market factors and COVID-19 pandemic, cellulosic bioethanol production ceased near the end of 2019 and remaining operations were idled June of 2020.

09 BIOMASS FUELS↗
DOE OSTI2023

Surface Reorganization of Transition Metal Dichalcogenide Nanoflowers for Efficient Electrochemical Coenzyme Regeneration

In the past 20 years, enzymatic conversions have been intensely examined as a practical and environmentally friendly alternative to traditional organocatalytic conversions for chemicals and pharmaceutical intermediate production. Out of all commercial enzymes, more than one-fourth are oxidoreductases that operate in tandem with coenzymes, typically nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). Enzymes utilize coenzymes as a source for electrons, protons, or holes. Unfortunately, coenzymes can be exorbitant; thus, recycling coenzymes is paramount to establishing a sustainable and affordable cell-free enzymatic catalyst system. Herein, cost-effective transition metal dichalcogenides (TMDCs), 2H-MoS 2 , 2H-WS 2 , and 2H-WSe 2 , were employed for the first time for direct electrochemical reduction of NAD + to the active form of the NADH (1,4-NADH). Of the three TMDCs, 2H-WSe 2 shows optimal activity, producing 1,4 NADH at a rate of 6.5 μmol cm –2 h –1 and a faradaic efficiency of 45% at -0.8 V vs Ag/AgCl. Interestingly, a self-induced surface reorganization process was identified, where the native surface oxide grown in the air was spontaneously removed in the electrochemical process, resulting in the activation of TMDCs.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. Here, the effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH 4 ) reduction and sodium chlorite-acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X-ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH 4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%–50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions.

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

Investigation of Cyrene organosolv fractionation of softwood biomass and alkaline post-incubation

Cyrene organosolv fractionation effectively extracted 78% of lignin from recalcitrant softwood biomass pine at a mild temperature of 120°C. However, the enzymatic conversion of the fractionated cellulose-rich solid did not improve significantly. Alkali post-incubation of the fractionated cellulose fraction notably enhanced the glucan conversion. This phenomenon has also been observed in other organosolv processes, but how this approach transforms pretreated biomass has not yet been comprehensively investigated. In this study, small-angle X-ray scattering (SAXS) was employed to understand the structural changes in biomass during fractionation and post-incubation at the nanometer scale. Further, no lignin aggregation was found on the microfibrils, whereas the distance between the microfibrils increased after pretreatment and decreased after alkaline post-incubation. These results suggests that the Cyrene molecules remained between the microfibrils and were removed by post-incubation. In addition, the pine lignin recovered after pretreatment was characterized by NMR to understand the impact of Cyrene pretreatment on the lignin structure.

09 BIOMASS FUELS↗
DOE OSTI2023

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C–C bond formation. Here, in this study, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions – the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard β-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Understanding the Influence of Water-Soluble Compounds from Unpretreated Corn Stover Pellets on Enzymatic Hydrolysis of Cellulose

Aqueous extractives are minor and non-structural compounds in biomass that can be extracted using solvents, while contributing significantly to lignocellulose characteristics. Yet their roles in enzyme-mediated processing of lignocellulosic biomass remain elusive. Here, we examine the composition and features of extractives derived from untreated pelleted corn stover as well as their effects on enzymatic hydrolysis. Unlike the observations described in previous reports, we find that water extractable material improves the enzymatic hydrolysis of extractive-free stover by 67% with a glucose yield increase from 12% to 20% with 6 FPU cellulase per g glucan, whereas the enzyme activities are diminished when using microcrystalline cellulose as a substrate. The different behavior of corn stover and microcrystalline cellulose is likely attributed to the presence of lignin, which may interact with inhibitory compounds such as phenolics, mitigating the detrimental impacts of soluble inhibitors or insoluble lignin or both. These findings advance our fundamental understanding of the intrinsic behavior of extractives and help to optimize the schemes for efficient and cost-competitive enzymatic conversion of lignocellulose.

09 BIOMASS FUELS↗
DOE OSTI2025

Process innovations to enable viable enzymatic poly(ethylene terephthalate) recycling

Enzymatic depolymerization of poly(ethylene terephthalate) (PET) has received considerable attention for closed-loop polyester recycling. However, current approaches for enzymatic PET recycling face challenges to achieve commercial viability with lower environmental impacts compared with virgin polyester manufacturing. Here, in this study, we present multiple process innovations for enzymatic PET recycling that enable economic and environmental feasibility. We show that substrate amorphization through extrusion and quenching is energy-efficient and enables near-quantitative enzymatic conversion in 50 h. Using ammonium hydroxide for pH control and thermolysis of the isolated diammonium terephthalate salt reduces the acid and base consumption by >99%, lowering annual operating expenses by 74%. Fed-batch processing increased ethylene glycol concentration, leading to a 65% reduction in energy consumption for ethylene glycol recovery. These improvements were modeled in an optimal process, with recycled PET estimated to be US$\$$1.51 kg -1 relative to US domestic virgin PET at US$\$$1.87 kg -1 and eliminating key life cycle obstacles to scale this technology.

36 MATERIALS SCIENCE↗