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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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
NASA NTRS2011

Production of functional proteins: balance of shear stress and gravity

A method for the production of functional proteins including hormones by renal cells in a three dimensional culturing process responsive to shear stress uses a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-.alpha.-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D.sub.3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating an in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

Goodwin, Thomas John↗
DOE OSTI2023

Reversal of Catalytic Material Substrate Selectivity through Partitioning of Polymers in Hierarchically Ordered Virus-like Particle Frameworks

Control over the selectivity of catalytic materials is a topic of growing interest. Virus-like particle (VLP) based materials such as protein macromolecular frameworks (PMFs) are promising for catalytic applications due to their ease of assembly, modular ability to encapsulate a variety of enzymes, and ease of separation from a reaction mixture. Here we demonstrate the reversal of the initially negative material charge through the titration of a positively charged polymer into the material, causing the reversal of guest molecule uptake and enzymatic activity of PMFs. The charge-inverse material partitions a charged enzyme substrate to concentrate the substrate near an enzyme incorporated within the material, generating up to 5.9-fold increases in enzyme activity toward the partitioned substrate over the excluded substrate. Here we also show that the polymer distributes heterogeneously through the material up to a point of saturation and the effects of guest macromolecules on the lattice parameters of PMFs.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 Å resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this work highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

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

A broad specificity β-propeller enzyme from Rhodopseudomonas palustris that hydrolyzes many lactones including γ-valerolactone

Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca 2+ -dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this β-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other β-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1982

Zoological effects of variations in atmospheric oxygen levels

The role of certain gene enzymes in survival in modified atmospheres was examined. Chromosome morphology was studied. Mortality and life span were measured. Equipment to deliver various gas mixtures to the flies was designed and fabricated. To study the gene enzymes a technique called starch gel electrophoresis was needed. Equipment and supplies for this work and the study of chromosome morphology was available on the market, although some of the equipment was fabricated to save the project money.

Kloek, G.↗
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↗
NASA NTRS1972

Enzyme activity in terrestrial soil in relation to exploration of the Martian surface

Urease activity in soil is persistent for long periods under low water, low temperature, and sterile regimes, and it was suggested that some form of enzyme-protective mechanism exists in soil. Dublin soil was extracted by sonication in water followed by adding a mixture of salts. Urease activity is associated with the organo-mineral complex thus obtained and is resistant to the activities of proteolytic enzymes. Clay free soil organic matter prepared subsequently by filtration also exhibits urease activity which is resistant to proteolysis. Models consisting of enzymes with bentonite and lignin were found to mimic this resistance to proteolysis. A model system is presented which suggests both the origin and location of soil ureases and a reason for their persistence in nature.

Ardakani, M. S.↗
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↗
NASA NTRS1993

Evolution in vitro of an RNA enzyme with altered metal dependence

The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

NASA Discipline Exobiology↗
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↗