Search NASA⌕ Search

SEARCH · Search NASA

Results for “Enzyme engineering”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

Development of thermostable carbonic anhydrases using structure-guided recombination for use in CO2 removal systems on spacecraft

Carbon capture and storage has been a research area of great interest in recent years asa method for mitigation of CO2emissions, due to the effects of climate change. The development of technologies for the efficient capture of CO2are also of great interest for human spaceflight applications. One of the most promising technologies in this area is CO2 scrubbing using liquid amines, unfortunately, liquid amines with low heats of desorption tend to have slow CO2binding kinetics. One potential solution to this problem is to use the enzyme carbonic anhydrase (CA) to enhance the kinetics of CO2binding to liquid amines, allowing the overall process to be more energy efficient. Interest in using carbonic anhydrase as a biocatalyst has led to a number of efforts to improve the thermostability and solvent tolerance of several distinct carbonic anhydrase enzymes. In the work described here, we screened through a diverse set of natural carbonic anhydrases to identify candidates for protein engineering aimed at increased stability and activity in various liquid amines. We then used SCHEMA structure-guided recombination to develop a set of chimeric carbonic anhydrases with high thermostability and activity. These chimeras were used as the starting points for further protein engineering work targeting activity in liquid amine systems. Our ultimate goal is to test the engineered enzymes in a liquid amine system for cabin air revitalization on ISS or other spacecraft.

Life Support↗
NASA NTRSDate not supplied

Development of Thermostable Carbonic Anhydrases Using Structure-Guided Recombination for Use in CO2 Removal Systems on Spacecraft

Carbon capture and storage has been a research area of great interest in recent years as a method for mitigation of CO2 emissions, due to the effects of climate change. The development of technologies for the efficient capture of CO2 are also of great interest for human spaceflight applications. One of the most promising technologies in this area is CO2 scrubbing using liquid amines, unfortunately, liquid amines with low heats of desorption tend to have slow CO2 binding kinetics. One potential solution to this problem is to use the enzyme carbonic anhydrase (CA) to enhance the kinetics of CO2 binding to liquid amines, allowing the overall process to be more energy efficient. Interest in using carbonic anhydrase as a biocatalyst has led to a number of efforts to improve the thermostability and solvent tolerance of several distinct carbonic anhydrase enzymes. In the work described here, we screened through a diverse set of natural carbonic anhydrases to identify candidates for protein engineering aimed at increased stability and activity in various liquid amines. We then used SCHEMA structure-guided recombination to develop a set of chimeric carbonic anhydrases with high thermostability and activity. These chimeras were used as the starting points for further protein engineering work targeting activity in liquid amine systems. Our ultimate goal is to test the engineered enzymes in a liquid amine system for cabin air revitalization on ISS or other spacecraft.

Life Support, Carbon dioxide, Carbonic anhydrase, ↗
NASA NTRS1988

Electronic materials high-T(sub c) superconductivity polymers and composites structural materials surface science and catalysts industry participation

The fifth year of the Center for Advanced Materials was marked primarily by the significant scientific accomplishments of the research programs. The Electronics Materials program continued its work on the growth and characterization of gallium arsenide crystals, and the development of theories to understand the nature and distribution of defects in the crystals. The High Tc Superconductivity Program continued to make significant contributions to the field in theoretical and experimental work on both bulk materials and thin films and devices. The Ceramic Processing group developed a new technique for cladding YBCO superconductors for high current applications in work with the Electric Power Research Institute. The Polymers and Composites program published a number of important studies involving atomistic simulations of polymer surfaces with excellent correlations to experimental results. The new Enzymatic Synthesis of Materials project produced its first fluorinated polymers and successfully began engineering enzymes designed for materials synthesis. The structural Materials Program continued work on novel alloys, development of processing methods for advanced ceramics, and characterization of mechanical properties of these materials, including the newly documented characterization of cyclic fatigue crack propagation behavior in toughened ceramics. Finally, the Surface Science and Catalysis program made significant contributions to the understanding of microporous catalysts and the nature of surface structures and interface compounds.

Source record↗
NASA NTRS2016

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

proteinogenic amino acids↗
NASA NTRS1993

Crystal growth of enzymes in low gravity (L-5)

Recent developments in protein engineering have expanded the possibilities of studies of enzymes and other proteins. Now such studies are not limited to the elucidation of the relationship between the structure and function of the protein. They also aim at the production of proteins with new and practical functions, based on results obtained during investigation of structure and function. For continuing research in this field, investigation of the tertiary structure of proteins is important. X-ray diffraction of single crystals of protein is usually used for this purpose. The main difficulty is the preparation of the crystals. The theme of the research is to prepare such crystals at very low gravity, with the main purpose being to obtain large single crystals of proteins suitable for x-ray diffraction studies.

Morita, Yuhei↗
NASA NTRS1995

Structurally complex and highly active RNA ligases derived from random RNA sequences

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Non-NASA Center↗
NASA NTRS2023

Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Bailey Marshall↗
NASA NTRS1972

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

Systems for artificial manufacture of starch and for delineation of technological areas, and the rationale for studying them are considered. A discussion of the enzyme-catalyzed routes of synthesis available and a choice as to the most promising route are presented. A discussion of the enzymes involved, of enzyme insolubilization technology, and of possible engineering approaches, with examples in the form of model calculations for both reactors and separators, are also presented.

Bassham, J. A.↗
NASA NTRS1986

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Source record↗
NASA NTRS1982

Isoelectric focusing in space

The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.

Bier, M.↗
NASA NTRS2015

Genetically Engineered Cyanobacteria

The disclosed embodiments provide cyanobacteria spp. that have been genetically engineered to have increased production of carbon-based products of interest. These genetically engineered hosts efficiently convert carbon dioxide and light into carbon-based products of interest such as long chained hydrocarbons. Several constructs containing polynucleotides encoding enzymes active in the metabolic pathways of cyanobacteria are disclosed. In many instances, the cyanobacteria strains have been further genetically modified to optimize production of the carbon-based products of interest. The optimization includes both up-regulation and down-regulation of particular genes.

Zhou, Ruanbao↗
NASA NTRS1984

ECUT: Energy Conversion and Utilization Technologies program - Biocatalysis research activity

The activities of the Biocatalysis Research Activity are organized into the Biocatalysis and Molecular Modeling work elements and a supporting planning and analysis function. In the Biocatalysis work element, progress is made in developing a method for stabilizing genetically engineered traits in microorganisms, refining a technique for monitoring cells that are genetically engineered, and identifying strains of fungi for highly efficient preprocessing of biomass for optimizing the efficiency of bioreactors. In the Molecular Modeling work element, a preliminary model of the behavior of enzymes is developed. A preliminary investigation of the potential for synthesizing enzymes for use in electrochemical processes is completed. Contact with industry and universities is made to define key biocatalysis technical issues and to broaden the range of potential participants in the activity. Analyses are conducted to identify and evaluate potential concepts for future research funding.

Wilcox, R.↗
NASA NTRS1988

ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis Project

Fiscal year 1987 research activities and accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division are presented. The project's technical activities were organized into three work elements. The Molecular Modeling and Applied Genetics work element includes modeling and simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid stabilization by chromosomal integration; growth and stability characteristics of plasmid-containing cells; and determination of optional production parameters for hyper-production of polyphenol oxidase. The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields, and lower separation energetics. The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the economics and energetics of a given biocatalyst process.

Source record↗
NASA NTRS1992

Advanced Manufacturing and Value-added Products from US Agriculture

An objective of the US Department of Agriculture (USDA) Agriculture Research Service (ARS) is to develop technology leading to a broad portfolio of value-added marketable products. Modern scientific disciplines such as chemical engineering are brought into play to develop processes for converting bulk commodities into high-margin products. To accomplish this, the extremely sophisticated processing devices which form the basis of modern biotechnology, namely, genes and enzymes, can be tailored to perform the required functions. The USDA/ARS is a leader in the development of intelligent processing equipment (IPE) for agriculture in the broadest sense. Applications of IPE are found in the production, processing, grading, and marketing aspects of agriculture. Various biotechnology applications of IPE are discussed.

Villet, Ruxton H.↗
NASA NTRS2022

Final Report - Hypergravity Effects on Chromatin Conformation and Nuclear Structure in Cultured Cells using High-throughput Sequencing

The conformation of the human genome is known to play an important role in transcriptional control of gene expression. Our aim is to assess whether exposure of cultured human cardiomyocytes to hypergravity can induce changes in chromatin organization as assayed by Hi-C proximity ligation technique. Hi-C is used to analyze chromatin interactions by using formaldehyde to crosslink regions of chromatin that are in close proximity. The DNA is then fragmented using a restriction enzyme and ligated under dilute conditions to favor intramolecular ligation of cross-linked fragments. Finally, the DNA is sequenced, allowing reconstruction of genomic structure. iCell cardiomyocyte cultures will be exposed to 40 g via an engineered device (incu-fuge) that allows for the chemical fixation of cells on a spinning centrifuge. Additionally, we plan on using fluorescence microscopy to analyze the nuclear and actin cytoskeletal conformation of cardiomyocytes post hypergravity exposure. We hypothesize that chromatin will rearrange in hypergravity conditions, both rapidly due to direct mechanical forces, as well as over longer time frames due to changes to structures necessary for transcriptional responses such as the de novo formation of promoter-enhancer loops. A negative result–no change between 1 g and hypergravity conditions–would nevertheless be an important data point in our understanding of the rheology of the nucleus, and how cells and cellular structures respond to different gravity fields. The cellular physiology of hypergravity has clinical relevance for spaceflight, and can further inform our understanding of microgravity physiology.

chromatin↗
NASA NTRSDate not supplied

Yeast strain development to test in-space bioproduction in the Lunar Explorer Instrument for space biology Applications (LEIA) mission

The Lunar Explorer Instrument for space biology Applications (LEIA) is investigating the effects of lunar radiation and gravity on yeast viability, growth, and metabolism. LEIA is part of the CP-22 Commercial Lunar Payload Services (CLPS) surface mission to the south polar region of the Moon. The biological payload will test genetic factors that are likely to influence the tolerance of yeast for deep space and lunar surface radiation. LEIA is also investigating in-space production of β-carotene in bioengineered yeast. This carotenoid is both an antioxidant and pro-vitamin A- an essential human micronutrient. We report progress on engineering carotenoid-expressing strains to test the impacts of strain background and specific genetic variants on growth and production of β-carotene. To test for enhanced sensitivity to the LEIA mission environment, we generated gene knockouts for the RAD51 DNA damage repair locus as well as the SOD1, SOD2, and TSA1 reactive oxygen species (ROS) defense enzymes. We are also generating strains expected to increase tolerance to abiotic stressors and ROS. To be included in the biology payload, each strain needs to satisfy a series of requirements to be compatible with the mission concept of operations. The LEIA mission will conduct experiments using an autonomous light emitting diode optical detection system and microfluidics incubator to quantify growth, metabolism, and carotenoid production. Strains must produce sufficient carotenoids for bioproduction to be detectable with this optical system. Cells will be loaded into microfluidics cards, desiccated, and stored for 8-12 months prior to the initiation of lunar surface operations. The CLPS lander will operate for one lunar day, and strains will need to grow to stationary phase within 96 hours to ensure that telemetry of LEIA data to Earth can be completed. Genetic variants also need to display expected phenotypes within these optical detection, storage, and growth cycle constraints.

Yeast Engineering↗
NASA NTRS1998

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Non-NASA Center↗
NASA NTRS2003

Development of a pilot system for converting sweet potato starch into glucose syrup

Sweet potato has been chosen as one of NASA's crops to support human beings in future space missions. One of the possible uses is to make syrup that can be used as a general sweetener. In this work a simple engineering system for converting sweet potato starch into glucose syrup was studied on a laboratory scale. The system comprises the following main units: a blender, continuous stirred tank reactor (CSTR), centrifugal and vacuum filters, deionization column and vacuum evaporator. The system was tested by carrying out conversion processes from fresh sweet potato roots. The roots were pealed, sliced, homogenized, heated and hydrolyzed by diastase of malt and Dextrozyme C (Novo Nordisk BioChem, North America, Inc.) enzymes in the CSTR. After hydrolysis the slurry was filtered, de-ionized and concentrated to get glucose syrup. The performance of the system was evaluated based on the quality of the conversion. The main factor was the level of reducing sugars except for the deionization where ash content and color were the main factors. Through careful control of the system units, good heating performance in the CSTR was obtained and the hydrolysis process attained sufficient conversion. The filtration process that incorporated the centrifuge was faster than when it was by-passed to the vacuum filter but losses in sugars were higher. Deionization removed more than 90% of the ash and reduced pigmentation, with probable insignificant losses in sugars during the deionization process. Recovery levels when the centrifuge was used and when it was by-passed could reach about 65% and 78%, respectively. These correspond to reducing sugar concentration of 259 and 310 mg/ml in 150-ml syrups from 300 g of sweet potatoes in each process. However, from concentration trials, syrups with volumes of 100 and 70 ml with the respective dextrose equivalence of 281 and 213 mg/ml were obtained. The syrups obtained were brownish in color and the process that employed centrifugal filtration gave a product with color that resembled the original color of the sweet potatoes. Further work is required to improve the overall system performance.

Non-NASA Center↗