Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.

PubMed

Styles, Matthew Q; Nesbitt, Edward A; Marr, Scott; Hutchby, Marc; Leak, David J

2017-06-01

The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class-I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ-muurolol at temperatures up to 78 °C. We also report an enzyme with a k cat value of 0.95 s -1 at 65 °C, the highest k cat recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class-I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. Model data are available in the PMDB database under the accession number PM0080780. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Experimental Design and Bioinformatics Analysis for the Application of Metagenomics in Environmental Sciences and Biotechnology.

PubMed

Ju, Feng; Zhang, Tong

2015-11-03

Recent advances in DNA sequencing technologies have prompted the widespread application of metagenomics for the investigation of novel bioresources (e.g., industrial enzymes and bioactive molecules) and unknown biohazards (e.g., pathogens and antibiotic resistance genes) in natural and engineered microbial systems across multiple disciplines. This review discusses the rigorous experimental design and sample preparation in the context of applying metagenomics in environmental sciences and biotechnology. Moreover, this review summarizes the principles, methodologies, and state-of-the-art bioinformatics procedures, tools and database resources for metagenomics applications and discusses two popular strategies (analysis of unassembled reads versus assembled contigs/draft genomes) for quantitative or qualitative insights of microbial community structure and functions. Overall, this review aims to facilitate more extensive application of metagenomics in the investigation of uncultured microorganisms, novel enzymes, microbe-environment interactions, and biohazards in biotechnological applications where microbial communities are engineered for bioenergy production, wastewater treatment, and bioremediation.

From 20th century metabolic wall charts to 21st century systems biology: database of mammalian metabolic enzymes.

PubMed

Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A

2017-03-01

The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database ( Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. Copyright © 2017 the American Physiological Society.

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii.

PubMed

Boyle, Nanette R; Morgan, John A

2009-01-07

Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of FBA to estimate intracellular fluxes also provides flux values that can be used as a starting point for rational engineering of C. reinhardtii. From these initial estimates, it is clear that aerobic heterotrophic growth on acetate has a low yield on carbon, while mixotrophically and autotrophically grown cells are significantly more carbon efficient.

Natural photosystems from an engineer's perspective: length, time, and energy scales of charge and energy transfer.

PubMed

Noy, Dror

2008-01-01

The vast structural and functional information database of photosynthetic enzymes includes, in addition to detailed kinetic records from decades of research on physical processes and chemical reaction-pathways, a variety of high and medium resolution crystal structures of key photosynthetic enzymes. Here, it is examined from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in novel biological and non-biological solar-energy conversion systems. This survey reveals that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Furthermore, the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. This underlines a critical challenge for projected de novo designed constructions, that is, the control of spatial organization of cofactor molecules within dense array of different cofactors, some well within 1 nm from each other.

The CoFactor database: organic cofactors in enzyme catalysis.

PubMed

Fischer, Julia D; Holliday, Gemma L; Thornton, Janet M

2010-10-01

Organic enzyme cofactors are involved in many enzyme reactions. Therefore, the analysis of cofactors is crucial to gain a better understanding of enzyme catalysis. To aid this, we have created the CoFactor database. CoFactor provides a web interface to access hand-curated data extracted from the literature on organic enzyme cofactors in biocatalysis, as well as automatically collected information. CoFactor includes information on the conformational and solvent accessibility variation of the enzyme-bound cofactors, as well as mechanistic and structural information about the hosting enzymes. The database is publicly available and can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/CoFactor.

Converting Enzymes into Tools of Industrial Importance.

PubMed

Prasad, Shivcharan; Roy, Ipsita

2018-01-01

Enzymes have applications in numerous biotechnological products and processes that are commonly used in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes, however, are optimized to function under physiological conditions. Any change in reaction conditions results in their activity as well as stability being compromised. Hence, most of the natural biomolecules are not suitable for industrial applications. Modifications are required to develop efficient and successful reagents as per demand. Protein engineering can be applied to cope up with these situations. This review describes some of the novel uses/unusual properties of enzymes as biological catalysts. It explains the different ways in which enzymes can be and have been used under non-native conditions. Different strategies have been discussed regarding stabilization of enzyme as well optimum conditions of its uses in different industries. The following patents databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. The review illustrates the width of the umbrella of applications covered by biocatalysts. Employing the tools of solvent and protein engineering, viz. non-aqueous media, additives, immobilization, mutagenesis, to name a few; biotechnology has been able to make enzyme catalyzed processes an essential components of the industrialist's armoury. The article lists a number of successful examples, both of patented technology as well as biocatalysts which are currently being used in the industry, to highlight the accomplishments of technologies which have been adopted till now for making enzyme technology industrially viable. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Thermodynamics of Enzyme-Catalyzed Reactions Database

National Institute of Standards and Technology Data Gateway

SRD 74 Thermodynamics of Enzyme-Catalyzed Reactions Database (Web, free access)   The Thermodynamics of Enzyme-Catalyzed Reactions Database contains thermodynamic data on enzyme-catalyzed reactions that have been recently published in the Journal of Physical and Chemical Reference Data (JPCRD). For each reaction the following information is provided: the reference for the data, the reaction studied, the name of the enzyme used and its Enzyme Commission number, the method of measurement, the data and an evaluation thereof.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karp, Peter D.

Pathway Tools is a systems-biology software package written by SRI International (SRI) that produces Pathway/Genome Databases (PGDBs) for organisms with a sequenced genome. Pathway Tools also provides a wide range of capabilities for analyzing predicted metabolic networks and user-generated omics data. More than 5,000 academic, industrial, and government groups have licensed Pathway Tools. This user community includes researchers at all three DOE bioenergy centers, as well as academic and industrial metabolic engineering (ME) groups. An integral part of the Pathway Tools software is MetaCyc, a large, multiorganism database of metabolic pathways and enzymes that SRI and its academic collaborators manuallymore » curate. This project included two main goals: I. Enhance the MetaCyc content of bioenergy-related enzymes and pathways. II. Develop computational tools for engineering metabolic pathways that satisfy specified design goals, in particular for bioenergy-related pathways. In part I, SRI proposed to significantly expand the coverage of bioenergy-related metabolic information in MetaCyc, followed by the generation of organism-specific PGDBs for all energy-relevant organisms sequenced at the DOE Joint Genome Institute (JGI). Part I objectives included: 1: Expand the content of MetaCyc to include bioenergy-related enzymes and pathways. 2: Enhance the Pathway Tools software to enable display of complex polymer degradation processes. 3: Create new PGDBs for the energy-related organisms sequenced by JGI, update existing PGDBs with new MetaCyc content, and make these data available to JBEI via the BioCyc website. In part II, SRI proposed to develop an efficient computational tool for the engineering of metabolic pathways. Part II objectives included: 4: Develop computational tools for generating metabolic pathways that satisfy specified design goals, enabling users to specify parameters such as starting and ending compounds, and preferred or disallowed intermediate compounds. The pathways were to be generated using metabolic reactions from a reference database (DB). 5: Develop computational tools for ranking the pathways generated in objective (4) according to their optimality. The ranking criteria include stoichiometric yield, the number and cost of additional inputs and the cofactor compounds required by the pathway, pathway length, and pathway energetics. 6: Develop tools for visualizing generated pathways to facilitate the evaluation of a large space of generated pathways.« less

Functional Enzyme-Based Approach for Linking Microbial Community Functions with Biogeochemical Process Kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Minjing; Qian, Wei-jun; Gao, Yuqian

The kinetics of biogeochemical processes in natural and engineered environmental systems are typically described using Monod-type or modified Monod-type models. These models rely on biomass as surrogates for functional enzymes in microbial community that catalyze biogeochemical reactions. A major challenge to apply such models is the difficulty to quantitatively measure functional biomass for constraining and validating the models. On the other hand, omics-based approaches have been increasingly used to characterize microbial community structure, functions, and metabolites. Here we proposed an enzyme-based model that can incorporate omics-data to link microbial community functions with biogeochemical process kinetics. The model treats enzymes asmore » time-variable catalysts for biogeochemical reactions and applies biogeochemical reaction network to incorporate intermediate metabolites. The sequences of genes and proteins from metagenomes, as well as those from the UniProt database, were used for targeted enzyme quantification and to provide insights into the dynamic linkage among functional genes, enzymes, and metabolites that are necessary to be incorporated in the model. The application of the model was demonstrated using denitrification as an example by comparing model-simulated with measured functional enzymes, genes, denitrification substrates and intermediates« less

Confronting the catalytic dark matter encoded by sequenced genomes

PubMed Central

Ellens, Kenneth W.; Christian, Nils; Singh, Charandeep; Satagopam, Venkata P.

2017-01-01

Abstract The post-genomic era has provided researchers with a deluge of protein sequences. However, a significant fraction of the proteins encoded by sequenced genomes remains without an identified function. Here, we aim at determining how many enzymes of uncertain or unknown function are still present in the Saccharomyces cerevisiae and human proteomes. Using information available in the Swiss-Prot, BRENDA and KEGG databases in combination with a Hidden Markov Model-based method, we estimate that >600 yeast and 2000 human proteins (>30% of their proteins of unknown function) are enzymes whose precise function(s) remain(s) to be determined. This illustrates the impressive scale of the ‘unknown enzyme problem’. We extensively review classical biochemical as well as more recent systematic experimental and computational approaches that can be used to support enzyme function discovery research. Finally, we discuss the possible roles of the elusive catalysts in light of recent developments in the fields of enzymology and metabolism as well as the significance of the unknown enzyme problem in the context of metabolic modeling, metabolic engineering and rare disease research. PMID:29059321

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES. PMID:22465851

BRENDA in 2013: integrated reactions, kinetic data, enzyme function data, improved disease classification: new options and contents in BRENDA.

PubMed

Schomburg, Ida; Chang, Antje; Placzek, Sandra; Söhngen, Carola; Rother, Michael; Lang, Maren; Munaretto, Cornelia; Ulas, Susanne; Stelzer, Michael; Grote, Andreas; Scheer, Maurice; Schomburg, Dietmar

2013-01-01

The BRENDA (BRaunschweig ENzyme DAtabase) enzyme portal (http://www.brenda-enzymes.org) is the main information system of functional biochemical and molecular enzyme data and provides access to seven interconnected databases. BRENDA contains 2.7 million manually annotated data on enzyme occurrence, function, kinetics and molecular properties. Each entry is connected to a reference and the source organism. Enzyme ligands are stored with their structures and can be accessed via their names, synonyms or via a structure search. FRENDA (Full Reference ENzyme DAta) and AMENDA (Automatic Mining of ENzyme DAta) are based on text mining methods and represent a complete survey of PubMed abstracts with information on enzymes in different organisms, tissues or organelles. The supplemental database DRENDA provides more than 910 000 new EC number-disease relations in more than 510 000 references from automatic search and a classification of enzyme-disease-related information. KENDA (Kinetic ENzyme DAta), a new amendment extracts and displays kinetic values from PubMed abstracts. The integration of the EnzymeDetector offers an automatic comparison, evaluation and prediction of enzyme function annotations for prokaryotic genomes. The biochemical reaction database BKM-react contains non-redundant enzyme-catalysed and spontaneous reactions and was developed to facilitate and accelerate the construction of biochemical models.

Molecular engineering of industrial enzymes: recent advances and future prospects.

PubMed

Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

2014-01-01

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.

Using the structure-function linkage database to characterize functional domains in enzymes.

PubMed

Brown, Shoshana; Babbitt, Patricia

2014-12-12

The Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu/) is a Web-accessible database designed to link enzyme sequence, structure, and functional information. This unit describes the protocols by which a user may query the database to predict the function of uncharacterized enzymes and to correct misannotated functional assignments. The information in this unit is especially useful in helping a user discriminate functional capabilities of a sequence that is only distantly related to characterized sequences in publicly available databases. Copyright © 2014 John Wiley & Sons, Inc.

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.

PubMed

Bernal, Claudia; Rodríguez, Karen; Martínez, Ronny

2018-06-09

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, -thus not reusable- and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts. In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel "immobilized biocatalyst engineering" research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications. Copyright © 2018. Published by Elsevier Inc.

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

PubMed

Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

2014-12-01

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Biocatalysts: application and engineering for industrial purposes.

PubMed

Jemli, Sonia; Ayadi-Zouari, Dorra; Hlima, Hajer Ben; Bejar, Samir

2016-01-01

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites.

PubMed

Ribeiro, António J M; Holliday, Gemma L; Furnham, Nicholas; Tyzack, Jonathan D; Ferris, Katherine; Thornton, Janet M

2018-01-04

M-CSA (Mechanism and Catalytic Site Atlas) is a database of enzyme active sites and reaction mechanisms that can be accessed at www.ebi.ac.uk/thornton-srv/m-csa. Our objectives with M-CSA are to provide an open data resource for the community to browse known enzyme reaction mechanisms and catalytic sites, and to use the dataset to understand enzyme function and evolution. M-CSA results from the merging of two existing databases, MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes. We are releasing M-CSA as a new website and underlying database architecture. At the moment, M-CSA contains 961 entries, 423 of these with detailed mechanism information, and 538 with information on the catalytic site residues only. In total, these cover 81% (195/241) of third level EC numbers with a PDB structure, and 30% (840/2793) of fourth level EC numbers with a PDB structure, out of 6028 in total. By searching for close homologues, we are able to extend M-CSA coverage of PDB and UniProtKB to 51 993 structures and to over five million sequences, respectively, of which about 40% and 30% have a conserved active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

76 FR 8707 - Syngenta Seeds, Inc.; Determination of Nonregulated Status for Corn Genetically Engineered To...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-15

... for Corn Genetically Engineered To Produce an Enzyme That Facilitates Ethanol Production AGENCY... event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates ethanol... transformation event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates...

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

PubMed

Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae; Lee, Chong-Soon; Yun, Hyungdon

2015-12-01

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Synthetic metabolism: metabolic engineering meets enzyme design.

PubMed

Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren

2017-04-01

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

novPTMenzy: a database for enzymes involved in novel post-translational modifications

PubMed Central

Khater, Shradha; Mohanty, Debasisa

2015-01-01

With the recent discoveries of novel post-translational modifications (PTMs) which play important roles in signaling and biosynthetic pathways, identification of such PTM catalyzing enzymes by genome mining has been an area of major interest. Unlike well-known PTMs like phosphorylation, glycosylation, SUMOylation, no bioinformatics resources are available for enzymes associated with novel and unusual PTMs. Therefore, we have developed the novPTMenzy database which catalogs information on the sequence, structure, active site and genomic neighborhood of experimentally characterized enzymes involved in five novel PTMs, namely AMPylation, Eliminylation, Sulfation, Hydroxylation and Deamidation. Based on a comprehensive analysis of the sequence and structural features of these known PTM catalyzing enzymes, we have created Hidden Markov Model profiles for the identification of similar PTM catalyzing enzymatic domains in genomic sequences. We have also created predictive rules for grouping them into functional subfamilies and deciphering their mechanistic details by structure-based analysis of their active site pockets. These analytical modules have been made available as user friendly search interfaces of novPTMenzy database. It also has a specialized analysis interface for some PTMs like AMPylation and Eliminylation. The novPTMenzy database is a unique resource that can aid in discovery of unusual PTM catalyzing enzymes in newly sequenced genomes. Database URL: http://www.nii.ac.in/novptmenzy.html PMID:25931459

Recent advances in rational approaches for enzyme engineering

PubMed Central

Steiner, Kerstin; Schwab, Helmut

2012-01-01

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications. PMID:24688651

Engineering Novel and Improved Biocatalysts by Cell Surface Display

PubMed Central

Smith, Mason R.; Khera, Eshita; Wen, Fei

2017-01-01

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use.

PubMed

Kataoka, Michihiko; Miyakawa, Takuya; Shimizu, Sakayu; Tanokura, Masaru

2016-07-01

Biocatalysts (enzymes) have many advantages as catalysts for the production of useful compounds as compared to chemical catalysts. The stereoselectivity of the enzymes is one advantage, and thus the stereoselective production of chiral compounds using enzymes is a promising approach. Importantly, industrial application of the enzymes for chiral compound production requires the discovery of a novel useful enzyme or enzyme function; furthermore, improving the enzyme properties through protein engineering and directed evolution approaches is significant. In this review, the significance of several enzymes showing stereoselectivity (quinuclidinone reductase, aminoalcohol dehydrogenase, old yellow enzyme, and threonine aldolase) in chiral compound production is described, and the improvement of these enzymes using protein engineering and directed evolution approaches for further usability is discussed. Currently, enzymes are widely used as catalysts for the production of chiral compounds; however, for further use of enzymes in chiral compound production, improvement of enzymes should be more essential, as well as discovery of novel enzymes and enzyme functions.

Adjacency and Proximity Searching in the Science Citation Index and Google

DTIC Science & Technology

2005-01-01

major database search engines , including commercial S&T database search engines (e.g., Science Citation Index (SCI), Engineering Compendex (EC...PubMed, OVID), Federal agency award database search engines (e.g., NSF, NIH, DOE, EPA, as accessed in Federal R&D Project Summaries), Web search Engines (e.g...searching. Some database search engines allow strict constrained co- occurrence searching as a user option (e.g., OVID, EC), while others do not (e.g., SCI

The Protein-DNA Interface database

PubMed Central

2010-01-01

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

The Protein-DNA Interface database.

PubMed

Norambuena, Tomás; Melo, Francisco

2010-05-18

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 A or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface.We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes.

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Engineering Ligninolytic Consortium for Bioconversion of Lignocelluloses to Ethanol and Chemicals.

PubMed

Bilal, Muhammad; Nawaz, Muhammad Zohaib; Iqbal, Hafiz M N; Hou, Jialin; Mahboob, Shahid; Al-Ghanim, Khalid A; Cheng, Hairong

2018-01-01

Rising environmental concerns and recent global scenario of cleaner production and consumption are leading to the design of green industrial processes to produce alternative fuels and chemicals. Although bioethanol is one of the most promising and eco-friendly alternatives to fossil fuels yet its production from food and feed has received much negative criticism. The main objective of this study was to present the noteworthy potentialities of lignocellulosic biomass as an enormous and renewable biological resource. The particular focus was also given on engineering ligninolytic consortium for bioconversion of lignocelluloses to ethanol and chemicals on sustainable and environmentally basis. Herein, an effort has been made to extensively review, analyze and compile salient information related to the topic of interest. Several authentic bibliographic databases including PubMed, Scopus, Elsevier, Springer, Bentham Science and other scientific databases were searched with utmost care, and inclusion/ exclusion criterion was adopted to appraise the quality of retrieved peer-reviewed research literature. Bioethanol production from lignocellulosic biomass can largely satisfy the possible inconsistency of first-generation ethanol since it utilizes inedible lignocellulosic feedstocks, primarily sourced from agriculture and forestry wastes. Two major polysaccharides in lignocellulosic biomass namely, cellulose and hemicellulose constitute a complex lignocellulosic network by connecting with lignin, which is highly recalcitrant to depolymerization. Several attempts have been made to reduce the cost involved in the process through improving the pretreatment process. While, the ligninolytic enzymes of white rot fungi (WRF) including laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) have appeared as versatile biocatalysts for delignification of several lignocellulosic residues. The first part of the review is mainly focused on engineering ligninolytic consortium. In the second part, WRF and its unique ligninolytic enzyme-based bio-delignification of lignocellulosic biomass, enzymatic hydrolysis, and fermentation of hydrolyzed feedstock are discussed. The metabolic engineering, enzymatic engineering, synthetic biology aspects for ethanol production and platform chemicals production are comprehensively reviewed in the third part. Towards the end information is also given on futuristic viewpoints. In conclusion, given the present unpredicted scenario of energy and fuel crisis accompanied by global warming, lignocellulosic bioethanol holds great promise as an alternative to petroleum. Apart from bioethanol, the simultaneous production of other value-added products may improve the economics of lignocellulosic bioethanol bioconversion process. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE PAGES

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan; ...

2017-04-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants1[OPEN

PubMed Central

Zhang, Peifen; Kim, Taehyong; Banf, Michael; Chavali, Arvind K.; Nilo-Poyanco, Ricardo; Bernard, Thomas

2017-01-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters. PMID:28228535

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants.

PubMed

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan; Kim, Taehyong; Banf, Michael; Chae, Lee; Dreher, Kate; Chavali, Arvind K; Nilo-Poyanco, Ricardo; Bernard, Thomas; Kahn, Daniel; Rhee, Seung Y

2017-04-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters. © 2017 American Society of Plant Biologists. All Rights Reserved.

A global characterization and identification of multifunctional enzymes.

PubMed

Cheng, Xian-Ying; Huang, Wei-Juan; Hu, Shi-Chang; Zhang, Hai-Lei; Wang, Hao; Zhang, Jing-Xian; Lin, Hong-Huang; Chen, Yu-Zong; Zou, Quan; Ji, Zhi-Liang

2012-01-01

Multi-functional enzymes are enzymes that perform multiple physiological functions. Characterization and identification of multi-functional enzymes are critical for communication and cooperation between different functions and pathways within a complex cellular system or between cells. In present study, we collected literature-reported 6,799 multi-functional enzymes and systematically characterized them in structural, functional, and evolutionary aspects. It was found that four physiochemical properties, that is, charge, polarizability, hydrophobicity, and solvent accessibility, are important for characterization of multi-functional enzymes. Accordingly, a combinational model of support vector machine and random forest model was constructed, based on which 6,956 potential novel multi-functional enzymes were successfully identified from the ENZYME database. Moreover, it was observed that multi-functional enzymes are non-evenly distributed in species, and that Bacteria have relatively more multi-functional enzymes than Archaebacteria and Eukaryota. Comparative analysis indicated that the multi-functional enzymes experienced a fluctuation of gene gain and loss during the evolution from S. cerevisiae to H. sapiens. Further pathway analyses indicated that a majority of multi-functional enzymes were well preserved in catalyzing several essential cellular processes, for example, metabolisms of carbohydrates, nucleotides, and amino acids. What's more, a database of known multi-functional enzymes and a server for novel multi-functional enzyme prediction were also constructed for free access at http://bioinf.xmu.edu.cn/databases/MFEs/index.htm.

Biocatalysis engineering: the big picture.

PubMed

Sheldon, Roger A; Pereira, Pedro C

2017-05-22

In this tutorial review we describe a holistic approach to the invention, development and optimisation of biotransformations utilising isolated enzymes. Increasing attention to applied biocatalysis is motivated by its numerous economic and environmental benefits. Biocatalysis engineering concerns the development of enzymatic systems as a whole, which entails engineering its different components: substrate engineering, medium engineering, protein (enzyme) engineering, biocatalyst (formulation) engineering, biocatalytic cascade engineering and reactor engineering.

Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K

2012-01-01

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted thatmore » mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.« less

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli.

PubMed

Rodriguez, Gabriel M; Atsumi, Shota

2014-09-01

Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

Identification of Functionally Related Enzymes by Learning-to-Rank Methods.

PubMed

Stock, Michiel; Fober, Thomas; Hüllermeier, Eyke; Glinca, Serghei; Klebe, Gerhard; Pahikkala, Tapio; Airola, Antti; De Baets, Bernard; Waegeman, Willem

2014-01-01

Enzyme sequences and structures are routinely used in the biological sciences as queries to search for functionally related enzymes in online databases. To this end, one usually departs from some notion of similarity, comparing two enzymes by looking for correspondences in their sequences, structures or surfaces. For a given query, the search operation results in a ranking of the enzymes in the database, from very similar to dissimilar enzymes, while information about the biological function of annotated database enzymes is ignored. In this work, we show that rankings of that kind can be substantially improved by applying kernel-based learning algorithms. This approach enables the detection of statistical dependencies between similarities of the active cleft and the biological function of annotated enzymes. This is in contrast to search-based approaches, which do not take annotated training data into account. Similarity measures based on the active cleft are known to outperform sequence-based or structure-based measures under certain conditions. We consider the Enzyme Commission (EC) classification hierarchy for obtaining annotated enzymes during the training phase. The results of a set of sizeable experiments indicate a consistent and significant improvement for a set of similarity measures that exploit information about small cavities in the surface of enzymes.

From sequence to enzyme mechanism using multi-label machine learning.

PubMed

De Ferrari, Luna; Mitchell, John B O

2014-05-19

In this work we predict enzyme function at the level of chemical mechanism, providing a finer granularity of annotation than traditional Enzyme Commission (EC) classes. Hence we can predict not only whether a putative enzyme in a newly sequenced organism has the potential to perform a certain reaction, but how the reaction is performed, using which cofactors and with susceptibility to which drugs or inhibitors, details with important consequences for drug and enzyme design. Work that predicts enzyme catalytic activity based on 3D protein structure features limits the prediction of mechanism to proteins already having either a solved structure or a close relative suitable for homology modelling. In this study, we evaluate whether sequence identity, InterPro or Catalytic Site Atlas sequence signatures provide enough information for bulk prediction of enzyme mechanism. By splitting MACiE (Mechanism, Annotation and Classification in Enzymes database) mechanism labels to a finer granularity, which includes the role of the protein chain in the overall enzyme complex, the method can predict at 96% accuracy (and 96% micro-averaged precision, 99.9% macro-averaged recall) the MACiE mechanism definitions of 248 proteins available in the MACiE, EzCatDb (Database of Enzyme Catalytic Mechanisms) and SFLD (Structure Function Linkage Database) databases using an off-the-shelf K-Nearest Neighbours multi-label algorithm. We find that InterPro signatures are critical for accurate prediction of enzyme mechanism. We also find that incorporating Catalytic Site Atlas attributes does not seem to provide additional accuracy. The software code (ml2db), data and results are available online at http://sourceforge.net/projects/ml2db/ and as supplementary files.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

Database Search Engines: Paradigms, Challenges and Solutions.

PubMed

Verheggen, Kenneth; Martens, Lennart; Berven, Frode S; Barsnes, Harald; Vaudel, Marc

2016-01-01

The first step in identifying proteins from mass spectrometry based shotgun proteomics data is to infer peptides from tandem mass spectra, a task generally achieved using database search engines. In this chapter, the basic principles of database search engines are introduced with a focus on open source software, and the use of database search engines is demonstrated using the freely available SearchGUI interface. This chapter also discusses how to tackle general issues related to sequence database searching and shows how to minimize their impact.

PlantCAZyme: a database for plant carbohydrate-active enzymes

PubMed Central

Ekstrom, Alexander; Taujale, Rahil; McGinn, Nathan; Yin, Yanbin

2014-01-01

PlantCAZyme is a database built upon dbCAN (database for automated carbohydrate active enzyme annotation), aiming to provide pre-computed sequence and annotation data of carbohydrate active enzymes (CAZymes) to plant carbohydrate and bioenergy research communities. The current version contains data of 43 790 CAZymes of 159 protein families from 35 plants (including angiosperms, gymnosperms, lycophyte and bryophyte mosses) and chlorophyte algae with fully sequenced genomes. Useful features of the database include: (i) a BLAST server and a HMMER server that allow users to search against our pre-computed sequence data for annotation purpose, (ii) a download page to allow batch downloading data of a specific CAZyme family or species and (iii) protein browse pages to provide an easy access to the most comprehensive sequence and annotation data. Database URL: http://cys.bios.niu.edu/plantcazyme/ PMID:25125445

Expanding P450 catalytic reaction space through evolution and engineering

PubMed Central

McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

2014-01-01

Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

Advances in the Engineering of the Gene Editing Enzymes and the Genomes: Understanding and Handling the Off-Target Effects of CRISPR/Cas9.

PubMed

Yin, Yufang; Wang, Qian; Xiao, Li; Wang, Fengjiao; Song, Zhuo; Zhou, Cuilan; Liu, Xuan; Xing, Chungen; He, Nongyue; Li, Kai; Feng, Yan; Zhang, Jia

2018-03-01

In the past decades, significant progresses have been achieved in genetic engineering of nucleases. Among the genetically engineered nucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and CRIPSPR/Cas9 system form a new field of gene editing. The gene editing efficiency or targeting effect and the off-target effect are the two major determinant factors in evaluating the usefulness of a new enzyme. Engineering strategies in improving these gene editing enzymes, particularly in minimizing their off-target effects, are the focus of this paper. Examples of using these genetically engineered enzymes in genome modification are discussed in order to better understand the requirement of engineering efforts in obtaining more powerful and useful gene editing enzymes. In addition, the identification of naturally existed anti-Cas proteins has been employed in minimizing off-target effects. Considering the future application in human gene therapy, optimization of these well recognized gene editing enzymes and exploration of more novel enzymes are both required. Before people find an ideal gene editing system having virtually no off-target effect, technologies used to screen and identify off-target effects are of importance in clinical trials employing gene therapy.

Optimization of enzyme parameters for fermentative production of biorenewable fuels and chemicals

PubMed Central

Jarboe, Laura R.; Liu, Ping; Kautharapu, Kumar Babu; Ingram, Lonnie O.

2012-01-01

Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene. PMID:24688665

ThermoData Engine Database

National Institute of Standards and Technology Data Gateway

SRD 103a NIST ThermoData Engine Database (PC database for purchase)   ThermoData Engine is the first product fully implementing all major principles of the concept of dynamic data evaluation formulated at NIST/TRC.

ORENZA: a web resource for studying ORphan ENZyme activities

PubMed Central

Lespinet, Olivier; Labedan, Bernard

2006-01-01

Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene sequences to the relevant enzymes. PMID:17026747

EFICAz2.5: application of a high-precision enzyme function predictor to 396 proteomes.

PubMed

Kumar, Narendra; Skolnick, Jeffrey

2012-10-15

High-quality enzyme function annotation is essential for understanding the biochemistry, metabolism and disease processes of organisms. Previously, we developed a multi-component high-precision enzyme function predictor, EFICAz(2) (enzyme function inference by a combined approach). Here, we present an updated improved version, EFICAz(2.5), that is trained on a significantly larger data set of enzyme sequences and PROSITE patterns. We also present the results of the application of EFICAz(2.5) to the enzyme reannotation of 396 genomes cataloged in the ENSEMBL database. The EFICAz(2.5) server and database is freely available with a use-friendly interface at http://cssb.biology.gatech.edu/EFICAz2.5.

ThermoData Engine Database - Pure Compounds and Binary Mixtures

National Institute of Standards and Technology Data Gateway

SRD 103b NIST ThermoData Engine Version 6.0 - Pure CompoThermoData Engine Database - Pure Compounds and Binary Mixtures (PC database for purchase)   This database contains property data for more than 21,000 pure compounds, 37,500 binary mixtures, 10,000 ternary mixtures, and 6,000 chemical reactions.

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

PubMed Central

2010-01-01

Background Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates. PMID:21083930

Elucidation of metabolic pathways from enzyme classification data.

PubMed

McDonald, Andrew G; Tipton, Keith F

2014-01-01

The IUBMB Enzyme List is widely used by other databases as a source for avoiding ambiguity in the recognition of enzymes as catalytic entities. However, it was not designed for metabolic pathway tracing, which has become increasingly important in systems biology. A Reactions Database has been created from the material in the Enzyme List to allow reactions to be searched by substrate/product, and pathways to be traced from any selected starting/seed substrate. An extensive synonym glossary allows searches by many of the alternative names, including accepted abbreviations, by which a chemical compound may be known. This database was necessary for the development of the application Reaction Explorer ( http://www.reaction-explorer.org ), which was written in Real Studio ( http://www.realsoftware.com/realstudio/ ) to search the Reactions Database and draw metabolic pathways from reactions selected by the user. Having input the name of the starting compound (the "seed"), the user is presented with a list of all reactions containing that compound and then selects the product of interest as the next point on the ensuing graph. The pathway diagram is then generated as the process iterates. A contextual menu is provided, which allows the user: (1) to remove a compound from the graph, along with all associated links; (2) to search the reactions database again for additional reactions involving the compound; (3) to search for the compound within the Enzyme List.

In-vitro engineering of novel bioactivity in the natural enzymes

NASA Astrophysics Data System (ADS)

Tiwari, Vishvanath

2016-10-01

Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

An approach in building a chemical compound search engine in oracle database.

PubMed

Wang, H; Volarath, P; Harrison, R

2005-01-01

A searching or identifying of chemical compounds is an important process in drug design and in chemistry research. An efficient search engine involves a close coupling of the search algorithm and database implementation. The database must process chemical structures, which demands the approaches to represent, store, and retrieve structures in a database system. In this paper, a general database framework for working as a chemical compound search engine in Oracle database is described. The framework is devoted to eliminate data type constrains for potential search algorithms, which is a crucial step toward building a domain specific query language on top of SQL. A search engine implementation based on the database framework is also demonstrated. The convenience of the implementation emphasizes the efficiency and simplicity of the framework.

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

Agrivida, Inc., is an agricultural biotechnology company developing industrial crop feedstocks for the fuel and chemical industries. Agrivida's crops have improved processing traits that enable efficient, low cost conversion of the crops' cellulosic components into fermentable sugars. Currently, pretreatment and enzymatic conversion of the major cell wall components, cellulose and hemicellulose, into fermentable sugars is the most expensive processing step that prevents widespread adoption of biomass in biofuels processes. To lower production costs we are consolidating pretreatment and enzyme production within the crop. In this strategy, transgenic plants express engineered cell wall degrading enzymes in an inactive form, which can be reactivated after harvest. We have engineered protein elements that disrupt enzyme activity during normal plant growth. Upon exposure to specific processing conditions, the engineered enzymes are converted into their active forms. This mechanism significantly lowers pretreatment costs and enzyme loadings (>75% reduction) below those currently available to the industry.

A World Wide Web (WWW) server database engine for an organelle database, MitoDat.

PubMed

Lemkin, P F; Chipperfield, M; Merril, C; Zullo, S

1996-03-01

We describe a simple database search engine "dbEngine" which may be used to quickly create a searchable database on a World Wide Web (WWW) server. Data may be prepared from spreadsheet programs (such as Excel, etc.) or from tables exported from relationship database systems. This Common Gateway Interface (CGI-BIN) program is used with a WWW server such as available commercially, or from National Center for Supercomputer Algorithms (NCSA) or CERN. Its capabilities include: (i) searching records by combinations of terms connected with ANDs or ORs; (ii) returning search results as hypertext links to other WWW database servers; (iii) mapping lists of literature reference identifiers to the full references; (iv) creating bidirectional hypertext links between pictures and the database. DbEngine has been used to support the MitoDat database (Mendelian and non-Mendelian inheritance associated with the Mitochondrion) on the WWW.

Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.

PubMed

Tong, Xiaoxue; Barberi, Tania Triscari; Botting, Catherine H; Sharma, Sunil V; Simmons, Mark J H; Overton, Tim W; Goss, Rebecca J M

2016-10-21

Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.

Rewiring Algae's Catalytic Circuits - Continuum Magazine | NREL

Science.gov Websites

with labels depicting the engineering of hydrogen-producing enzyme to create a hydrogen production circuit to increase hydrogen during photosynthesis. Engineering of the hydrogen-producing enzyme to create circuits, or pathways. To do so, they would replace the normal hydrogen-producing enzyme, hydrogenase

BioProspecting: novel marker discovery obtained by mining the bibleome.

PubMed

Elkin, Peter L; Tuttle, Mark S; Trusko, Brett E; Brown, Steven H

2009-02-05

BioProspecting is a novel approach that enabled our team to mine data related to genetic markers from the New England Journal of Medicine (NEJM) utilizing SNOMED CT and the Human Gene Onotology (HUGO). The Biomedical Informatics Research Collaborative was able to link genes and disorders using the Multi-threaded Clinical Vocabulary Server (MCVS) and natural language processing engine, whose output creates an ontology-network using the semantic encodings of the literature that is organized by these two terminologies. We identified relationships between (genes or proteins) and (diseases or drugs) as linked by metabolic functions and identified potentially novel functional relationships between, for example, genes and diseases (e.g. Article #1 ([Gene - IL27] = > {Enzyme - Dipeptidyl Carboxypeptidase 1}) and Article #2 ({Enzyme - Dipeptidyl Carboxypeptidase 1} < = [Disorder - Type II DM]) showing a metabolic link between IL27 and Type II DM). In this manuscript we describe our method for developing the database and its content as well as its potential to assist in the discovery of novel markers and drugs.

BioCarian: search engine for exploratory searches in heterogeneous biological databases.

PubMed

Zaki, Nazar; Tennakoon, Chandana

2017-10-02

There are a large number of biological databases publicly available for scientists in the web. Also, there are many private databases generated in the course of research projects. These databases are in a wide variety of formats. Web standards have evolved in the recent times and semantic web technologies are now available to interconnect diverse and heterogeneous sources of data. Therefore, integration and querying of biological databases can be facilitated by techniques used in semantic web. Heterogeneous databases can be converted into Resource Description Format (RDF) and queried using SPARQL language. Searching for exact queries in these databases is trivial. However, exploratory searches need customized solutions, especially when multiple databases are involved. This process is cumbersome and time consuming for those without a sufficient background in computer science. In this context, a search engine facilitating exploratory searches of databases would be of great help to the scientific community. We present BioCarian, an efficient and user-friendly search engine for performing exploratory searches on biological databases. The search engine is an interface for SPARQL queries over RDF databases. We note that many of the databases can be converted to tabular form. We first convert the tabular databases to RDF. The search engine provides a graphical interface based on facets to explore the converted databases. The facet interface is more advanced than conventional facets. It allows complex queries to be constructed, and have additional features like ranking of facet values based on several criteria, visually indicating the relevance of a facet value and presenting the most important facet values when a large number of choices are available. For the advanced users, SPARQL queries can be run directly on the databases. Using this feature, users will be able to incorporate federated searches of SPARQL endpoints. We used the search engine to do an exploratory search on previously published viral integration data and were able to deduce the main conclusions of the original publication. BioCarian is accessible via http://www.biocarian.com . We have developed a search engine to explore RDF databases that can be used by both novice and advanced users.

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown.

PubMed

Seppälä, Susanna; Wilken, St Elmo; Knop, Doriv; Solomon, Kevin V; O'Malley, Michelle A

2017-11-01

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging 'omics'-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

PubMed

Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

2013-04-19

Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

BioFuelDB: a database and prediction server of enzymes involved in biofuels production.

PubMed

Chaudhary, Nikhil; Gupta, Ankit; Gupta, Sudheer; Sharma, Vineet K

2017-01-01

In light of the rapid decrease in fossils fuel reserves and an increasing demand for energy, novel methods are required to explore alternative biofuel production processes to alleviate these pressures. A wide variety of molecules which can either be used as biofuels or as biofuel precursors are produced using microbial enzymes. However, the common challenges in the industrial implementation of enzyme catalysis for biofuel production are the unavailability of a comprehensive biofuel enzyme resource, low efficiency of known enzymes, and limited availability of enzymes which can function under extreme conditions in the industrial processes. We have developed a comprehensive database of known enzymes with proven or potential applications in biofuel production through text mining of PubMed abstracts and other publicly available information. A total of 131 enzymes with a role in biofuel production were identified and classified into six enzyme classes and four broad application categories namely 'Alcohol production', 'Biodiesel production', 'Fuel Cell' and 'Alternate biofuels'. A prediction tool 'Benz' was developed to identify and classify novel homologues of the known biofuel enzyme sequences from sequenced genomes and metagenomes. 'Benz' employs a hybrid approach incorporating HMMER 3.0 and RAPSearch2 programs to provide high accuracy and high speed for prediction. Using the Benz tool, 153,754 novel homologues of biofuel enzymes were identified from 23 diverse metagenomic sources. The comprehensive data of curated biofuel enzymes, their novel homologs identified from diverse metagenomes, and the hybrid prediction tool Benz are presented as a web server which can be used for the prediction of biofuel enzymes from genomic and metagenomic datasets. The database and the Benz tool is publicly available at http://metabiosys.iiserb.ac.in/biofueldb& http://metagenomics.iiserb.ac.in/biofueldb.

Systematization of the protein sequence diversity in enzymes related to secondary metabolic pathways in plants, in the context of big data biology inspired by the KNApSAcK motorcycle database.

PubMed

Ikeda, Shun; Abe, Takashi; Nakamura, Yukiko; Kibinge, Nelson; Hirai Morita, Aki; Nakatani, Atsushi; Ono, Naoaki; Ikemura, Toshimichi; Nakamura, Kensuke; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

2013-05-01

Biology is increasingly becoming a data-intensive science with the recent progress of the omics fields, e.g. genomics, transcriptomics, proteomics and metabolomics. The species-metabolite relationship database, KNApSAcK Core, has been widely utilized and cited in metabolomics research, and chronological analysis of that research work has helped to reveal recent trends in metabolomics research. To meet the needs of these trends, the KNApSAcK database has been extended by incorporating a secondary metabolic pathway database called Motorcycle DB. We examined the enzyme sequence diversity related to secondary metabolism by means of batch-learning self-organizing maps (BL-SOMs). Initially, we constructed a map by using a big data matrix consisting of the frequencies of all possible dipeptides in the protein sequence segments of plants and bacteria. The enzyme sequence diversity of the secondary metabolic pathways was examined by identifying clusters of segments associated with certain enzyme groups in the resulting map. The extent of diversity of 15 secondary metabolic enzyme groups is discussed. Data-intensive approaches such as BL-SOM applied to big data matrices are needed for systematizing protein sequences. Handling big data has become an inevitable part of biology.

Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

DTIC Science & Technology

2011-10-30

stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low- cost high-capacity celiulosic...NOVEL ENZYMATIC CARBOHYDRATE-TO-HYDROGEN TECHNOLOGY BY ENZYME ENGINEERING Grant/Contract Number: FA9550-08-1-0145 Program Manager: Dr. Walt...bbtransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembly of enzyme and coenzymes. Different from in vivo

Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function.

PubMed

Busk, P K; Pilgaard, B; Lezyk, M J; Meyer, A S; Lange, L

2017-04-12

Carbohydrate-active enzymes are found in all organisms and participate in key biological processes. These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis for prediction of enzyme function. A fast and reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interest as demonstrated for the glycosyl hydrolase and the lytic polysaccharide monooxygenase families. This approach not only assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy. We identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition. The conserved peptides were matched to protein sequence for de novo annotation and functional prediction of carbohydrate-active enzymes with the Hotpep method. Annotation of protein sequences from 12 bacterial and 16 fungal genomes to families with Hotpep had an accuracy of 0.84 (measured as F1-score) compared to semiautomatic annotation by the CAZy database whereas the dbCAN HMM-based method had an accuracy of 0.77 with optimized parameters. Furthermore, Hotpep provided a functional prediction with 86% accuracy for the annotated genes. Hotpep is available as a stand-alone application for MS Windows. Hotpep is a state-of-the-art method for automatic annotation and functional prediction of carbohydrate-active enzymes.

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

DTIC Science & Technology

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to... Engineering and Single-Molecule Techniques The views, opinions and/or findings contained in this report are those of the author(s) and should not...Status: Technology Transfer: Report Date: 1 FINAL REPORT Project Title: Probing Enzyme-Surface Interactions via Protein Engineering and

[Application of bioinformatics in researches of industrial biocatalysis].

PubMed

Yu, Hui-Min; Luo, Hui; Shi, Yue; Sun, Xu-Dong; Shen, Zhong-Yao

2004-05-01

Industrial biocatalysis is currently attracting much attention to rebuild or substitute traditional producing process of chemicals and drugs. One of key focuses in industrial biocatalysis is biocatalyst, which is usually one kind of microbial enzyme. In the recent, new technologies of bioinformatics have played and will continue to play more and more significant roles in researches of industrial biocatalysis in response to the waves of genomic revolution. One of the key applications of bioinformatics in biocatalysis is the discovery and identification of the new biocatalyst through advanced DNA and protein sequence search, comparison and analyses in Internet database using different algorithm and software. The unknown genes of microbial enzymes can also be simply harvested by primer design on the basis of bioinformatics analyses. The other key applications of bioinformatics in biocatalysis are the modification and improvement of existing industrial biocatalyst. In this aspect, bioinformatics is of great importance in both rational design and directed evolution of microbial enzymes. Based on the successful prediction of tertiary structures of enzymes using the tool of bioinformatics, the undermentioned experiments, i.e. site-directed mutagenesis, fusion protein construction, DNA family shuffling and saturation mutagenesis, etc, are usually of very high efficiency. On all accounts, bioinformatics will be an essential tool for either biologist or biological engineer in the future researches of industrial biocatalysis, due to its significant function in guiding and quickening the step of discovery and/or improvement of novel biocatalysts.

Biofuel Database

National Institute of Standards and Technology Data Gateway

Biofuel Database (Web, free access)   This database brings together structural, biological, and thermodynamic data for enzymes that are either in current use or are being considered for use in the production of biofuels.

Maximizing the efficiency of multienzyme process by stoichiometry optimization.

PubMed

Dvorak, Pavel; Kurumbang, Nagendra P; Bendl, Jaroslav; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri

2014-09-05

Multienzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of the stoichiometry of the biocatalysts. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalyzing a five-step chemical conversion. Kinetic models of pathways with wild-type or engineered enzymes were built, and the enzyme stoichiometry of each pathway was optimized. Mathematical modeling and one-pot multienzyme experiments provided detailed insights into pathway dynamics, enabled the selection of a suitable engineered enzyme, and afforded high efficiency while minimizing biocatalyst loadings. Optimizing the stoichiometry in a pathway with an engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multienzyme processes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

GALT protein database: querying structural and functional features of GALT enzyme.

PubMed

d'Acierno, Antonio; Facchiano, Angelo; Marabotti, Anna

2014-09-01

Knowledge of the impact of variations on protein structure can enhance the comprehension of the mechanisms of genetic diseases related to that protein. Here, we present a new version of GALT Protein Database, a Web-accessible data repository for the storage and interrogation of structural effects of variations of the enzyme galactose-1-phosphate uridylyltransferase (GALT), the impairment of which leads to classic Galactosemia, a rare genetic disease. This new version of this database now contains the models of 201 missense variants of GALT enzyme, including heterozygous variants, and it allows users not only to retrieve information about the missense variations affecting this protein, but also to investigate their impact on substrate binding, intersubunit interactions, stability, and other structural features. In addition, it allows the interactive visualization of the models of variants collected into the database. We have developed additional tools to improve the use of the database by nonspecialized users. This Web-accessible database (http://bioinformatica.isa.cnr.it/GALT/GALT2.0) represents a model of tools potentially suitable for application to other proteins that are involved in human pathologies and that are subjected to genetic variations. © 2014 WILEY PERIODICALS, INC.

Protein Engineering Towards Natural Product Synthesis and Diversification

PubMed Central

Zabala, Angelica O.; Cacho, Ralph A.; Tang, Yi

2014-01-01

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products. PMID:22006344

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase.

PubMed

Nandy, Suman Kumar; Bhuyan, Rajabrata; Seal, Alpana

2012-01-01

Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. The database is available for free at http://www.bifku.in/DBD/

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

PubMed Central

Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

2013-01-01

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases

PubMed Central

Caspi, Ron; Altman, Tomer; Dale, Joseph M.; Dreher, Kate; Fulcher, Carol A.; Gilham, Fred; Kaipa, Pallavi; Karthikeyan, Athikkattuvalasu S.; Kothari, Anamika; Krummenacker, Markus; Latendresse, Mario; Mueller, Lukas A.; Paley, Suzanne; Popescu, Liviu; Pujar, Anuradha; Shearer, Alexander G.; Zhang, Peifen; Karp, Peter D.

2010-01-01

The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible resource for metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are experimentally determined, small-molecule metabolic pathways and are curated from the primary scientific literature. With more than 1400 pathways, MetaCyc is the largest collection of metabolic pathways currently available. Pathways reactions are linked to one or more well-characterized enzymes, and both pathways and enzymes are annotated with reviews, evidence codes, and literature citations. BioCyc (BioCyc.org) is a collection of more than 500 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the full genome and predicted metabolic network of one organism. The network, which is predicted by the Pathway Tools software using MetaCyc as a reference, consists of metabolites, enzymes, reactions and metabolic pathways. BioCyc PGDBs also contain additional features, such as predicted operons, transport systems, and pathway hole-fillers. The BioCyc Web site offers several tools for the analysis of the PGDBs, including Omics Viewers that enable visualization of omics datasets on two different genome-scale diagrams and tools for comparative analysis. The BioCyc PGDBs generated by SRI are offered for adoption by any party interested in curation of metabolic, regulatory, and genome-related information about an organism. PMID:19850718

Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications.

PubMed

Pyzocha, Neena K; Chen, Sidi

2018-02-16

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

SSER: Species specific essential reactions database.

PubMed

Labena, Abraham A; Ye, Yuan-Nong; Dong, Chuan; Zhang, Fa-Z; Guo, Feng-Biao

2017-04-19

Essential reactions are vital components of cellular networks. They are the foundations of synthetic biology and are potential candidate targets for antimetabolic drug design. Especially if a single reaction is catalyzed by multiple enzymes, then inhibiting the reaction would be a better option than targeting the enzymes or the corresponding enzyme-encoding gene. The existing databases such as BRENDA, BiGG, KEGG, Bio-models, Biosilico, and many others offer useful and comprehensive information on biochemical reactions. But none of these databases especially focus on essential reactions. Therefore, building a centralized repository for this class of reactions would be of great value. Here, we present a species-specific essential reactions database (SSER). The current version comprises essential biochemical and transport reactions of twenty-six organisms which are identified via flux balance analysis (FBA) combined with manual curation on experimentally validated metabolic network models. Quantitative data on the number of essential reactions, number of the essential reactions associated with their respective enzyme-encoding genes and shared essential reactions across organisms are the main contents of the database. SSER would be a prime source to obtain essential reactions data and related gene and metabolite information and it can significantly facilitate the metabolic network models reconstruction and analysis, and drug target discovery studies. Users can browse, search, compare and download the essential reactions of organisms of their interest through the website http://cefg.uestc.edu.cn/sser .

Effective Engineering Outreach through an Undergraduate Mentoring Team and Module Database

ERIC Educational Resources Information Center

Young, Colin; Butterfield, Anthony E.

2014-01-01

The rising need for engineers has led to increased interest in community outreach in engineering departments nationwide. We present a sustainable outreach model involving trained undergraduate mentors to build ties with K-12 teachers and students. An associated online module database of chemical engineering demonstrations, available to educators…

Technology Prospecting on Enzymes: Application, Marketing and Engineering

PubMed Central

Li, Shuang; Yang, Xiaofeng; Yang, Shuai; Zhu, Muzi; Wang, Xiaoning

2012-01-01

Enzymes are protein molecules functioning as specialized catalysts for chemical reactions. They have contributed greatly to the traditional and modern chemical industry by improving existing processes. In this article, we first give a survey of representative industrial applications of enzymes, focusing on the technical applications, feed industry, food processing and cosmetic products. The recent important developments and applications of enzymes in industry are reviewed. Then large efforts are dedicated to the worldwide enzyme market from the demand and production perspectives. Special attention is laid on the Chinese enzyme market. Although enzyme applications are being developed in full swing, breakthroughs are needed to overcome their weaknesses in maintaining activities during the catalytic processes. Strategies of metagomic analysis, cell surface display technology and cell-free system might give valuable solutions in novel enzyme exploiting and enzyme engineering. PMID:24688658

Anatomy and evolution of database search engines-a central component of mass spectrometry based proteomic workflows.

PubMed

Verheggen, Kenneth; Raeder, Helge; Berven, Frode S; Martens, Lennart; Barsnes, Harald; Vaudel, Marc

2017-09-13

Sequence database search engines are bioinformatics algorithms that identify peptides from tandem mass spectra using a reference protein sequence database. Two decades of development, notably driven by advances in mass spectrometry, have provided scientists with more than 30 published search engines, each with its own properties. In this review, we present the common paradigm behind the different implementations, and its limitations for modern mass spectrometry datasets. We also detail how the search engines attempt to alleviate these limitations, and provide an overview of the different software frameworks available to the researcher. Finally, we highlight alternative approaches for the identification of proteomic mass spectrometry datasets, either as a replacement for, or as a complement to, sequence database search engines. © 2017 Wiley Periodicals, Inc.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE PAGES

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

2016-10-24

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, J. Vincent; Chen, Long; Whitaker, W. Brian

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

PubMed Central

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

2016-01-01

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. PMID:27791059

dbHiMo: a web-based epigenomics platform for histone-modifying enzymes

PubMed Central

Choi, Jaeyoung; Kim, Ki-Tae; Huh, Aram; Kwon, Seomun; Hong, Changyoung; Asiegbu, Fred O.; Jeon, Junhyun; Lee, Yong-Hwan

2015-01-01

Over the past two decades, epigenetics has evolved into a key concept for understanding regulation of gene expression. Among many epigenetic mechanisms, covalent modifications such as acetylation and methylation of lysine residues on core histones emerged as a major mechanism in epigenetic regulation. Here, we present the database for histone-modifying enzymes (dbHiMo; http://hme.riceblast.snu.ac.kr/) aimed at facilitating functional and comparative analysis of histone-modifying enzymes (HMEs). HMEs were identified by applying a search pipeline built upon profile hidden Markov model (HMM) to proteomes. The database incorporates 11 576 HMEs identified from 603 proteomes including 483 fungal, 32 plants and 51 metazoan species. The dbHiMo provides users with web-based personalized data browsing and analysis tools, supporting comparative and evolutionary genomics. With comprehensive data entries and associated web-based tools, our database will be a valuable resource for future epigenetics/epigenomics studies. Database URL: http://hme.riceblast.snu.ac.kr/ PMID:26055100

Effect of Additives on the Selectivity and Reactivity of Enzymes.

PubMed

Liang, Yi-Ru; Wu, Qi; Lin, Xian-Fu

2017-01-01

Enzymes have been widely used as efficient, eco-friendly, and biodegradable catalysts in organic chemistry due to their mild reaction conditions and high selectivity and efficiency. In recent years, the catalytic promiscuity of many enzymes in unnatural reactions has been revealed and studied by chemists and biochemists, which has expanded the application potential of enzymes. To enhance the selectivity and activity of enzymes in their natural or promiscuous reactions, many methods have been recommended, such as protein engineering, process engineering, and media engineering. Among them, the additive approach is very attractive because of its simplicity to use and high efficiency. In this paper, we will review the recent developments about the applications of additives to improve the catalytic performances of enzymes in their natural and promiscuous reactions. These additives include water, organic bases, water mimics, cosolvents, crown ethers, salts, surfactants, and some particular molecular additives. © 2017 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Therapeutic Enzymes: Applications and Approaches to Pharmacological Improvement.

PubMed

Yari, Maryam; Ghoshoon, Mohammad B; Vakili, Bahareh; Ghasemi, Younes

2017-01-01

Among therapeutic proteins, enzymes represent small and of course profitable market. They can be used to treat important, rare, and deadly diseases. Enzyme therapy is the only available treatment for certain disorders. Here, pharmaceutical enzymes are reviewed. They are categorized in four main groups, enzymes in replacement therapy, enzymes in cancer treatment, enzymes for fibrinolysis, and finally enzymes that are used topically for various treatments. Furthermore, enzyme gene therapy and future perspective of therapeutic enzymes are mentioned in brief. There are many important approved enzymes in pharmaceutical market. Several approaches such as point mutation, fusion protein designing, glycoengineering, and PEGylation were used to achieve improved enzymes. Although sometimes enzymes were engineered to facilitate production and purification process, appropriate delivery to target sites, extending half-life, and reducing immunogenicity are among the main goals of engineering approaches. Overall, enzymes play a critical role in treatment of common and rare diseases. Evaluation of new enzymes as well as improvement of approved enzymes are of the most important challenges in biotechnology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Enzyme dynamics and engineering: one step at a time.

PubMed

Tokuriki, Nobuhiko; Jackson, Colin J

2014-10-23

Although protein dynamics are accepted as being essential for enzyme function, their effects are not fully understood. In this issue of Chemistry and Biology, Gobeil and coworkers describe how engineered changes in the millisecond motions of a mutant TEM-1 β-lactamase do not significantly affect substrate turnover. This mutational robustness has implications for protein engineering and design strategies.

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.

2010-09-03

The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired themore » enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.« less

BrEPS 2.0: Optimization of sequence pattern prediction for enzyme annotation.

PubMed

Dudek, Christian-Alexander; Dannheim, Henning; Schomburg, Dietmar

2017-01-01

The prediction of gene functions is crucial for a large number of different life science areas. Faster high throughput sequencing techniques generate more and larger datasets. The manual annotation by classical wet-lab experiments is not suitable for these large amounts of data. We showed earlier that the automatic sequence pattern-based BrEPS protocol, based on manually curated sequences, can be used for the prediction of enzymatic functions of genes. The growing sequence databases provide the opportunity for more reliable patterns, but are also a challenge for the implementation of automatic protocols. We reimplemented and optimized the BrEPS pattern generation to be applicable for larger datasets in an acceptable timescale. Primary improvement of the new BrEPS protocol is the enhanced data selection step. Manually curated annotations from Swiss-Prot are used as reliable source for function prediction of enzymes observed on protein level. The pool of sequences is extended by highly similar sequences from TrEMBL and SwissProt. This allows us to restrict the selection of Swiss-Prot entries, without losing the diversity of sequences needed to generate significant patterns. Additionally, a supporting pattern type was introduced by extending the patterns at semi-conserved positions with highly similar amino acids. Extended patterns have an increased complexity, increasing the chance to match more sequences, without losing the essential structural information of the pattern. To enhance the usability of the database, we introduced enzyme function prediction based on consensus EC numbers and IUBMB enzyme nomenclature. BrEPS is part of the Braunschweig Enzyme Database (BRENDA) and is available on a completely redesigned website and as download. The database can be downloaded and used with the BrEPScmd command line tool for large scale sequence analysis. The BrEPS website and downloads for the database creation tool, command line tool and database are freely accessible at http://breps.tu-bs.de.

BrEPS 2.0: Optimization of sequence pattern prediction for enzyme annotation

PubMed Central

Schomburg, Dietmar

2017-01-01

The prediction of gene functions is crucial for a large number of different life science areas. Faster high throughput sequencing techniques generate more and larger datasets. The manual annotation by classical wet-lab experiments is not suitable for these large amounts of data. We showed earlier that the automatic sequence pattern-based BrEPS protocol, based on manually curated sequences, can be used for the prediction of enzymatic functions of genes. The growing sequence databases provide the opportunity for more reliable patterns, but are also a challenge for the implementation of automatic protocols. We reimplemented and optimized the BrEPS pattern generation to be applicable for larger datasets in an acceptable timescale. Primary improvement of the new BrEPS protocol is the enhanced data selection step. Manually curated annotations from Swiss-Prot are used as reliable source for function prediction of enzymes observed on protein level. The pool of sequences is extended by highly similar sequences from TrEMBL and SwissProt. This allows us to restrict the selection of Swiss-Prot entries, without losing the diversity of sequences needed to generate significant patterns. Additionally, a supporting pattern type was introduced by extending the patterns at semi-conserved positions with highly similar amino acids. Extended patterns have an increased complexity, increasing the chance to match more sequences, without losing the essential structural information of the pattern. To enhance the usability of the database, we introduced enzyme function prediction based on consensus EC numbers and IUBMB enzyme nomenclature. BrEPS is part of the Braunschweig Enzyme Database (BRENDA) and is available on a completely redesigned website and as download. The database can be downloaded and used with the BrEPScmd command line tool for large scale sequence analysis. The BrEPS website and downloads for the database creation tool, command line tool and database are freely accessible at http://breps.tu-bs.de. PMID:28750104

The BRENDA enzyme information system-From a database to an expert system.

PubMed

Schomburg, I; Jeske, L; Ulbrich, M; Placzek, S; Chang, A; Schomburg, D

2017-11-10

Enzymes, representing the largest and by far most complex group of proteins, play an essential role in all processes of life, including metabolism, gene expression, cell division, the immune system, and others. Their function, also connected to most diseases or stress control makes them interesting targets for research and applications in biotechnology, medical treatments, or diagnosis. Their functional parameters and other properties are collected, integrated, and made available to the scientific community in the BRaunschweig ENzyme DAtabase (BRENDA). In the last 30 years BRENDA has developed into one of the most highly used biological databases worldwide. The data contents, the process of data acquisition, data integration and control, the ways to access the data, and visualizations provided by the website are described and discussed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

PubMed

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching.

PubMed

Boonyapakron, Katewadee; Jaruwat, Aritsara; Liwnaree, Benjamas; Nimchua, Thidarat; Champreda, Verawat; Chitnumsub, Penchit

2017-10-10

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the k cat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pK a and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Software Engineering Laboratory (SEL) database organization and user's guide, revision 2

NASA Technical Reports Server (NTRS)

Morusiewicz, Linda; Bristow, John

1992-01-01

The organization of the Software Engineering Laboratory (SEL) database is presented. Included are definitions and detailed descriptions of the database tables and views, the SEL data, and system support data. The mapping from the SEL and system support data to the base table is described. In addition, techniques for accessing the database through the Database Access Manager for the SEL (DAMSEL) system and via the ORACLE structured query language (SQL) are discussed.

Software Engineering Laboratory (SEL) database organization and user's guide

NASA Technical Reports Server (NTRS)

So, Maria; Heller, Gerard; Steinberg, Sandra; Spiegel, Douglas

1989-01-01

The organization of the Software Engineering Laboratory (SEL) database is presented. Included are definitions and detailed descriptions of the database tables and views, the SEL data, and system support data. The mapping from the SEL and system support data to the base tables is described. In addition, techniques for accessing the database, through the Database Access Manager for the SEL (DAMSEL) system and via the ORACLE structured query language (SQL), are discussed.

Bioinformatic perspectives on NRPS/PKS megasynthases: advances and challenges.

PubMed

Jenke-Kodama, Holger; Dittmann, Elke

2009-07-01

The increased understanding of both fundamental principles and mechanistic variations of NRPS/PKS megasynthases along with the unprecedented availability of microbial sequences has inspired a number of in silico studies of both enzyme families. The insights that can be extracted from these analyses go far beyond a rough classification of data and have turned bioinformatics into a frontier field of natural products research. As databases are flooded with NRPS/PKS gene sequence of microbial genomes and metagenomes, increasingly reliable structural prediction methods can help to uncover hidden treasures. Already, phylogenetic analyses have revealed that NRPS/PKS pathways should not simply be regarded as enzyme complexes, specifically evolved to product a selected natural product. Rather, they represent a collection of genetic opinions, allowing biosynthetic pathways to be shuffled in a process of perpetual chemical innovations and pathways diversification in nature can give impulses for specificities, protein interactions and genetic engineering of libraries of novel peptides and polyketides. The successful translation of the knowledge obtained from bioinformatic dissection of NRPS/PKS megasynthases into new techniques for drug discovery and design remain challenges for the future.

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

PubMed

Zafred, Domen; Steiner, Barbara; Teufelberger, Andrea R; Hromic, Altijana; Karplus, P Andrew; Schofield, Christopher J; Wallner, Silvia; Macheroux, Peter

2015-08-01

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF. © 2015 FEBS.

TRENDS: The aeronautical post-test database management system

NASA Technical Reports Server (NTRS)

Bjorkman, W. S.; Bondi, M. J.

1990-01-01

TRENDS, an engineering-test database operating system developed by NASA to support rotorcraft flight tests, is described. Capabilities and characteristics of the system are presented, with examples of its use in recalling and analyzing rotorcraft flight-test data from a TRENDS database. The importance of system user-friendliness in gaining users' acceptance is stressed, as is the importance of integrating supporting narrative data with numerical data in engineering-test databases. Considerations relevant to the creation and maintenance of flight-test database are discussed and TRENDS' solutions to database management problems are described. Requirements, constraints, and other considerations which led to the system's configuration are discussed and some of the lessons learned during TRENDS' development are presented. Potential applications of TRENDS to a wide range of aeronautical and other engineering tests are identified.

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

PubMed

Chánique, Andrea M; Parra, Loreto P

2018-01-01

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa . Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

Directed evolution: an approach to engineer enzymes.

PubMed

Kaur, Jasjeet; Sharma, Rohit

2006-01-01

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

Accessing Nature’s diversity through metabolic engineering and synthetic biology

PubMed Central

King, Jason R.; Edgar, Steven; Qiao, Kangjian; Stephanopoulos, Gregory

2016-01-01

In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents. PMID:27081481

Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.

PubMed

Lv, Xiaomei; Gu, Jiali; Wang, Fan; Xie, Wenping; Liu, Min; Ye, Lidan; Yu, Hongwei

2016-12-01

Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein Design for Pathway Engineering

PubMed Central

Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

2013-01-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

Protein design for pathway engineering.

PubMed

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

DockScreen: A database of in silico biomolecular interactions to support computational toxicology

EPA Science Inventory

We have developed DockScreen, a database of in silico biomolecular interactions designed to enable rational molecular toxicological insight within a computational toxicology framework. This database is composed of chemical/target (receptor and enzyme) binding scores calculated by...

The LAILAPS search engine: a feature model for relevance ranking in life science databases.

PubMed

Lange, Matthias; Spies, Karl; Colmsee, Christian; Flemming, Steffen; Klapperstück, Matthias; Scholz, Uwe

2010-03-25

Efficient and effective information retrieval in life sciences is one of the most pressing challenge in bioinformatics. The incredible growth of life science databases to a vast network of interconnected information systems is to the same extent a big challenge and a great chance for life science research. The knowledge found in the Web, in particular in life-science databases, are a valuable major resource. In order to bring it to the scientist desktop, it is essential to have well performing search engines. Thereby, not the response time nor the number of results is important. The most crucial factor for millions of query results is the relevance ranking. In this paper, we present a feature model for relevance ranking in life science databases and its implementation in the LAILAPS search engine. Motivated by the observation of user behavior during their inspection of search engine result, we condensed a set of 9 relevance discriminating features. These features are intuitively used by scientists, who briefly screen database entries for potential relevance. The features are both sufficient to estimate the potential relevance, and efficiently quantifiable. The derivation of a relevance prediction function that computes the relevance from this features constitutes a regression problem. To solve this problem, we used artificial neural networks that have been trained with a reference set of relevant database entries for 19 protein queries. Supporting a flexible text index and a simple data import format, this concepts are implemented in the LAILAPS search engine. It can easily be used both as search engine for comprehensive integrated life science databases and for small in-house project databases. LAILAPS is publicly available for SWISSPROT data at http://lailaps.ipk-gatersleben.de.

NSWC Crane Aerospace Cell Test History Database

NASA Technical Reports Server (NTRS)

Brown, Harry; Moore, Bruce

1994-01-01

The Aerospace Cell Test History Database was developed to provide project engineers and scientists ready access to the data obtained from testing of aerospace cell designs at Naval Surface Warfare Center, Crane Division. The database is intended for use by all aerospace engineers and scientists involved in the design of power systems for satellites. Specifically, the database will provide a tool for project engineers to review the progress of their test at Crane and to have ready access to data for evaluation. Additionally, the database will provide a history of test results that designers can draw upon to answer questions about cell performance under certain test conditions and aid in selection of a cell for a satellite battery. Viewgraphs are included.

Cazymes Analysis Toolkit (CAT): Webservice for searching and analyzing carbohydrateactive enzymes in a newly sequenced organism using CAZy database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpinets, Tatiana V; Park, Byung; Syed, Mustafa H

2010-01-01

The Carbohydrate-Active Enzyme (CAZy) database provides a rich set of manually annotated enzymes that degrade, modify, or create glycosidic bonds. Despite rich and invaluable information stored in the database, software tools utilizing this information for annotation of newly sequenced genomes by CAZy families are limited. We have employed two annotation approaches to fill the gap between manually curated high-quality protein sequences collected in the CAZy database and the growing number of other protein sequences produced by genome or metagenome sequencing projects. The first approach is based on a similarity search against the entire non-redundant sequences of the CAZy database. Themore » second approach performs annotation using links or correspondences between the CAZy families and protein family domains. The links were discovered using the association rule learning algorithm applied to sequences from the CAZy database. The approaches complement each other and in combination achieved high specificity and sensitivity when cross-evaluated with the manually curated genomes of Clostridium thermocellum ATCC 27405 and Saccharophagus degradans 2-40. The capability of the proposed framework to predict the function of unknown protein domains (DUF) and of hypothetical proteins in the genome of Neurospora crassa is demonstrated. The framework is implemented as a Web service, the CAZymes Analysis Toolkit (CAT), and is available at http://cricket.ornl.gov/cgi-bin/cat.cgi.« less

CAZymes Analysis Toolkit (CAT): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using CAZy database.

PubMed

Park, Byung H; Karpinets, Tatiana V; Syed, Mustafa H; Leuze, Michael R; Uberbacher, Edward C

2010-12-01

The Carbohydrate-Active Enzyme (CAZy) database provides a rich set of manually annotated enzymes that degrade, modify, or create glycosidic bonds. Despite rich and invaluable information stored in the database, software tools utilizing this information for annotation of newly sequenced genomes by CAZy families are limited. We have employed two annotation approaches to fill the gap between manually curated high-quality protein sequences collected in the CAZy database and the growing number of other protein sequences produced by genome or metagenome sequencing projects. The first approach is based on a similarity search against the entire nonredundant sequences of the CAZy database. The second approach performs annotation using links or correspondences between the CAZy families and protein family domains. The links were discovered using the association rule learning algorithm applied to sequences from the CAZy database. The approaches complement each other and in combination achieved high specificity and sensitivity when cross-evaluated with the manually curated genomes of Clostridium thermocellum ATCC 27405 and Saccharophagus degradans 2-40. The capability of the proposed framework to predict the function of unknown protein domains and of hypothetical proteins in the genome of Neurospora crassa is demonstrated. The framework is implemented as a Web service, the CAZymes Analysis Toolkit, and is available at http://cricket.ornl.gov/cgi-bin/cat.cgi.

Comet: an open-source MS/MS sequence database search tool.

PubMed

Eng, Jimmy K; Jahan, Tahmina A; Hoopmann, Michael R

2013-01-01

Proteomics research routinely involves identifying peptides and proteins via MS/MS sequence database search. Thus the database search engine is an integral tool in many proteomics research groups. Here, we introduce the Comet search engine to the existing landscape of commercial and open-source database search tools. Comet is open source, freely available, and based on one of the original sequence database search tools that has been widely used for many years. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

PubMed

Ye, Lidan; Yang, Chengcheng; Yu, Hongwei

2018-01-01

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

PubMed

Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

2013-01-01

In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

Computer-assisted engineering of the synthetic pathway for biodegradation of a toxic persistent pollutant.

PubMed

Kurumbang, Nagendra Prasad; Dvorak, Pavel; Bendl, Jaroslav; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri

2014-03-21

Anthropogenic halogenated compounds were unknown to nature until the industrial revolution, and microorganisms have not had sufficient time to evolve enzymes for their degradation. The lack of efficient enzymes and natural pathways can be addressed through a combination of protein and metabolic engineering. We have assembled a synthetic route for conversion of the highly toxic and recalcitrant 1,2,3-trichloropropane to glycerol in Escherichia coli, and used it for a systematic study of pathway bottlenecks. Optimal ratios of enzymes for the maximal production of glycerol, and minimal toxicity of metabolites were predicted using a mathematical model. The strains containing the expected optimal ratios of enzymes were constructed and characterized for their viability and degradation efficiency. Excellent agreement between predicted and experimental data was observed. The validated model was used to quantitatively describe the kinetic limitations of currently available enzyme variants and predict improvements required for further pathway optimization. This highlights the potential of forward engineering of microorganisms for the degradation of toxic anthropogenic compounds.

Enzyme Engineering for In Situ Immobilization.

PubMed

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

NASA Astrophysics Data System (ADS)

Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

2015-12-01

Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

Blending Education and Polymer Science: Semiautomated Creation of a Thermodynamic Property Database

ERIC Educational Resources Information Center

Tchoua, Roselyne B.; Qin, Jian; Audus, Debra J.; Chard, Kyle; Foster, Ian T.; de Pablo, Juan

2016-01-01

Structured databases of chemical and physical properties play a central role in the everyday research activities of scientists and engineers. In materials science, researchers and engineers turn to these databases to quickly query, compare, and aggregate various properties, thereby allowing for the development or application of new materials. The…

Digging Deeper: The Deep Web.

ERIC Educational Resources Information Center

Turner, Laura

2001-01-01

Focuses on the Deep Web, defined as Web content in searchable databases of the type that can be found only by direct query. Discusses the problems of indexing; inability to find information not indexed in the search engine's database; and metasearch engines. Describes 10 sites created to access online databases or directly search them. Lists ways…

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

PubMed Central

Chánique, Andrea M.; Parra, Loreto P.

2018-01-01

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa. Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity. PMID:29491854

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence-Function Space and Genome Context to Discover Novel Functions.

PubMed

Gerlt, John A

2017-08-22

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of "genomic enzymology" web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence-function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems.

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

PubMed Central

2017-01-01

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of “genomic enzymology” web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence–function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems. PMID:28826221

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

PubMed

Wieczorek, Andrew S; Martin, Vincent J J

2012-12-15

The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

Common Database Interface for Heterogeneous Software Engineering Tools.

DTIC Science & Technology

1987-12-01

SUB-GROUP Database Management Systems ;Programming(Comuters); 1e 05 Computer Files;Information Transfer;Interfaces; 19. ABSTRACT (Continue on reverse...Air Force Institute of Technology Air University In Partial Fulfillment of the Requirements for the Degree of Master of Science in Information Systems ...Literature ..... 8 System 690 Configuration ......... 8 Database Functionis ............ 14 Software Engineering Environments ... 14 Data Manager

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis.

PubMed

Wang, Xiaoqiang

2011-03-01

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.

Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds.

PubMed

Dai, Lu; Tao, Fei; Tang, Hongzhi; Guo, Yali; Shen, Yaling; Xu, Ping

2017-11-01

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as "specialists". However, some enzymes are "generalists" that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineering fungal morphology for enhanced production of hydrolytic enzymes by Aspergillus oryzae SBS50 using microparticles.

PubMed

Singh, Bijender

2018-06-01

Effect of microparticles and silver nanoparticles was studied on the production of hydrolytic enzymes by a potent phytase-producing mould, Aspergillus oryzae SBS50. Addition of microparticles, viz. talc powder and aluminum oxide enhanced phytase production from 2894 to 3903 and 2847 to 4204 U/L, cellulase from 2529 to 4931 and 2455 to 3444 U/L, xylanase from 9067 to 9642 and 9994 to 14,783 U/L, amylase from 5880 to 11,000 and 6130 to 13,145 U/L, respectively. Fungal morphology was also engineered by the use of microparticles. Fungal pellet size was significantly reduced (~ 90%) by the addition of microparticles. Fermentation time was reduced from 4 to 3 days after the addition of microparticles, thus increasing the productivity of the enzymes significantly. These results confirmed the importance of microparticles in engineering fungal morphology for enhanced production of hydrolytic enzymes.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Biological production of organic compounds

DOEpatents

Yu, Jianping; Wang, Bo; Paddock, Troy; Carrieri, Damian; Maness, Pin-Ching; Seibert, Michael

2018-03-13

Methods of producing ethylene oxide and ethylene glycol are disclosed herein. Ethylene produced by cyanobacteria engineered to express ethylene-forming enzymes may be converted to ethylene oxide by bacteria engineered to express a monooxygenase enzyme. Ethylene oxide may be converted to ethylene glycol by exposure to an acidic solution. The methods may be performed in a bioreactor.

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

PubMed

Munawar, Nayla; Engel, Paul C

2013-01-01

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Representing metabolic pathway information: an object-oriented approach.

PubMed

Ellis, L B; Speedie, S M; McLeish, R

1998-01-01

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD) is a website providing information and dynamic links for microbial metabolic pathways, enzyme reactions, and their substrates and products. The Compound, Organism, Reaction and Enzyme (CORE) object-oriented database management system was developed to contain and serve this information. CORE was developed using Java, an object-oriented programming language, and PSE persistent object classes from Object Design, Inc. CORE dynamically generates descriptive web pages for reactions, compounds and enzymes, and reconstructs ad hoc pathway maps starting from any UM-BBD reaction. CORE code is available from the authors upon request. CORE is accessible through the UM-BBD at: http://www. labmed.umn.edu/umbbd/index.html.

Determination of enzyme thermal parameters for rational enzyme engineering and environmental/evolutionary studies.

PubMed

Lee, Charles K; Monk, Colin R; Daniel, Roy M

2013-01-01

Of the two independent processes by which enzymes lose activity with increasing temperature, irreversible thermal inactivation and rapid reversible equilibration with an inactive form, the latter is only describable by the Equilibrium Model. Any investigation of the effect of temperature upon enzymes, a mandatory step in rational enzyme engineering and study of enzyme temperature adaptation, thus requires determining the enzymes' thermodynamic parameters as defined by the Equilibrium Model. The necessary data for this procedure can be collected by carrying out multiple isothermal enzyme assays at 3-5°C intervals over a suitable temperature range. If the collected data meet requirements for V max determination (i.e., if the enzyme kinetics are "ideal"), then the enzyme's Equilibrium Model parameters (ΔH eq, T eq, ΔG (‡) cat, and ΔG (‡) inact) can be determined using a freely available iterative model-fitting software package designed for this purpose.Although "ideal" enzyme reactions are required for determination of all four Equilibrium Model parameters, ΔH eq, T eq, and ΔG (‡) cat can be determined from initial (zero-time) rates for most nonideal enzyme reactions, with substrate saturation being the only requirement.

Database on Demand: insight how to build your own DBaaS

NASA Astrophysics Data System (ADS)

Gaspar Aparicio, Ruben; Coterillo Coz, Ignacio

2015-12-01

At CERN, a number of key database applications are running on user-managed MySQL, PostgreSQL and Oracle database services. The Database on Demand (DBoD) project was born out of an idea to provide CERN user community with an environment to develop and run database services as a complement to the central Oracle based database service. The Database on Demand empowers the user to perform certain actions that had been traditionally done by database administrators, providing an enterprise platform for database applications. It also allows the CERN user community to run different database engines, e.g. presently three major RDBMS (relational database management system) vendors are offered. In this article we show the actual status of the service after almost three years of operations, some insight of our new redesign software engineering and near future evolution.

Algorithms for database-dependent search of MS/MS data.

PubMed

Matthiesen, Rune

2013-01-01

The frequent used bottom-up strategy for identification of proteins and their associated modifications generate nowadays typically thousands of MS/MS spectra that normally are matched automatically against a protein sequence database. Search engines that take as input MS/MS spectra and a protein sequence database are referred as database-dependent search engines. Many programs both commercial and freely available exist for database-dependent search of MS/MS spectra and most of the programs have excellent user documentation. The aim here is therefore to outline the algorithm strategy behind different search engines rather than providing software user manuals. The process of database-dependent search can be divided into search strategy, peptide scoring, protein scoring, and finally protein inference. Most efforts in the literature have been put in to comparing results from different software rather than discussing the underlining algorithms. Such practical comparisons can be cluttered by suboptimal implementation and the observed differences are frequently caused by software parameters settings which have not been set proper to allow even comparison. In other words an algorithmic idea can still be worth considering even if the software implementation has been demonstrated to be suboptimal. The aim in this chapter is therefore to split the algorithms for database-dependent searching of MS/MS data into the above steps so that the different algorithmic ideas become more transparent and comparable. Most search engines provide good implementations of the first three data analysis steps mentioned above, whereas the final step of protein inference are much less developed for most search engines and is in many cases performed by an external software. The final part of this chapter illustrates how protein inference is built into the VEMS search engine and discusses a stand-alone program SIR for protein inference that can import a Mascot search result.

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering.

PubMed

Nagao, Jun-ichi; Aso, Yuji; Shioya, Kouki; Nakayama, Jiro; Sonomoto, Kenji

2007-01-01

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.

EFFECTS OF X-IRRADIATION ON THE HEXOBARBITAL METABOLIZING ENZYME SYSTEM OF RAT LIVER MICROSOMES.

DTIC Science & Technology

RADIATION EFFECTS , *ENZYME INHIBITORS, *HYPNOTICS AND SEDATIVES, ENZYMES, BIOSYNTHESIS, METABOLISM, DETOXIFICATION, BARBITURATES, OXIDATION...MICROSOMES, LIVER, REGENERATION(ENGINEERING), EXCISION, SUBLETHAL DOSAGE, TOXICITY , HYPNOSIS, SLEEP, HEAD(ANATOMY), MALES, FEMALES, RATS.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

Image Engine: an object-oriented multimedia database for storing, retrieving and sharing medical images and text.

PubMed Central

Lowe, H. J.

1993-01-01

This paper describes Image Engine, an object-oriented, microcomputer-based, multimedia database designed to facilitate the storage and retrieval of digitized biomedical still images, video, and text using inexpensive desktop computers. The current prototype runs on Apple Macintosh computers and allows network database access via peer to peer file sharing protocols. Image Engine supports both free text and controlled vocabulary indexing of multimedia objects. The latter is implemented using the TView thesaurus model developed by the author. The current prototype of Image Engine uses the National Library of Medicine's Medical Subject Headings (MeSH) vocabulary (with UMLS Meta-1 extensions) as its indexing thesaurus. PMID:8130596

Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

PubMed

García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

2017-08-01

Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

PubMed

Karim, Ashty S; Jewett, Michael C

2016-07-01

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

dbHiMo: a web-based epigenomics platform for histone-modifying enzymes.

PubMed

Choi, Jaeyoung; Kim, Ki-Tae; Huh, Aram; Kwon, Seomun; Hong, Changyoung; Asiegbu, Fred O; Jeon, Junhyun; Lee, Yong-Hwan

2015-01-01

Over the past two decades, epigenetics has evolved into a key concept for understanding regulation of gene expression. Among many epigenetic mechanisms, covalent modifications such as acetylation and methylation of lysine residues on core histones emerged as a major mechanism in epigenetic regulation. Here, we present the database for histone-modifying enzymes (dbHiMo; http://hme.riceblast.snu.ac.kr/) aimed at facilitating functional and comparative analysis of histone-modifying enzymes (HMEs). HMEs were identified by applying a search pipeline built upon profile hidden Markov model (HMM) to proteomes. The database incorporates 11,576 HMEs identified from 603 proteomes including 483 fungal, 32 plants and 51 metazoan species. The dbHiMo provides users with web-based personalized data browsing and analysis tools, supporting comparative and evolutionary genomics. With comprehensive data entries and associated web-based tools, our database will be a valuable resource for future epigenetics/epigenomics studies. © The Author(s) 2015. Published by Oxford University Press.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

PubMed

Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2016-03-28

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

Development of a One-Stop Data Search and Discovery Engine using Ontologies for Semantic Mappings (HydroSeek)

NASA Astrophysics Data System (ADS)

Piasecki, M.; Beran, B.

2007-12-01

Search engines have changed the way we see the Internet. The ability to find the information by just typing in keywords was a big contribution to the overall web experience. While the conventional search engine methodology worked well for textual documents, locating scientific data remains a problem since they are stored in databases not readily accessible by search engine bots. Considering different temporal, spatial and thematic coverage of different databases, especially for interdisciplinary research it is typically necessary to work with multiple data sources. These sources can be federal agencies which generally offer national coverage or regional sources which cover a smaller area with higher detail. However for a given geographic area of interest there often exists more than one database with relevant data. Thus being able to query multiple databases simultaneously is a desirable feature that would be tremendously useful for scientists. Development of such a search engine requires dealing with various heterogeneity issues. In scientific databases, systems often impose controlled vocabularies which ensure that they are generally homogeneous within themselves but are semantically heterogeneous when moving between different databases. This defines the boundaries of possible semantic related problems making it easier to solve than with the conventional search engines that deal with free text. We have developed a search engine that enables querying multiple data sources simultaneously and returns data in a standardized output despite the aforementioned heterogeneity issues between the underlying systems. This application relies mainly on metadata catalogs or indexing databases, ontologies and webservices with virtual globe and AJAX technologies for the graphical user interface. Users can trigger a search of dozens of different parameters over hundreds of thousands of stations from multiple agencies by providing a keyword, a spatial extent, i.e. a bounding box, and a temporal bracket. As part of this development we have also added an environment that allows users to do some of the semantic tagging, i.e. the linkage of a variable name (which can be anything they desire) to defined concepts in the ontology structure which in turn provides the backbone of the search engine.

Reducing Enzyme Costs Increases Market Potential of Biofuels; The Spectrum of Clean Energy Innovation (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Fact sheet describing NREL's work with enzyme producers Novozymes and Genencor to engineer new cellulase enzymes to breakdown cellulosic ethanol into fermentable sugars that can be converted into biofuels.

Configuration management program plan for Hanford site systems engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kellie, C.L.

This plan establishes the integrated management program for the evolving technical baseline developed through the systems engineering process. This configuration management program aligns with the criteria identified in the DOE Standard, DOE-STD-1073-93. Included are specific requirements for control of the systems engineering RDD-100 database, and electronic data incorporated in the database that establishes the Hanford Site Technical Baseline.

Correlated Attack Modeling (CAM)

DTIC Science & Technology

2003-10-01

describing attack models to a scenario recognition engine, a prototype of such an engine was developed, using components of the EMERALD intrusion...content. Results – The attacker gains information enabling remote access to database (i.e., privileged login information, database layout to allow...engine that uses attack specifications written in CAML. The implementation integrates two advanced technologies devel- oped in the EMERALD program [27, 31

Biochemical Engineering Fundamentals

ERIC Educational Resources Information Center

Bailey, J. E.; Ollis, D. F.

1976-01-01

Discusses a biochemical engineering course that is offered as part of a chemical engineering curriculum and includes topics that influence the behavior of man-made or natural microbial or enzyme reactors. (MLH)

de novo computational enzyme design.

PubMed

Zanghellini, Alexandre

2014-10-01

Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

State analysis requirements database for engineering complex embedded systems

NASA Technical Reports Server (NTRS)

Bennett, Matthew B.; Rasmussen, Robert D.; Ingham, Michel D.

2004-01-01

It has become clear that spacecraft system complexity is reaching a threshold where customary methods of control are no longer affordable or sufficiently reliable. At the heart of this problem are the conventional approaches to systems and software engineering based on subsystem-level functional decomposition, which fail to scale in the tangled web of interactions typically encountered in complex spacecraft designs. Furthermore, there is a fundamental gap between the requirements on software specified by systems engineers and the implementation of these requirements by software engineers. Software engineers must perform the translation of requirements into software code, hoping to accurately capture the systems engineer's understanding of the system behavior, which is not always explicitly specified. This gap opens up the possibility for misinterpretation of the systems engineer's intent, potentially leading to software errors. This problem is addressed by a systems engineering tool called the State Analysis Database, which provides a tool for capturing system and software requirements in the form of explicit models. This paper describes how requirements for complex aerospace systems can be developed using the State Analysis Database.

A case study for a digital seabed database: Bohai Sea engineering geology database

NASA Astrophysics Data System (ADS)

Tianyun, Su; Shikui, Zhai; Baohua, Liu; Ruicai, Liang; Yanpeng, Zheng; Yong, Wang

2006-07-01

This paper discusses the designing plan of ORACLE-based Bohai Sea engineering geology database structure from requisition analysis, conceptual structure analysis, logical structure analysis, physical structure analysis and security designing. In the study, we used the object-oriented Unified Modeling Language (UML) to model the conceptual structure of the database and used the powerful function of data management which the object-oriented and relational database ORACLE provides to organize and manage the storage space and improve its security performance. By this means, the database can provide rapid and highly effective performance in data storage, maintenance and query to satisfy the application requisition of the Bohai Sea Oilfield Paradigm Area Information System.

Software Re-Engineering of the Human Factors Analysis and Classification System - (Maintenance Extension) Using Object Oriented Methods in a Microsoft Environment

DTIC Science & Technology

2001-09-01

replication) -- all from Visual Basic and VBA . In fact, we found that the SQL Server engine actually had a plethora of options, most formidable of...2002, the new SQL Server 2000 database engine, and Microsoft Visual Basic.NET. This thesis describes our use of the Spiral Development Model to...versions of Microsoft products? Specifically, the pending release of Microsoft Office 2002, the new SQL Server 2000 database engine, and Microsoft

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

PubMed

Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

2018-08-01

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

Configuration management program plan for Hanford site systems engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffman, A.G.

This plan establishes the integrated configuration management program for the evolving technical baseline developed through the systems engineering process. This configuration management program aligns with the criteria identified in the DOE Standard, DOE-STD-1073-93. Included are specific requirements for control of the systems engineering RDD-100 database, and electronic data incorporated in the database that establishes the Hanford site technical baseline.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Advanced Transportation System Studies. Technical Area 3: Alternate Propulsion Subsystem Concepts. Volume 1; Executive Summary

NASA Technical Reports Server (NTRS)

Levack, Daniel J. H.

2000-01-01

The Alternate Propulsion Subsystem Concepts contract had seven tasks defined that are reported under this contract deliverable. The tasks were: FAA Restart Study, J-2S Restart Study, Propulsion Database Development. SSME Upper Stage Use. CERs for Liquid Propellant Rocket Engines. Advanced Low Cost Engines, and Tripropellant Comparison Study. The two restart studies, F-1A and J-2S, generated program plans for restarting production of each engine. Special emphasis was placed on determining changes to individual parts due to obsolete materials, changes in OSHA and environmental concerns, new processes available, and any configuration changes to the engines. The Propulsion Database Development task developed a database structure and format which is easy to use and modify while also being comprehensive in the level of detail available. The database structure included extensive engine information and allows for parametric data generation for conceptual engine concepts. The SSME Upper Stage Use task examined the changes needed or desirable to use the SSME as an upper stage engine both in a second stage and in a translunar injection stage. The CERs for Liquid Engines task developed qualitative parametric cost estimating relationships at the engine and major subassembly level for estimating development and production costs of chemical propulsion liquid rocket engines. The Advanced Low Cost Engines task examined propulsion systems for SSTO applications including engine concept definition, mission analysis. trade studies. operating point selection, turbomachinery alternatives, life cycle cost, weight definition. and point design conceptual drawings and component design. The task concentrated on bipropellant engines, but also examined tripropellant engines. The Tripropellant Comparison Study task provided an unambiguous comparison among various tripropellant implementation approaches and cycle choices, and then compared them to similarly designed bipropellant engines in the SSTO mission This volume overviews each of the tasks giving its objectives, main results. and conclusions. More detailed Final Task Reports are available on each individual task.

Microbial chemical factories: recent advances in pathway engineering for synthesis of value added chemicals.

PubMed

Dhamankar, Himanshu; Prather, Kristala L J

2011-08-01

The dwindling nature of petroleum and other fossil reserves has provided impetus towards microbial synthesis of fuels and value added chemicals from biomass-derived sugars as a renewable resource. Microbes have naturally evolved enzymes and pathways that can convert biomass into hundreds of unique chemical structures, a property that can be effectively exploited for their engineering into Microbial Chemical Factories (MCFs). De novo pathway engineering facilitates expansion of the repertoire of microbially synthesized compounds beyond natural products. In this review, we visit some recent successes in such novel pathway engineering and optimization, with particular emphasis on the selection and engineering of pathway enzymes and balancing of their accessory cofactors. Copyright © 2011 Elsevier Ltd. All rights reserved.

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis.

PubMed

Singh, Satendra; Singh, Dev Bukhsh; Singh, Anamika; Gautam, Budhayash; Ram, Gurudayal; Dwivedi, Seema; Ramteke, Pramod W

2016-12-01

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

PubMed

Coppella, S J; DelaCruz, N; Payne, G F; Pogell, B M; Speedie, M K; Karns, J S; Sybert, E M; Connor, M A

1990-01-01

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

Biocatalysis in the Pharmaceutical Industry: The Need for Speed

PubMed Central

2017-01-01

The use of biocatalysis in the pharmaceutical industry continues to expand as a result of increased access to enzymes and the ability to engineer those enzymes to meet the demands of industrial processes. However, we are still just scratching the surface of potential biocatalytic applications. The time pressures present in pharmaceutical process development are incompatible with the long lead times required for engineering a suitable biocatalyst. Dramatic increases in the speed of protein engineering are needed to deliver on the ever increasing opportunities for industrial biocatalytic processes. PMID:28523096

Biocatalysis in the Pharmaceutical Industry: The Need for Speed.

PubMed

Truppo, Matthew D

2017-05-11

The use of biocatalysis in the pharmaceutical industry continues to expand as a result of increased access to enzymes and the ability to engineer those enzymes to meet the demands of industrial processes. However, we are still just scratching the surface of potential biocatalytic applications. The time pressures present in pharmaceutical process development are incompatible with the long lead times required for engineering a suitable biocatalyst. Dramatic increases in the speed of protein engineering are needed to deliver on the ever increasing opportunities for industrial biocatalytic processes.

Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

PubMed

Liu, Jia; Yang, Qihua; Li, Can

2015-09-18

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

PubMed Central

Lawton, Thomas J.; Rosenzweig, Amy C.

2017-01-01

Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16–13 s−1, these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock. PMID:27366961

Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion.

PubMed

Lawton, Thomas J; Rosenzweig, Amy C

2016-08-03

Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16-13 s(-1), these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock.

Thermophilic lignocellulose deconstruction.

PubMed

Blumer-Schuette, Sara E; Brown, Steven D; Sander, Kyle B; Bayer, Edward A; Kataeva, Irina; Zurawski, Jeffrey V; Conway, Jonathan M; Adams, Michael W W; Kelly, Robert M

2014-05-01

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Change, exchange, and rearrange: protein engineering for the biotechnological production of fuels, pharmaceuticals, and other chemicals.

PubMed

Fisher, Michael A; Tullman-Ercek, Danielle

2013-12-01

Enzymes are indispensable in the effort to produce chemicals from fuels to pharmaceuticals in an ecologically friendly manner. They have the potential to catalyze reactions with high specificity and efficiency without the use of hazardous chemicals. Nature provides an extensive collection of enzymes, but often these must be altered to perform desired functions under required conditions. Advances in protein engineering permit the design and/or directed evolution of enzymes specifically tailored for such industrial applications. Recent years have seen the development of improved enzymes to assist in both the conversion of biomass into fuels and chemicals, and the creation of key intermediates in pharmaceutical production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure-based Engineering of a Plant-Fungal Hybrid Peroxidase for Enhanced Temperature and pH Tolerance.

PubMed

Kohler, Amanda C; Simmons, Blake A; Sale, Kenneth L

2018-04-28

In an age of ever-increasing biotechnological and industrial demand for new and specialized biocatalysts, rational protein engineering offers a direct approach to enzyme design and innovation. Heme peroxidases, as indispensable oxidative biocatalysts, provide a relatively mild alternative to the traditional harsh, and often toxic, chemical catalysts, and subsequently, have found widespread application throughout industry. However, the potential for these enzymes is far greater than their present use, as processes are currently restricted to the more stable, but less catalytically powerful, subset of peroxidases. Here we describe the structure-guided, rational engineering of a plant-fungal hybrid peroxidase built to overcome the application barrier of these high-reduction potential peroxidases. This engineered enzyme has the catalytic versatility and oxidative ability of a high-reduction potential versatile peroxidase, with enhanced temperature and pH tolerance similar to that of a highly stable plant peroxidase. Copyright © 2018 Elsevier Ltd. All rights reserved.

CropEx Web-Based Agricultural Monitoring and Decision Support

NASA Technical Reports Server (NTRS)

Harvey. Craig; Lawhead, Joel

2011-01-01

CropEx is a Web-based agricultural Decision Support System (DSS) that monitors changes in crop health over time. It is designed to be used by a wide range of both public and private organizations, including individual producers and regional government offices with a vested interest in tracking vegetation health. The database and data management system automatically retrieve and ingest data for the area of interest. Another stores results of the processing and supports the DSS. The processing engine will allow server-side analysis of imagery with support for image sub-setting and a set of core raster operations for image classification, creation of vegetation indices, and change detection. The system includes the Web-based (CropEx) interface, data ingestion system, server-side processing engine, and a database processing engine. It contains a Web-based interface that has multi-tiered security profiles for multiple users. The interface provides the ability to identify areas of interest to specific users, user profiles, and methods of processing and data types for selected or created areas of interest. A compilation of programs is used to ingest available data into the system, classify that data, profile that data for quality, and make data available for the processing engine immediately upon the data s availability to the system (near real time). The processing engine consists of methods and algorithms used to process the data in a real-time fashion without copying, storing, or moving the raw data. The engine makes results available to the database processing engine for storage and further manipulation. The database processing engine ingests data from the image processing engine, distills those results into numerical indices, and stores each index for an area of interest. This process happens each time new data is ingested and processed for the area of interest, and upon subsequent database entries, the database processing engine qualifies each value for each area of interest and conducts a logical processing of results indicating when and where thresholds are exceeded. Reports are provided at regular, operator-determined intervals that include variances from thresholds and links to view raw data for verification, if necessary. The technology and method of development allow the code base to easily be modified for varied use in the real-time and near-real-time processing environments. In addition, the final product will be demonstrated as a means for rapid draft assessment of imagery.

SSME environment database development

NASA Technical Reports Server (NTRS)

Reardon, John

1987-01-01

The internal environment of the Space Shuttle Main Engine (SSME) is being determined from hot firings of the prototype engines and from model tests using either air or water as the test fluid. The objectives are to develop a database system to facilitate management and analysis of test measurements and results, to enter available data into the the database, and to analyze available data to establish conventions and procedures to provide consistency in data normalization and configuration geometry references.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

PubMed

Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Zimmermann, Wolfgang; Wei, Ren; Leggewie, Christian; Li, Xiangzhen; Hazen, Terry; Streit, Wolfgang R

2018-04-15

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria , Proteobacteria , and Bacteroidetes Within the Proteobacteria , the Betaproteobacteria , Deltaproteobacteria , and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria , as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases. Copyright © 2018 Danso et al.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes

PubMed Central

Danso, Dominik; Schmeisser, Christel; Chow, Jennifer; Wei, Ren; Leggewie, Christian; Li, Xiangzhen

2018-01-01

ABSTRACT Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases. PMID:29427431

DRUMS: a human disease related unique gene mutation search engine.

PubMed

Li, Zuofeng; Liu, Xingnan; Wen, Jingran; Xu, Ye; Zhao, Xin; Li, Xuan; Liu, Lei; Zhang, Xiaoyan

2011-10-01

With the completion of the human genome project and the development of new methods for gene variant detection, the integration of mutation data and its phenotypic consequences has become more important than ever. Among all available resources, locus-specific databases (LSDBs) curate one or more specific genes' mutation data along with high-quality phenotypes. Although some genotype-phenotype data from LSDB have been integrated into central databases little effort has been made to integrate all these data by a search engine approach. In this work, we have developed disease related unique gene mutation search engine (DRUMS), a search engine for human disease related unique gene mutation as a convenient tool for biologists or physicians to retrieve gene variant and related phenotype information. Gene variant and phenotype information were stored in a gene-centred relational database. Moreover, the relationships between mutations and diseases were indexed by the uniform resource identifier from LSDB, or another central database. By querying DRUMS, users can access the most popular mutation databases under one interface. DRUMS could be treated as a domain specific search engine. By using web crawling, indexing, and searching technologies, it provides a competitively efficient interface for searching and retrieving mutation data and their relationships to diseases. The present system is freely accessible at http://www.scbit.org/glif/new/drums/index.html. © 2011 Wiley-Liss, Inc.

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

NASA Technical Reports Server (NTRS)

Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

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.

A Taxonomic Search Engine: Federating taxonomic databases using web services

PubMed Central

Page, Roderic DM

2005-01-01

Background The taxonomic name of an organism is a key link between different databases that store information on that organism. However, in the absence of a single, comprehensive database of organism names, individual databases lack an easy means of checking the correctness of a name. Furthermore, the same organism may have more than one name, and the same name may apply to more than one organism. Results The Taxonomic Search Engine (TSE) is a web application written in PHP that queries multiple taxonomic databases (ITIS, Index Fungorum, IPNI, NCBI, and uBIO) and summarises the results in a consistent format. It supports "drill-down" queries to retrieve a specific record. The TSE can optionally suggest alternative spellings the user can try. It also acts as a Life Science Identifier (LSID) authority for the source taxonomic databases, providing globally unique identifiers (and associated metadata) for each name. Conclusion The Taxonomic Search Engine is available at and provides a simple demonstration of the potential of the federated approach to providing access to taxonomic names. PMID:15757517

In vitro metabolic engineering for the salvage synthesis of NAD(.).

PubMed

Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

2016-05-01

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A Summary of the Naval Postgraduate School Research Program

DTIC Science & Technology

1989-08-30

5 Fundamental Theory for Automatically Combining Changes to Software Systems ............................ 6 Database -System Approach to...Software Engineering Environments(SEE’s) .................................. 10 Multilevel Database Security .......................... 11 Temporal... Database Management and Real-Time Database Computers .................................... 12 The Multi-lingual, Multi Model, Multi-Backend Database

Evaluating Functional Annotations of Enzymes Using the Gene Ontology.

PubMed

Holliday, Gemma L; Davidson, Rebecca; Akiva, Eyal; Babbitt, Patricia C

2017-01-01

The Gene Ontology (GO) (Ashburner et al., Nat Genet 25(1):25-29, 2000) is a powerful tool in the informatics arsenal of methods for evaluating annotations in a protein dataset. From identifying the nearest well annotated homologue of a protein of interest to predicting where misannotation has occurred to knowing how confident you can be in the annotations assigned to those proteins is critical. In this chapter we explore what makes an enzyme unique and how we can use GO to infer aspects of protein function based on sequence similarity. These can range from identification of misannotation or other errors in a predicted function to accurate function prediction for an enzyme of entirely unknown function. Although GO annotation applies to any gene products, we focus here a describing our approach for hierarchical classification of enzymes in the Structure-Function Linkage Database (SFLD) (Akiva et al., Nucleic Acids Res 42(Database issue):D521-530, 2014) as a guide for informed utilisation of annotation transfer based on GO terms.

Profiling the orphan enzymes.

PubMed

Sorokina, Maria; Stam, Mark; Médigue, Claudine; Lespinet, Olivier; Vallenet, David

2014-06-06

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

Entamoeba histolytica: construction and applications of subgenomic databases.

PubMed

Hofer, Margit; Duchêne, Michael

2005-07-01

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.

Decision making in family medicine: randomized trial of the effects of the InfoClinique and Trip database search engines.

PubMed

Labrecque, Michel; Ratté, Stéphane; Frémont, Pierre; Cauchon, Michel; Ouellet, Jérôme; Hogg, William; McGowan, Jessie; Gagnon, Marie-Pierre; Njoya, Merlin; Légaré, France

2013-10-01

To compare the ability of users of 2 medical search engines, InfoClinique and the Trip database, to provide correct answers to clinical questions and to explore the perceived effects of the tools on the clinical decision-making process. Randomized trial. Three family medicine units of the family medicine program of the Faculty of Medicine at Laval University in Quebec city, Que. Fifteen second-year family medicine residents. Residents generated 30 structured questions about therapy or preventive treatment (2 questions per resident) based on clinical encounters. Using an Internet platform designed for the trial, each resident answered 20 of these questions (their own 2, plus 18 of the questions formulated by other residents, selected randomly) before and after searching for information with 1 of the 2 search engines. For each question, 5 residents were randomly assigned to begin their search with InfoClinique and 5 with the Trip database. The ability of residents to provide correct answers to clinical questions using the search engines, as determined by third-party evaluation. After answering each question, participants completed a questionnaire to assess their perception of the engine's effect on the decision-making process in clinical practice. Of 300 possible pairs of answers (1 answer before and 1 after the initial search), 254 (85%) were produced by 14 residents. Of these, 132 (52%) and 122 (48%) pairs of answers concerned questions that had been assigned an initial search with InfoClinique and the Trip database, respectively. Both engines produced an important and similar absolute increase in the proportion of correct answers after searching (26% to 62% for InfoClinique, for an increase of 36%; 24% to 63% for the Trip database, for an increase of 39%; P = .68). For all 30 clinical questions, at least 1 resident produced the correct answer after searching with either search engine. The mean (SD) time of the initial search for each question was 23.5 (7.6) minutes with InfoClinique and 22.3 (7.8) minutes with the Trip database (P = .30). Participants' perceptions of each engine's effect on the decision-making process were very positive and similar for both search engines. Family medicine residents' ability to provide correct answers to clinical questions increased dramatically and similarly with the use of both InfoClinique and the Trip database. These tools have strong potential to increase the quality of medical care.

An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge

PubMed Central

Onwubiko, J.; Zaru, R.; Rosanoff, S.; Antunes, R.; Bingley, M.; Watkins, X.; O'Donovan, C.; Martin, M. J.

2017-01-01

Abstract Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al., (2013), BMC Bioinformatics, 14 (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al., Nucleic Acids Res.2016; 44, D385–D395) Rhea—a database of enzyme-catalysed reactions (Morgat, A., et al., Nucleic Acids Res. 2015; 43, D459-D464), Reactome—a database of biochemical pathways (Fabregat, A., et al., Nucleic Acids Res. 2016; 44, D481–D487), IntEnz—a resource with enzyme nomenclature information (Fleischmann, A., et al., Nucleic Acids Res. 2004 32, D434–D437) and ChEBI (Hastings, J., et al., Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al., Nucleic Acids Res. 201442, 1083–1090)—resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. PMID:28158609

An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge.

PubMed

Pundir, S; Onwubiko, J; Zaru, R; Rosanoff, S; Antunes, R; Bingley, M; Watkins, X; O'Donovan, C; Martin, M J

2017-03-01

Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al. , (2013), BMC Bioinformatics , (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al ., Nucleic Acids Res. 2016; , D385-D395) Rhea-a database of enzyme-catalysed reactions (Morgat, A., et al .,  Nucleic Acids Res.  2015; , D459-D464), Reactome-a database of biochemical pathways (Fabregat, A., et al ., Nucleic Acids Res. 2016;  , D481-D487), IntEnz-a resource with enzyme nomenclature information (Fleischmann, A., et al ., Nucleic Acids Res.  2004 , D434-D437) and ChEBI (Hastings, J., et al .,  Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al ., Nucleic Acids Res.  2014 , 1083-1090)-resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Metabolite damage and repair in metabolic engineering design.

PubMed

Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

PubMed Central

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra

2017-01-01

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. PMID:28455329

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

PubMed

Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

2017-07-01

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named Ef Amy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel Ef Amy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications. Copyright © 2017 Yang et al.

Reducing Enzyme Costs Increases the Market Potential of Biofuels (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Cellulosic ethanol prices depend heavily on the cost of the cellulase enzymes used to break down the biomass into fermentable sugars. To reduce these costs, NREL partnered with two leading enzyme companies, Novozymes and Genencor, to engineer new cellulase enzymes that are exceptionally good at breaking down cellulose. Genencor is now part of DuPont Industrial Biosciences.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

Engineered enzymatically active bacteriophages and methods of uses thereof

DOEpatents

Collins, James J [Newton, MA; Kobayashi, Hideki [Yokohama, JP; Kearn, Mads [Ottawa, CA; Araki, Michihiro [Minatoku, JP; Friedland, Ari [Boston, MA; Lu, Timothy Kuan-Ta [Palo Alto, CA

2012-05-22

The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.

PubMed

Tan, Zaigao; Chen, Jing; Zhang, Xueli

2016-01-01

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

Finding Sequences for over 270 Orphan Enzymes

PubMed Central

Shearer, Alexander G.; Altman, Tomer; Rhee, Christine D.

2014-01-01

Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These ‘orphan enzymes’ represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes. PMID:24826896

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradationmore » pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.« less

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

PubMed

Mäkelä, Miia R; Dilokpimol, Adiphol; Koskela, Salla M; Kuuskeri, Jaana; de Vries, Ronald P; Hildén, Kristiina

2018-04-26

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

PubMed

Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

2014-11-01

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

Bioenergy News | Bioenergy | NREL

Science.gov Websites

. April 13, 2018 News Release: Research Team Engineers a Better Plastic-Degrading Enzyme A breakthrough in enzyme research led by the U.S. Department of Energy's National Renewable Energy Laboratory (NREL) and the United Kingdom's University of Portsmouth has led to an improved variant of an enzyme that can

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Molecular Bases of Enantioselectivity of Haloalkane Dehalogenase DbjA

NASA Astrophysics Data System (ADS)

Sato, Yukari; Natsume, Ryo; Prokop, Zbynek; Brezovsky, Jan; Chaloupkova, Radka; Damborsky, Jiri; Nagata, Yuji; Senda, Toshiya

Enzymes are widely used for the synthesis of pharmaceuticals, agrochemicals, and food additives because they can catalyze high enantioselective transformations. In order to construct selective enzymes by protein engineering, it is important to understand the molecular basis of enzyme-substrate interactions that contribute to enantioselectivity. The haloalkane dehalogenase DbjA showed high enantioselectivity for two racemic mixtures: α-bromoesters and β-bromoalkanes. Thermodynamic analysis, protein crystallography, and computer simulations indicated that DbjA carries two bases for the enantiodiscrimination of each racemic mixture. This study helps us understand the molecular basis of the enantioselectivity and opens up new possibilities for constructing enantiospecific biocatalysts through protein engineering.

HEMD: an integrated tool of human epigenetic enzymes and chemical modulators for therapeutics.

PubMed

Huang, Zhimin; Jiang, Haiming; Liu, Xinyi; Chen, Yingyi; Wong, Jiemin; Wang, Qi; Huang, Wenkang; Shi, Ting; Zhang, Jian

2012-01-01

Epigenetic mechanisms mainly include DNA methylation, post-translational modifications of histones, chromatin remodeling and non-coding RNAs. All of these processes are mediated and controlled by enzymes. Abnormalities of the enzymes are involved in a variety of complex human diseases. Recently, potent natural or synthetic chemicals are utilized to establish the quantitative contributions of epigenetic regulation through the enzymes and provide novel insight for developing new therapeutics. However, the development of more specific and effective epigenetic therapeutics requires a more complete understanding of the chemical epigenomic landscape. Here, we present a human epigenetic enzyme and modulator database (HEMD), the database which provides a central resource for the display, search, and analysis of the structure, function, and related annotation for human epigenetic enzymes and chemical modulators focused on epigenetic therapeutics. Currently, HEMD contains 269 epigenetic enzymes and 4377 modulators in three categories (activators, inhibitors, and regulators). Enzymes are annotated with detailed description of epigenetic mechanisms, catalytic processes, and related diseases, and chemical modulators with binding sites, pharmacological effect, and therapeutic uses. Integrating the information of epigenetic enzymes in HEMD should allow for the prediction of conserved features for proteins and could potentially classify them as ideal targets for experimental validation. In addition, modulators curated in HEMD can be used to investigate potent epigenetic targets for the query compound and also help chemists to implement structural modifications for the design of novel epigenetic drugs. HEMD could be a platform and a starting point for biologists and medicinal chemists for furthering research on epigenetic therapeutics. HEMD is freely available at http://mdl.shsmu.edu.cn/HEMD/.

Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn: A theoretical N-glycan structure database.

PubMed

Akune, Yukie; Lin, Chi-Hung; Abrahams, Jodie L; Zhang, Jingyu; Packer, Nicolle H; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P

2016-08-05

Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database. Copyright © 2016 Elsevier Ltd. All rights reserved.

Jet aircraft engine exhaust emissions database development: Year 1990 and 2015 scenarios

NASA Technical Reports Server (NTRS)

Landau, Z. Harry; Metwally, Munir; Vanalstyne, Richard; Ward, Clay A.

1994-01-01

Studies relating to environmental emissions associated with the High Speed Civil Transport (HSCT) military jet and charter jet aircraft were conducted by McDonnell Douglas Aerospace Transport Aircraft. The report includes engine emission results for baseline 1990 charter and military scenario and the projected jet engine emissions results for a 2015 scenario for a Mach 1.6 HSCT charter and military fleet. Discussions of the methodology used in formulating these databases are provided.

[Application of atomic absorption spectrometry in the engine knock detection].

PubMed

Chen, Li-Dan

2013-02-01

Because existing human experience diagnosis method and apparatus for auxiliary diagnosis method are difficult to diagnose quickly engine knock. Atomic absorption spectrometry was used to detect the automobile engine knock in in innovative way. After having determined Fe, Al, Cu, Cr and Pb content in the 35 groups of Audi A6 engine oil whose travel course is 2 000 -70 000 kilometers and whose sampling interval is 2 000 kilometers by atomic absorption spectrometry, the database of primary metal content in the same automobile engine at different mileage was established. The research shows that the main metal content fluctuates within a certain range. In practical engineering applications, after the determination of engine oil main metal content and comparison with its database value, it can not only help to diagnose the type and location of engine knock without the disintegration and reduce vehicle maintenance costs and improve the accuracy of engine knock fault diagnosis.

Rational assignment of key motifs for function guides in silico enzyme identification.

PubMed

Höhne, Matthias; Schätzle, Sebastian; Jochens, Helge; Robins, Karen; Bornscheuer, Uwe T

2010-11-01

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

Database Deposit Service through JOIS : JAFIC File on Food Industry and Osaka Urban Engineering File

NASA Astrophysics Data System (ADS)

Kataoka, Akihiro

JICST has launched the database deposit service for the excellent quality in small-and medium size, both of which have no dissemination network. JAFIC File on Food Industry produced by the Japan Food Industry Center and Osaka Urban Engineering File by Osaka City have been in service by JOIS since March 2, 1987. In this paper the outline of the above databases is introduced in focussing on the items covered and retrieved by JOIS.

[Fermentation production of microbial catalase and its application in textile industry].

PubMed

Zhang, Dongxu; Du, Guocheng; Chen, Jian

2010-11-01

Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.

Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.

PubMed

Moraïs, Sarah; Stern, Johanna; Kahn, Amaranta; Galanopoulou, Anastasia P; Yoav, Shahar; Shamshoum, Melina; Smith, Matthew A; Hatzinikolaou, Dimitris G; Arnold, Frances H; Bayer, Edward A

2016-01-01

The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomes.

Increments and duplication events of enzymes and transcription factors influence metabolic and regulatory diversity in prokaryotes.

PubMed

Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

2013-01-01

In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.

Increments and Duplication Events of Enzymes and Transcription Factors Influence Metabolic and Regulatory Diversity in Prokaryotes

PubMed Central

Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto

2013-01-01

In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected. PMID:23922780

Data collection procedures for the Software Engineering Laboratory (SEL) database

NASA Technical Reports Server (NTRS)

Heller, Gerard; Valett, Jon; Wild, Mary

1992-01-01

This document is a guidebook to collecting software engineering data on software development and maintenance efforts, as practiced in the Software Engineering Laboratory (SEL). It supersedes the document entitled Data Collection Procedures for the Rehosted SEL Database, number SEL-87-008 in the SEL series, which was published in October 1987. It presents procedures to be followed on software development and maintenance projects in the Flight Dynamics Division (FDD) of Goddard Space Flight Center (GSFC) for collecting data in support of SEL software engineering research activities. These procedures include detailed instructions for the completion and submission of SEL data collection forms.

Profiling the orphan enzymes

PubMed Central

2014-01-01

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities. Reviewers This article was reviewed by Michael Galperin, Daniel Haft and Daniel Kahn. PMID:24906382

Integrating the intrinsic conformational preferences of non-coded α-amino acids modified at the peptide bond into the NCAD database

PubMed Central

Revilla-López, Guillem; Rodríguez-Ropero, Francisco; Curcó, David; Torras, Juan; Calaza, M. Isabel; Zanuy, David; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos

2011-01-01

Recently, we reported a database (NCAD, Non-Coded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of non-proteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally-established conformational propensities, and applications (J. Phys. Chem. B 2010, 114, 7413). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the –NHCO– peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 310-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database. PMID:21491493

Streptomyces spp. in the biocatalysis toolbox.

PubMed

Spasic, Jelena; Mandic, Mina; Djokic, Lidija; Nikodinovic-Runic, Jasmina

2018-04-01

About 20,100 research publications dated 2000-2017 were recovered searching the PubMed and Web of Science databases for Streptomyces, which are the richest known source of bioactive molecules. However, these bacteria with versatile metabolism are powerful suppliers of biocatalytic tools (enzymes) for advanced biotechnological applications such as green chemical transformations and biopharmaceutical and biofuel production. The recent technological advances, especially in DNA sequencing coupled with computational tools for protein functional and structural prediction, and the improved access to microbial diversity enabled the easier access to enzymes and the ability to engineer them to suit a wider range of biotechnological processes. The major driver behind a dramatic increase in the utilization of biocatalysis is sustainable development and the shift toward bioeconomy that will, in accordance to the UN policy agenda "Bioeconomy to 2030," become a global effort in the near future. Streptomyces spp. already play a significant role among industrial microorganisms. The intention of this minireview is to highlight the presence of Streptomyces in the toolbox of biocatalysis and to give an overview of the most important advances in novel biocatalyst discovery and applications. Judging by the steady increase in a number of recent references (228 for the 2000-2017 period), it is clear that biocatalysts from Streptomyces spp. hold promises in terms of valuable properties and applicative industrial potential.

Advanced transportation system studies. Alternate propulsion subsystem concepts: Propulsion database

NASA Technical Reports Server (NTRS)

Levack, Daniel

1993-01-01

The Advanced Transportation System Studies alternate propulsion subsystem concepts propulsion database interim report is presented. The objective of the database development task is to produce a propulsion database which is easy to use and modify while also being comprehensive in the level of detail available. The database is to be available on the Macintosh computer system. The task is to extend across all three years of the contract. Consequently, a significant fraction of the effort in this first year of the task was devoted to the development of the database structure to ensure a robust base for the following years' efforts. Nonetheless, significant point design propulsion system descriptions and parametric models were also produced. Each of the two propulsion databases, parametric propulsion database and propulsion system database, are described. The descriptions include a user's guide to each code, write-ups for models used, and sample output. The parametric database has models for LOX/H2 and LOX/RP liquid engines, solid rocket boosters using three different propellants, a hybrid rocket booster, and a NERVA derived nuclear thermal rocket engine.

What are the Limitations of Enzymes in Synthetic Organic Chemistry?

PubMed

Reetz, Manfred T

2016-12-01

Enzymes have been used in organic chemistry and biotechnology for 100 years, but their widespread application has been prevented by a number of limitations, including the often-observed limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, especially since recent methodology development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asymmetric catalysis has led to a number of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermentation are likewise enzyme-based techniques that enrich chemistry. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

PubMed

Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

2016-01-01

Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

PubMed

Kürten, Charlotte; Syrén, Per-Olof

2016-01-16

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Directed evolution: tailoring biocatalysts for industrial applications.

PubMed

Kumar, Ashwani; Singh, Suren

2013-12-01

Current challenges and promises of white biotechnology encourage protein engineers to use a directed evolution approach to generate novel and useful biocatalysts for various sets of applications. Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. This review covers fundamental and major development of directed evolution techniques, and highlights the advances in mutagenesis, screening and selection methods with examples of enzymes developed by using these approaches. Several commonly used methods for creating molecular diversity with their advantages and disadvantages including some recently used strategies are also discussed.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

PubMed

Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross

2017-08-25

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

PubMed

Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

2018-01-29

Amino acid biosynthesis pathways observed in nature typically 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: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

PubMed Central

Bokinsky, Gregory; Peralta-Yahya, Pamela P.; George, Anthe; Holmes, Bradley M.; Steen, Eric J.; Dietrich, Jeffrey; Soon Lee, Taek; Tullman-Ercek, Danielle; Voigt, Christopher A.; Simmons, Blake A.; Keasling, Jay D.

2011-01-01

One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels. PMID:22123987

The Multiple-Institution Database for Investigating Engineering Longitudinal Development: An Experiential Case Study of Data Sharing and Reuse

ERIC Educational Resources Information Center

Ohland, Matthew W.; Long, Russell A.

2016-01-01

Sharing longitudinal student record data and merging data from different sources is critical to addressing important questions being asked of higher education. The Multiple-Institution Database for Investigating Engineering Longitudinal Development (MIDFIELD) is a multi-institution, longitudinal, student record level dataset that is used to answer…

Enhanced plasmid DNA production by enzyme-controlled glucose release and an engineered Escherichia coli.

PubMed

Ramírez, Elisa A; Velázquez, Daniela; Lara, Alvaro R

2016-04-01

To evaluate the combination of a culture medium employing glucoamylase-mediated glucose reléase from a gluco-polysaccharide and an E. coli strain engineered in its glucose transport system for improving plasmid DNA (pDNA) production. The production of pDNA was tested using E. coli DH5α grown in shake-flasks and the recently developed VH33 Δ(recA deoR)-engineered strain, which utilizes glucose more efficiently than wild type strains. Three glucoamylase concentrations for releasing glucose from the polysaccharide carbon source were used: 1, 2 and 3 U l(-1). Both strains reached similar cell densities ranging from 5 to 8.8 g l(-1) under the different conditions. The highest pDNA yields on biomass (YpDNA/X) for both strains were obtained when 3 U enzyme l(-1)were used. Under these conditions, 35 ± 3 mgof pDNA l(-1) were produced by DH5α after 24 h of culture. Under the same conditions, the engineered strain produced 66 ± 1 mgpDNAl(-1) after 20 h. pDNA supercoiled fractionswere close to 80 % for both strains. The pDNA concentration achieved by the engineered E. coli was 89 % higher than that of DH5α. The combination of the engineered strain and enzyme-controlled glucose release is an attractive alternative for pDNA production in shake-flasks.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2016-03-01

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Enzyme as catalytic wheel powered by a Markovian engine: conformational coupling and barrier surfing models

NASA Astrophysics Data System (ADS)

Tsong, Tian Yow; Chang, Cheng-Hung

2005-05-01

We examine a typical Michaelis-Menten Enzyme (MME) and redress it to form a transducer of free energy, and electric, acoustic, or other types of energy. This amendment and extension is necessary in lieu of recent experiments in which enzymes are shown to perform pump, motor, and locomotion functions resembling their macroscopic counterparts. Classical textbook depicts enzyme, or an MME, as biocatalyst which can enhance the rate of a chemical reaction by lowering the activation barrier but cannot shift the thermodynamic equilibrium of the biochemical reaction. An energy transducer, on the other hand, must also be able to harvest, store, or divert energy and in doing so alter the chemical equilibrium, change the energy form, fuel an energy consuming process, or perform all these functions stepwise in one catalytic turnover. The catalytic wheel presented in this communication is both a catalyst and an energy transducer and can perform all these tasks with ease. A Conformational Coupling Model for the rotary motors and a Barrier Surfing Model for the track-guided stepping motors and transporters, are presented and compared. It is shown that the core engine of the catalytic wheel, or a Brownian motor, is a Markovian engine. It remains to be seen if this core engine is the basic mechanism for a wide variety of bio-molecular energy transducers, as well as certain other dynamic systems, for example, the Parrondo's Games.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rayl, K.D.; Gaasterland, T.

This paper presents an overview of the purpose, content, and design of a subset of the currently available biological databases, with an emphasis on protein databases. Databases included in this summary are 3D-ALI, Berlin RNA databank, Blocks, DSSP, EMBL Nucleotide Database, EMP, ENZYME, FSSP, GDB, GenBank, HSSP, LiMB, PDB, PIR, PKCDD, ProSite, and SWISS-PROT. The goal is to provide a starting point for researchers who wish to take advantage of the myriad available databases. Rather than providing a complete explanation of each database, we present its content and form by explaining the details of typical entries. Pointers to more completemore » ``user guides`` are included, along with general information on where to search for a new database.« less

Research status and development of application fields in enzyme technology

NASA Astrophysics Data System (ADS)

Ji, Y. B.; Wang, S. W.; Yu, M.; Ru, X.; Wei, C.; Zhu, H. J.; Li, Z. Y.; Zhao, H.; Qiao, A. N.; Guo, S. Z.; Lu, L.

2018-01-01

Biological enzymes are catalyzed by living cells, most of which are proteins, and very few are RNA. Biological engineering as a new high-tech has been rapid development, Enzyme manufacturing and application areas are gradually expanding, In this paper, the status and progress of the application of enzyme technology are reviewed by reviewing the literature. and aims to provide reference for the application of enzyme technology and provide scientific basis for its future research and development in new field.

Industrial applications of enzyme biocatalysis: Current status and future aspects.

PubMed

Choi, Jung-Min; Han, Sang-Soo; Kim, Hak-Sung

2015-11-15

Enzymes are the most proficient catalysts, offering much more competitive processes compared to chemical catalysts. The number of industrial applications for enzymes has exploded in recent years, mainly owing to advances in protein engineering technology and environmental and economic necessities. Herein, we review recent progress in enzyme biocatalysis, and discuss the trends and strategies that are leading to broader industrial enzyme applications. The challenges and opportunities in developing biocatalytic processes are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Biocuration in the structure-function linkage database: the anatomy of a superfamily.

PubMed

Holliday, Gemma L; Brown, Shoshana D; Akiva, Eyal; Mischel, David; Hicks, Michael A; Morris, John H; Huang, Conrad C; Meng, Elaine C; Pegg, Scott C-H; Ferrin, Thomas E; Babbitt, Patricia C

2017-01-01

With ever-increasing amounts of sequence data available in both the primary literature and sequence repositories, there is a bottleneck in annotating molecular function to a sequence. This article describes the biocuration process and methods used in the structure-function linkage database (SFLD) to help address some of the challenges. We discuss how the hierarchy within the SFLD allows us to infer detailed functional properties for functionally diverse enzyme superfamilies in which all members are homologous, conserve an aspect of their chemical function and have associated conserved structural features that enable the chemistry. Also presented is the Enzyme Structure-Function Ontology (ESFO), which has been designed to capture the relationships between enzyme sequence, structure and function that underlie the SFLD and is used to guide the biocuration processes within the SFLD. http://sfld.rbvi.ucsf.edu/. © The Author 2017. Published by Oxford University Press.

Astronomical databases of Nikolaev Observatory

NASA Astrophysics Data System (ADS)

Protsyuk, Y.; Mazhaev, A.

2008-07-01

Several astronomical databases were created at Nikolaev Observatory during the last years. The databases are built by using MySQL search engine and PHP scripts. They are available on NAO web-site http://www.mao.nikolaev.ua.

Structural Ceramics Database

National Institute of Standards and Technology Data Gateway

SRD 30 NIST Structural Ceramics Database (Web, free access)   The NIST Structural Ceramics Database (WebSCD) provides evaluated materials property data for a wide range of advanced ceramics known variously as structural ceramics, engineering ceramics, and fine ceramics.

GALT protein database, a bioinformatics resource for the management and analysis of structural features of a galactosemia-related protein and its mutants.

PubMed

d'Acierno, Antonio; Facchiano, Angelo; Marabotti, Anna

2009-06-01

We describe the GALT-Prot database and its related web-based application that have been developed to collect information about the structural and functional effects of mutations on the human enzyme galactose-1-phosphate uridyltransferase (GALT) involved in the genetic disease named galactosemia type I. Besides a list of missense mutations at gene and protein sequence levels, GALT-Prot reports the analysis results of mutant GALT structures. In addition to the structural information about the wild-type enzyme, the database also includes structures of over 100 single point mutants simulated by means of a computational procedure, and the analysis to each mutant was made with several bioinformatics programs in order to investigate the effect of the mutations. The web-based interface allows querying of the database, and several links are also provided in order to guarantee a high integration with other resources already present on the web. Moreover, the architecture of the database and the web application is flexible and can be easily adapted to store data related to other proteins with point mutations. GALT-Prot is freely available at http://bioinformatica.isa.cnr.it/GALT/.

Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community.

PubMed

Zhou, Man; Guo, Peng; Wang, Tao; Gao, Lina; Yin, Huijun; Cai, Cheng; Gu, Jie; Lü, Xin

2017-01-01

Degradation of pectin in lignocellulosic materials is one of the key steps for biofuel production. Biological hydrolysis of pectin, i.e., degradation by pectinolytic microbes and enzymes, is an attractive paradigm because of its obvious advantages, such as environmentally friendly procedures, low in energy demand for lignin removal, and the possibility to be integrated in consolidated process. In this study, a metagenomics sequence-guided strategy coupled with enrichment culture technique was used to facilitate targeted discovery of pectinolytic microbes and enzymes. An apple pomace-adapted compost (APAC) habitat was constructed to boost the enrichment of pectinolytic microorganisms. Analyses of 16S rDNA high-throughput sequencing revealed that microbial communities changed dramatically during composting with some bacterial populations being greatly enriched. Metagenomics data showed that apple pomace-adapted compost microbial community (APACMC) was dominated by Proteobacteria and Bacteroidetes . Functional analysis and carbohydrate-active enzyme profiles confirmed that APACMC had been successfully enriched for the targeted functions. Among the 1756 putative genes encoding pectinolytic enzymes, 129 were predicted as novel (with an identity <30% to any CAZy database entry) and only 1.92% were more than 75% identical with proteins in NCBI environmental database, demonstrating that they have not been observed in previous metagenome projects. Phylogenetic analysis showed that APACMC harbored a broad range of pectinolytic bacteria and many of them were previously unrecognized. The immensely diverse pectinolytic microbes and enzymes found in our study will expand the arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. Our study provides a powerful approach for targeted mining microbes and enzymes in numerous industries.

Engineering Review Information System

NASA Technical Reports Server (NTRS)

Grems, III, Edward G. (Inventor); Henze, James E. (Inventor); Bixby, Jonathan A. (Inventor); Roberts, Mark (Inventor); Mann, Thomas (Inventor)

2015-01-01

A disciplinal engineering review computer information system and method by defining a database of disciplinal engineering review process entities for an enterprise engineering program, opening a computer supported engineering item based upon the defined disciplinal engineering review process entities, managing a review of the opened engineering item according to the defined disciplinal engineering review process entities, and closing the opened engineering item according to the opened engineering item review.

Protein engineering in designing tailored enzymes and microorganisms for biofuels production

PubMed Central

Wen, Fei; Nair, Nikhil U; Zhao, Huimin

2009-01-01

Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues.

PubMed

Ben Taher, Imen; Fickers, Patrick; Chniti, Sofien; Hassouna, Mnasser

2017-03-01

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L -1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L -1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397-406, 2017. © 2017 American Institute of Chemical Engineers.

Metabolite damage and repair in metabolic engineering design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields,more » and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.« less

One step DNA assembly for combinatorial metabolic engineering.

PubMed

Coussement, Pieter; Maertens, Jo; Beauprez, Joeri; Van Bellegem, Wouter; De Mey, Marjan

2014-05-01

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Cell-free metabolic engineering: Biomanufacturing beyond the cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dudley, QM; Karim, AS; Jewett, MC

2014-10-15

Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedentedmore » level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L-1 h(-1), reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.« less

Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell

PubMed Central

Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.

2014-01-01

Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. PMID:25319678

Characterising Complex Enzyme Reaction Data

PubMed Central

Rahman, Syed Asad; Thornton, Janet M.

2016-01-01

The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy

NASA Astrophysics Data System (ADS)

Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

2015-05-01

It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme D-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces D-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased D-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced D-lactate productivity. Using this combinational strategy, increased D-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals.

Hyper-X Engine Testing in the NASA Langley 8-Foot High Temperature Tunnel

NASA Technical Reports Server (NTRS)

Huebner, Lawrence D.; Rock, Kenneth E.; Witte, David W.; Ruf, Edward G.; Andrews, Earl H., Jr.

2000-01-01

Airframe-integrated scramjet engine tests have 8 completed at Mach 7 in the NASA Langley 8-Foot High Temperature Tunnel under the Hyper-X program. These tests provided critical engine data as well as design and database verification for the Mach 7 flight tests of the Hyper-X research vehicle (X-43), which will provide the first-ever airframe- integrated scramjet flight data. The first model tested was the Hyper-X Engine Model (HXEM), and the second was the Hyper-X Flight Engine (HXFE). The HXEM, a partial-width, full-height engine that is mounted on an airframe structure to simulate the forebody features of the X-43, was tested to provide data linking flowpath development databases to the complete airframe-integrated three-dimensional flight configuration and to isolate effects of ground testing conditions and techniques. The HXFE, an exact geometric representation of the X-43 scramjet engine mounted on an airframe structure that duplicates the entire three-dimensional propulsion flowpath from the vehicle leading edge to the vehicle base, was tested to verify the complete design as it will be flight tested. This paper presents an overview of these two tests, their importance to the Hyper-X program, and the significance of their contribution to scramjet database development.

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

PubMed Central

Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

2015-01-01

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. PMID:26404235

An Improved Database System for Program Assessment

ERIC Educational Resources Information Center

Haga, Wayne; Morris, Gerard; Morrell, Joseph S.

2011-01-01

This research paper presents a database management system for tracking course assessment data and reporting related outcomes for program assessment. It improves on a database system previously presented by the authors and in use for two years. The database system presented is specific to assessment for ABET (Accreditation Board for Engineering and…

Optimization of yeast-based production of medicinal protoberberine alkaloids.

PubMed

Galanie, Stephanie; Smolke, Christina D

2015-09-16

Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine. This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.

Development of an Engineering Soil Database

DTIC Science & Technology

2017-12-27

systems such as agricultural and geological soil classifications and soil parameters. Tier 3 Data were converted into equivalent USCS classification...14 2.7 U.S. Department of Agriculture (USDA) textural soil classification ............................ 16 2.7.1 Properties of USDA textural...Defense ERDC U.S. Army Engineer Research and Development Center ESDB European Soil Database FAO Food and Agriculture Organization (of the United

Implementing Relational Operations in an Object-Oriented Database

DTIC Science & Technology

1992-03-01

computer aided software engineering (CASE) and computer aided design (CAD) tools. There has been some research done in the area of combining...35 2. Prograph Database Engine .................................................................. 38 III. W HY A N R/O...in most business applications where the bulk of data being stored and manipulated is simply textual or numeric data that can be stored and manipulated

State Analysis Database Tool

NASA Technical Reports Server (NTRS)

Rasmussen, Robert; Bennett, Matthew

2006-01-01

The State Analysis Database Tool software establishes a productive environment for collaboration among software and system engineers engaged in the development of complex interacting systems. The tool embodies State Analysis, a model-based system engineering methodology founded on a state-based control architecture (see figure). A state represents a momentary condition of an evolving system, and a model may describe how a state evolves and is affected by other states. The State Analysis methodology is a process for capturing system and software requirements in the form of explicit models and states, and defining goal-based operational plans consistent with the models. Requirements, models, and operational concerns have traditionally been documented in a variety of system engineering artifacts that address different aspects of a mission s lifecycle. In State Analysis, requirements, models, and operations information are State Analysis artifacts that are consistent and stored in a State Analysis Database. The tool includes a back-end database, a multi-platform front-end client, and Web-based administrative functions. The tool is structured to prompt an engineer to follow the State Analysis methodology, to encourage state discovery and model description, and to make software requirements and operations plans consistent with model descriptions.

[Scientometrics and bibliometrics of biomedical engineering periodicals and papers].

PubMed

Zhao, Ping; Xu, Ping; Li, Bingyan; Wang, Zhengrong

2003-09-01

This investigation was made to reveal the current status, research trend and research level of biomedical engineering in Chinese mainland by means of scientometrics and to assess the quality of the four domestic publications by bibliometrics. We identified all articles of four related publications by searching Chinese and foreign databases from 1997 to 2001. All articles collected or cited by these databases were searched and statistically analyzed for finding out the relevant distributions, including databases, years, authors, institutions, subject headings and subheadings. The source of sustentation funds and the related articles were analyzed too. The results showed that two journals were cited by two foreign databases and five Chinese databases simultaneously. The output of Journal of Biomedical Engineering was the highest. Its quantity of original papers cited by EI, CA and the totality of papers sponsored by funds were higher than those of the others, but the quantity and percentage per year of biomedical articles cited by EI were decreased in all. Inland core authors and institutions had come into being in the field of biomedical engineering. Their research topics were mainly concentrated on ten subject headings which included biocompatible materials, computer-assisted signal processing, electrocardiography, computer-assisted image processing, biomechanics, algorithms, electroencephalography, automatic data processing, mechanical stress, hemodynamics, mathematical computing, microcomputers, theoretical models, etc. The main subheadings were concentrated on instrumentation, physiopathology, diagnosis, therapy, ultrasonography, physiology, analysis, surgery, pathology, method, etc.

The synthesis of starch from carbon dioxide using isolubilized stabilized enzymes

NASA Technical Reports Server (NTRS)

Bassham, J. A.; Bearden, L.; Wilke, C.; Carroad, P.; Mitra, G.; Ige, R.

1972-01-01

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.

HIV Structural Database using Chem BLAST for all classes of AIDS inhibitors

National Institute of Standards and Technology Data Gateway

SRD 155 HIV Structural Database using Chem BLAST for all classes of AIDS inhibitors (Web, free access)   The HIV structural database (HIVSDB) is a comprehensive collection of the structures of HIV protease, both of unliganded enzyme and of its inhibitor complexes. It contains abstracts and crystallographic data such as inhibitor and protein coordinates for 248 data sets, of which only 141 are from the Protein Data Bank (PDB).

Investigating a Bio-Engineered Enzyme.

ERIC Educational Resources Information Center

Bullerwell, Lornie; And Others

1994-01-01

Describes science experiments with the enzyme lactose, which is available commercially as Lactaid and Dairy Ease. Experiments show how the rate of reaction of lactose converted to glucose and galactose is influenced by temperature, pH, and substrate concentration. (PR)

Microbial synthesis of medium-chain chemicals from renewables.

PubMed

Sarria, Stephen; Kruyer, Nicholas S; Peralta-Yahya, Pamela

2017-12-01

Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

PubMed

Matuskova, Miroslava; Kozovska, Zuzana; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Cierna, Zuzana; Bohovic, Roman; Kucerova, Lucia

2015-04-09

Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

PubMed

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-11-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified.

First Look--The Aerospace Database.

ERIC Educational Resources Information Center

Kavanagh, Stephen K.; Miller, Jay G.

1986-01-01

Presents overview prepared by producer of database newly available in 1985 that covers 10 subject categories: engineering, geosciences, chemistry and materials, space sciences, aeronautics, astronautics, mathematical and computer sciences, physics, social sciences, and life sciences. Database development, unique features, document delivery, sample…

The role of arginine and the modified arginine deiminase enzyme ADI-PEG 20 in cancer therapy with special emphasis on Phase I/II clinical trials.

PubMed

Synakiewicz, Anna; Stachowicz-Stencel, Teresa; Adamkiewicz-Drozynska, Elzbieta

2014-11-01

The metabolic differences between normal, healthy cells and neoplastic cells have been exploited by anticancer therapies targeting metabolic pathways. Various studies of malignant processes have demonstrated disturbances in both arginine synthesis and metabolism that enhance or inhibit tumor cell growth. Consequently, there has been an increased interest in the arginine-depleting enzyme arginine deiminase (ADI) as a potential antineoplastic therapy. This review summarizes the literature on the potential anti-cancer therapeutics arginine and ADI, an arginine-catabolizing enzyme. The authors searched the MEDLINE database PubMed using the key words: 'arginine, 'ADI', 'arginine in cancer' and 'ADI and cancer'. The authors evaluate prospective randomized studies on cancer patients between 2004 and 2013 as well as ongoing research found through the US National Institutes of Health trial database. The results of current studies are promising but do not give unequivocal answers and so it is impossible to recommend arginine or its enzyme ADI as a therapeutic. In the opinion of the authors, further identification of arginine-dependent malignant tumors and their metabolism should be investigated. Furthermore, the use of these chemicals, in combination with other chemotherapeutics drugs, should be investigated and indeed may improve the success of arginine-depleting enzymes such as pegylated ADI (ADI-PEG20).

Blending Education and Polymer Science: Semi Automated Creation of a Thermodynamic Property Database.

PubMed

Tchoua, Roselyne B; Qin, Jian; Audus, Debra J; Chard, Kyle; Foster, Ian T; de Pablo, Juan

2016-09-13

Structured databases of chemical and physical properties play a central role in the everyday research activities of scientists and engineers. In materials science, researchers and engineers turn to these databases to quickly query, compare, and aggregate various properties, thereby allowing for the development or application of new materials. The vast majority of these databases have been generated manually, through decades of labor-intensive harvesting of information from the literature; yet, while there are many examples of commonly used databases, a significant number of important properties remain locked within the tables, figures, and text of publications. The question addressed in our work is whether, and to what extent, the process of data collection can be automated. Students of the physical sciences and engineering are often confronted with the challenge of finding and applying property data from the literature, and a central aspect of their education is to develop the critical skills needed to identify such data and discern their meaning or validity. To address shortcomings associated with automated information extraction, while simultaneously preparing the next generation of scientists for their future endeavors, we developed a novel course-based approach in which students develop skills in polymer chemistry and physics and apply their knowledge by assisting with the semi-automated creation of a thermodynamic property database.

Blending Education and Polymer Science: Semiautomated Creation of a Thermodynamic Property Database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tchoua, Roselyne B.; Qin, Jian; Audus, Debra J.

Structured databases of chemical and physical properties play a central role in the everyday research activities of scientists and engineers. In materials science, researchers and engineers turn to these databases to quickly query, compare, and aggregate various properties, thereby allowing for the development or application of new materials. The vast majority of these databases have been generated manually, through decades of labor-intensive harvesting of information from the literature, yet while there are many examples of commonly used databases, a significant number of important properties remain locked within the tables, figures, and text of publications. The question addressed in our workmore » is whether and to what extent the process of data collection can be automated. Students of the physical sciences and engineering are often confronted with the challenge of finding and applying property data from the literature, and a central aspect of their education is to develop the critical skills needed to identify such data and discern their meaning or validity. To address shortcomings associated with automated information extraction while simultaneously preparing the next generation of scientists for their future endeavors, we developed a novel course-based approach in which students develop skills in polymer chemistry and physics and apply their knowledge by assisting with the semiautomated creation of a thermodynamic property database.« less

Developing a Global Database of Historic Flood Events to Support Machine Learning Flood Prediction in Google Earth Engine

NASA Astrophysics Data System (ADS)

Tellman, B.; Sullivan, J.; Kettner, A.; Brakenridge, G. R.; Slayback, D. A.; Kuhn, C.; Doyle, C.

2016-12-01

There is an increasing need to understand flood vulnerability as the societal and economic effects of flooding increases. Risk models from insurance companies and flood models from hydrologists must be calibrated based on flood observations in order to make future predictions that can improve planning and help societies reduce future disasters. Specifically, to improve these models both traditional methods of flood prediction from physically based models as well as data-driven techniques, such as machine learning, require spatial flood observation to validate model outputs and quantify uncertainty. A key dataset that is missing for flood model validation is a global historical geo-database of flood event extents. Currently, the most advanced database of historical flood extent is hosted and maintained at the Dartmouth Flood Observatory (DFO) that has catalogued 4320 floods (1985-2015) but has only mapped 5% of these floods. We are addressing this data gap by mapping the inventory of floods in the DFO database to create a first-of- its-kind, comprehensive, global and historical geospatial database of flood events. To do so, we combine water detection algorithms on MODIS and Landsat 5,7 and 8 imagery in Google Earth Engine to map discrete flood events. The created database will be available in the Earth Engine Catalogue for download by country, region, or time period. This dataset can be leveraged for new data-driven hydrologic modeling using machine learning algorithms in Earth Engine's highly parallelized computing environment, and we will show examples for New York and Senegal.

"Trojan Horse" strategy for deconstruction of biomass for biofuels production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinclair, Michael B.; Hadi, Masood Z.; Timlin, Jerilyn Ann

2008-08-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multi-agency national priority. Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyze themore » cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive and cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology; they propose to engineer plants that self-produce a suite of cellulase enzymes targeted to the apoplast for cleaving the linkages between lignin and cellulosic fibers; the genes encoding the degradation enzymes, also known as cellulases, are obtained from extremophilic organisms that grow at high temperatures (60-100 C) and acidic pH levels (<5). These enzymes will remain inactive during the life cycle of the plant but become active during hydrothermal pretreatment i.e., elevated temperatures. Deconstruction can be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The proposed disruptive technologies address biomass deconstruction processes by developing transgenic plants encoding a suite of enzymes used in cellulosic deconstruction. The unique aspects of this technology are the rationally engineered, highly productive extremophilic enzymes, targeted to specific cellular locations (apoplast) and their dormancy during normal plant proliferation, which become Trojan horses during pretreatment conditions. They have been leveraging established Sandia's enzyme-engineering and imaging capabilities. Their technical approach not only targets the recalcitrance and mass-transfer problem during biomass degradation but also eliminates the costs associated with industrial-scale production of microbial enzymes added during processing.« less

Engineering self-assembled bioreactors from protein microcompartments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savage, David

2016-10-12

The goals of this research are to understand how organisms such as bacteria segregate certain metabolic processes inside of specific structures, or “microcompartments,” in the cell and apply this knowledge to develop novel engineered microcompartments for use in nanotechnology and metabolic engineering. For example, in some bacteria, self-assembling protein microcompartments called carboxysomes encapsulate the enzymes involved in carbon fixation, enabling the cell to utilize carbon dioxide more effectively than if the enzymes were free in the cell. The proposed research will determine how structures such as carboxysomes assemble and function in bacteria and develop a means for creating novel, syntheticmore » microcompartments for optimizing production of specific energy-rich compounds.« less

Engineering Technology Of Fish Farming Floating Nets Cages On Polka Dot Grouper (Cromileptes Altivelis) Used Artificial Feed Enriched Phytase Enzyme

NASA Astrophysics Data System (ADS)

Samidjan, Istiyanto; Rachmawati, Diana

2018-02-01

One solution is to utilize engineering technology cultivation floating cage net polka dot grouper (ducker grouper), which is given artificial feed enriched with phytase enzymes. The objectives of this study was to examine the use of technology engineering floating net on ducker grouper on artificial feed that is enriched with different dose phytase enzymes to accelerate growth and survival. The research method used ducker grouper fish size 15,5 ± 0,5 cm in the net cages unit (1 m x 1 m x 1 m), 250 fish per cage, using 12 cages. Each net-cages was made of polyethylens netting, mesh size 12.5 mm. with complete randomized design (CRD) 4 treatment and 3 replication were feed Artificial enriched of phytase enzyme with the doses of A (0 FTU · kg-1 diet), B (200 FTU · kg-1 diet), C (500 FTU · kg-1 diet), and D (800 FTU · kg-1 diet) phytase enzyme. Feed was given 2 times a day in the morning and afternoon with 5% biomass per day. Data includes the growth of absolute weight polka dot grouper, FCR, and survival rate analyzed variety and Test Tukey.The result of the research showed that the difference of artificial feeding enriched phytase enzyme significantly (P <0,05) to growth, food conversion ratio (FCR), survival rete of polka dot grouper. The best treatment at C (500 mg / kg of feed) increase growth of absolute weight of 128.75 g, 1.75 (FCR), and a survival rate of 93.5%.

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology1[C][W][OA

PubMed Central

Sarrion-Perdigones, Alejandro; Vazquez-Vilar, Marta; Palací, Jorge; Castelijns, Bas; Forment, Javier; Ziarsolo, Peio; Blanca, José; Granell, Antonio; Orzaez, Diego

2013-01-01

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects. PMID:23669743

GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology.

PubMed

Sarrion-Perdigones, Alejandro; Vazquez-Vilar, Marta; Palací, Jorge; Castelijns, Bas; Forment, Javier; Ziarsolo, Peio; Blanca, José; Granell, Antonio; Orzaez, Diego

2013-07-01

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

The Phanerochaete chrysosporium secretome : database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium

Treesearch

Amber J. Vanden Wymelenberg; Grzegorz Sabat; Diego Martinez; Alex S. Rajangam; Tuula T. Teeri; Jill A. Gaskell; Philip J. Kersten; Daniel Cullen

2005-01-01

The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood : cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome...

Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes

PubMed Central

Baker, Perrin; Seah, Stephen Y. K.

2012-01-01

Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed. PMID:24688644

Strategies for discovery and improvement of enzyme function: state of the art and opportunities

PubMed Central

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Summary Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. PMID:21883976

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

PubMed

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

A Framework for Mapping User-Designed Forms to Relational Databases

ERIC Educational Resources Information Center

Khare, Ritu

2011-01-01

In the quest for database usability, several applications enable users to design custom forms using a graphical interface, and forward engineer the forms into new databases. The path-breaking aspect of such applications is that users are completely shielded from the technicalities of database creation. Despite this innovation, the process of…

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates.

PubMed

Addington, Trevor; Calisto, Barbara; Alfonso-Prieto, Mercedes; Rovira, Carme; Fita, Ignasi; Planas, Antoni

2011-02-01

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity. © 2010 Wiley-Liss, Inc.

Sagace: A web-based search engine for biomedical databases in Japan

PubMed Central

2012-01-01

Background In the big data era, biomedical research continues to generate a large amount of data, and the generated information is often stored in a database and made publicly available. Although combining data from multiple databases should accelerate further studies, the current number of life sciences databases is too large to grasp features and contents of each database. Findings We have developed Sagace, a web-based search engine that enables users to retrieve information from a range of biological databases (such as gene expression profiles and proteomics data) and biological resource banks (such as mouse models of disease and cell lines). With Sagace, users can search more than 300 databases in Japan. Sagace offers features tailored to biomedical research, including manually tuned ranking, a faceted navigation to refine search results, and rich snippets constructed with retrieved metadata for each database entry. Conclusions Sagace will be valuable for experts who are involved in biomedical research and drug development in both academia and industry. Sagace is freely available at http://sagace.nibio.go.jp/en/. PMID:23110816

Self-Assembling Protein Nanostructures - Towards Active Functionality

DTIC Science & Technology

2011-04-22

the (alpha-beta)8 barrel. A model thermophilic enzyme , natively a tetramer, was engineered as a monomer, a dimer (submitted to Biochimica et...problem. Objective one successfully built high surface area nanoscaffolds from amyloid fibrils, and demonstrated that enzyme activity could be attached...quaternary structure, amyloid fibril, organophosphorous hydrolase, enzyme Juliet Gerrard University of Canterbury 20 Kirkwood Ave Ilam 8041 - REPORT

Self-Reporting and Detoxifying Materials Based on Extremophilic Proteins

DTIC Science & Technology

2010-01-01

thermosome (Therm), a chaperonin from the thermophilic organism Thermoplasma acidophilum, with the spectral properties of fluorescent...We have developed a new approach to enzyme immobilization/stabilization in which an enzyme - chaperone chimera is engineered to attach a functional...chaperone domain (in this case, a subunit of the recombinant thermosome from Methanocaldococcus jannaschii) to the enzyme of interest (the model

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

PubMed

Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

2018-01-01

Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Engineering strategies for the fermentative production of plant alkaloids in yeast.

PubMed

Trenchard, Isis J; Smolke, Christina D

2015-07-01

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 μg/L stylopine, 548 μg/L cis-N-methylstylopine, 252 μg/L protopine, and 80 μg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

PubMed

Oh, Doo-Byoung

2015-08-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

An Overview of the Literature: Research in P-12 Engineering Education

ERIC Educational Resources Information Center

Mendoza Díaz, Noemi V.; Cox, Monica F.

2012-01-01

This paper presents an extensive overview of preschool to 12th grade (P-12) engineering education literature published between 2001 and 2011. Searches were conducted through education and engineering library engines and databases as well as queries in established publications in engineering education. More than 50 publications were found,…

Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol.

PubMed

Suzuki, Tadashi; Yano, Keiichi; Sugimoto, Seiji; Kitajima, Ken; Lennarz, William J; Inoue, Sadako; Inoue, Yasuo; Emori, Yasufumi

2002-07-23

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

Designing Allosteric Control into Enzymes by Chemical Rescue of Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deckert, Katelyn; Budiardjo, S. Jimmy; Brunner, Luke C.

2012-08-07

Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a {beta}-glycosidase enzyme ({beta}-gly) and W492Gmore » in a {beta}-glucuronidase enzyme ({beta}-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of {beta}-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of {beta}-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate {beta}-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Tianyong; Olson, Daniel G.; Tian, Liang

Clostridium thermocellum and Thermoanaerobacterium saccharolyticumare thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticumproduce ethanol with a yield of 90% of the theoretical maximum, engineered strains ofC. thermocellumproduce ethanol at lower yields (~50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in theiradhEgenes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, theadhEgenes from six strains ofC. thermocellumandT. saccharolyticumwere cloned and expressed inEscherichia coli, the enzymesmore » produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains ofT. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain ofC. thermocellumhas acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced intoC. thermocellumandT. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. This work describes the characterization of the AdhE enzyme from different strains ofC. thermocellumandT. saccharolyticum.C. thermocellumandT. saccharolyticumare thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production byC. thermocellumandT. saccharolyticum.« less

Collaborative and Multilingual Approach to Learn Database Topics Using Concept Maps

PubMed Central

Calvo, Iñaki

2014-01-01

Authors report on a study using the concept mapping technique in computer engineering education for learning theoretical introductory database topics. In addition, the learning of multilingual technical terminology by means of the collaborative drawing of a concept map is also pursued in this experiment. The main characteristics of a study carried out in the database subject at the University of the Basque Country during the 2011/2012 course are described. This study contributes to the field of concept mapping as these kinds of cognitive tools have proved to be valid to support learning in computer engineering education. It contributes to the field of computer engineering education, providing a technique that can be incorporated with several educational purposes within the discipline. Results reveal the potential that a collaborative concept map editor offers to fulfil the above mentioned objectives. PMID:25538957

Strategies for design of improved biocatalysts for industrial applications.

PubMed

Madhavan, Aravind; Sindhu, Raveendran; Binod, Parameswaran; Sukumaran, Rajeev K; Pandey, Ashok

2017-12-01

Biocatalysts are creating increased interest among researchers due to their unique properties. Several enzymes are efficiently produced by microorganisms. However, the use of natural enzymes as biocatalysts is hindered by low catalytic efficiency and stability during various industrial processes. Many advanced enzyme technologies have been developed to reshape the existing natural enzymes to reduce these limitations and prospecting of novel enzymes. Frequently used enzyme technologies include protein engineering by directed evolution, immobilisation techniques, metagenomics etc. This review summarizes recent and emerging advancements in the area of enzyme technologies for the development of novel biocatalysts and further discusses the future directions in this field. Copyright © 2017 Elsevier Ltd. All rights reserved.

How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?

PubMed

Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin

2017-11-01

Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of volatile and semivolatile compounds in chemical ionization GC-MS using a mass-to-structure (MTS) Search Engine with integral isotope pattern ranking.

PubMed

Liao, Wenta; Draper, William M

2013-02-21

The mass-to-structure or MTS Search Engine is an Access 2010 database containing theoretical molecular mass information for 19,438 compounds assembled from common sources such as the Merck Index, pesticide and pharmaceutical compilations, and chemical catalogues. This database, which contains no experimental mass spectral data, was developed as an aid to identification of compounds in atmospheric pressure ionization (API)-LC-MS. This paper describes a powerful upgrade to this database, a fully integrated utility for filtering or ranking candidates based on isotope ratios and patterns. The new MTS Search Engine is applied here to the identification of volatile and semivolatile compounds including pesticides, nitrosoamines and other pollutants. Methane and isobutane chemical ionization (CI) GC-MS spectra were obtained from unit mass resolution mass spectrometers to determine MH(+) masses and isotope ratios. Isotopes were measured accurately with errors of <4% and <6%, respectively, for A + 1 and A + 2 peaks. Deconvolution of interfering isotope clusters (e.g., M(+) and [M - H](+)) was required for accurate determination of the A + 1 isotope in halogenated compounds. Integrating the isotope data greatly improved the speed and accuracy of the database identifications. The database accurately identified unknowns from isobutane CI spectra in 100% of cases where as many as 40 candidates satisfied the mass tolerance. The paper describes the development and basic operation of the new MTS Search Engine and details performance testing with over 50 model compounds.

Multi-source and ontology-based retrieval engine for maize mutant phenotypes

PubMed Central

Green, Jason M.; Harnsomburana, Jaturon; Schaeffer, Mary L.; Lawrence, Carolyn J.; Shyu, Chi-Ren

2011-01-01

Model Organism Databases, including the various plant genome databases, collect and enable access to massive amounts of heterogeneous information, including sequence data, gene product information, images of mutant phenotypes, etc, as well as textual descriptions of many of these entities. While a variety of basic browsing and search capabilities are available to allow researchers to query and peruse the names and attributes of phenotypic data, next-generation search mechanisms that allow querying and ranking of text descriptions are much less common. In addition, the plant community needs an innovative way to leverage the existing links in these databases to search groups of text descriptions simultaneously. Furthermore, though much time and effort have been afforded to the development of plant-related ontologies, the knowledge embedded in these ontologies remains largely unused in available plant search mechanisms. Addressing these issues, we have developed a unique search engine for mutant phenotypes from MaizeGDB. This advanced search mechanism integrates various text description sources in MaizeGDB to aid a user in retrieving desired mutant phenotype information. Currently, descriptions of mutant phenotypes, loci and gene products are utilized collectively for each search, though expansion of the search mechanism to include other sources is straightforward. The retrieval engine, to our knowledge, is the first engine to exploit the content and structure of available domain ontologies, currently the Plant and Gene Ontologies, to expand and enrich retrieval results in major plant genomic databases. Database URL: http:www.PhenomicsWorld.org/QBTA.php PMID:21558151

ODP Legacy

Science.gov Websites

Legacy: Scientific results ODP Legacy: Engineering and science operations ODP Legacy: Samples & ; databases ODP Legacy: Outreach Overview Program Administration | Scientific Results | Engineering &

Histoplasma capsulatum proteome response to decreased iron availability

PubMed Central

Winters, Michael S; Spellman, Daniel S; Chan, Qilin; Gomez, Francisco J; Hernandez, Margarita; Catron, Brittany; Smulian, Alan G; Neubert, Thomas A; Deepe, George S

2008-01-01

Background A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis. Results To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown. Conclusion We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis. PMID:19108728

Jet aircraft engine emissions database development: 1992 military, charter, and nonscheduled traffic

NASA Technical Reports Server (NTRS)

Metwally, Munir

1995-01-01

Studies relating to environmental emissions database for the military, charter, and non-scheduled traffic for the year 1992 were conducted by McDonnell Douglas Aerospace Transport Aircraft. The report also includes a comparison with a previous emission database for year 1990. Discussions of the methodology used in formulating these databases are provided.

Biosynthetic engineering of nonribosomal peptide synthetases.

PubMed

Kries, Hajo

2016-09-01

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Transporter engineering in biomass utilization by yeast.

PubMed

Hara, Kiyotaka Y; Kobayashi, Jyumpei; Yamada, Ryosuke; Sasaki, Daisuke; Kuriya, Yuki; Hirono-Hara, Yoko; Ishii, Jun; Araki, Michihiro; Kondo, Akihiko

2017-11-01

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

PubMed

Whitaker, W Brian; Jones, J Andrew; Bennett, R Kyle; Gonzalez, Jacqueline E; Vernacchio, Victoria R; Collins, Shannon M; Palmer, Michael A; Schmidt, Samuel; Antoniewicz, Maciek R; Koffas, Mattheos A; Papoutsakis, Eleftherios T

2017-01-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13 C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts.

PubMed

Ye, Victor M; Bhatia, Sujata K

2012-08-01

Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.

Engineering a monolignol 4- O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

DOE PAGES

Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; ...

2015-09-16

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create anmore » enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.« less

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol*

PubMed Central

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

2015-01-01

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

Development of a Dynamic Visco-elastic Vehicle-Soil Interaction Model for Rut Depth, and Power Determinations

DTIC Science & Technology

2011-09-06

Presentation Outline A) Review of Soil Model governing equations B) Development of pedo -transfer functions (terrain database to engineering properties) C...lateral earth pressure) UNCLASSIFIED B) Development of pedo -transfer functions Engineering parameters needed by soil model - compression index - rebound...inches, RCI for fine- grained soils, CI for coarse-grained soils. UNCLASSIFIED Pedo -transfer function • Need to transfer existing terrain database

Requirements, Verification, and Compliance (RVC) Database Tool

NASA Technical Reports Server (NTRS)

Rainwater, Neil E., II; McDuffee, Patrick B.; Thomas, L. Dale

2001-01-01

This paper describes the development, design, and implementation of the Requirements, Verification, and Compliance (RVC) database used on the International Space Welding Experiment (ISWE) project managed at Marshall Space Flight Center. The RVC is a systems engineer's tool for automating and managing the following information: requirements; requirements traceability; verification requirements; verification planning; verification success criteria; and compliance status. This information normally contained within documents (e.g. specifications, plans) is contained in an electronic database that allows the project team members to access, query, and status the requirements, verification, and compliance information from their individual desktop computers. Using commercial-off-the-shelf (COTS) database software that contains networking capabilities, the RVC was developed not only with cost savings in mind but primarily for the purpose of providing a more efficient and effective automated method of maintaining and distributing the systems engineering information. In addition, the RVC approach provides the systems engineer the capability to develop and tailor various reports containing the requirements, verification, and compliance information that meets the needs of the project team members. The automated approach of the RVC for capturing and distributing the information improves the productivity of the systems engineer by allowing that person to concentrate more on the job of developing good requirements and verification programs and not on the effort of being a "document developer".

Metabolic pathway reconstruction of eugenol to vanillin bioconversion in Aspergillus niger

PubMed Central

Srivastava, Suchita; Luqman, Suaib; Khan, Feroz; Chanotiya, Chandan S; Darokar, Mahendra P

2010-01-01

Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques. PMID:20978605

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions.

PubMed

Beld, Joris; Blatti, Jillian L; Behnke, Craig; Mendez, Michael; Burkart, Michael D

2014-08-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

PubMed Central

Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

2014-01-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2: insights into engineering thermostable enzymes for CO2 sequestration.

PubMed

Díaz-Torres, Natalia A; Mahon, Brian P; Boone, Christopher D; Pinard, Melissa A; Tu, Chingkuang; Ng, Robert; Agbandje-McKenna, Mavis; Silverman, David; Scott, Kathleen; McKenna, Robert

2015-08-01

Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.

Engineering bacteriophage for a pragmatic low-resource setting bacterial diagnostic platform.

PubMed

Talbert, Joey N; Alcaine, Samuel D; Nugen, Sam R

2016-04-01

Bacteriophages represent multifaceted building blocks that can be incorporated as substitutes for, or in unison with other detection methods, to create powerful new diagnostics for the detection of bacteria. The ease of phage manipulation, production, and detection speed clearly highlights that there remains unrealized opportunities to leverage these phage-based components in diagnostics amenable to resource-limited settings. The passage of regulations like the Food Safety Modernization act, and the ever increasing extent of global trade and travel, will create further demand for these types of diagnostics. While phage-based diagnostics have begun to entering the market place, further research is needed to ensure the potential benefits of phage-based technologies for public health are fully realized. We are just beginning to explore the possibilities that phage-based detection can offer us in the future. The combination of engineered phages as well as engineered enzymes could result in ultrasensitive detection systems for low-resource settings. Because the reporter enzyme is synthesized in vivo, we need to consider the options outside of normal enzyme reporters. In this case, common enzyme issues such as purification and long-term stability are less important. Phage-based diagnostics were conceptualized from out-of-the box thinking and the evolution of these systems should be as well.

JPRS Report, Science & Technology, Europe & Latin America

DTIC Science & Technology

1988-04-06

courses and in polytechnics a growing number of undergraduate research theses [ tesi di laurea] are increasingly coming to resemble authentic feasibility...Information Science Eleven Priorities Research Priority Actions — Microbiological engineering —Enzyme engineering —Biotechnological engineering —Food...Foodstuffs Medicine Human and social sciences Technology, computer-integrated manufacturing Electronics, data processing Microbiological

78 FR 15110 - Aviation Rulemaking Advisory Committee; Engine Bird Ingestion Requirements-New Task

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-08

...: During the bird-ingestion rulemaking database (BRDB) working group`s reevaluation of the current engine... engine core ingestion. If the BRDB working group`s reevaluation determines that such requirements are... Task ARAC accepted the task and will establish the Engine Harmonization Working Group (EHWG), under the...

Seonah Kim, Ph.D. | NREL

Science.gov Websites

lignocellulosic biomass enzymes Design of new catalysts for vapor phase upgrading of biomass pyrolysis Enzymatic Engineering for Continuous Hydrocarbon Fuel Production (PI) Computational Pyrolysis Consortium Zeolite design Celluase enzyme structure-function relationships to design enhanced cellulose systems Catalyst

Deactivating Chemical Agents Using Enzyme-Coated Nanofibers Formed by Electrospinning

DTIC Science & Technology

2011-11-16

method we have previously explored the formation of specialized fibers for several applications, including tissue engineering,28 superhydrophobic fab...dissolved during the first exposure and subsequent Figure 9. Multilayer fiber mat with outer superhydrophobic fibers and inner enzyme containing fibers

Custom Search Engines: Tools & Tips

ERIC Educational Resources Information Center

Notess, Greg R.

2008-01-01

Few have the resources to build a Google or Yahoo! from scratch. Yet anyone can build a search engine based on a subset of the large search engines' databases. Use Google Custom Search Engine or Yahoo! Search Builder or any of the other similar programs to create a vertical search engine targeting sites of interest to users. The basic steps to…

Space Launch System Booster Separation Aerodynamic Database Development and Uncertainty Quantification

NASA Technical Reports Server (NTRS)

Chan, David T.; Pinier, Jeremy T.; Wilcox, Floyd J., Jr.; Dalle, Derek J.; Rogers, Stuart E.; Gomez, Reynaldo J.

2016-01-01

The development of the aerodynamic database for the Space Launch System (SLS) booster separation environment has presented many challenges because of the complex physics of the ow around three independent bodies due to proximity e ects and jet inter- actions from the booster separation motors and the core stage engines. This aerodynamic environment is dicult to simulate in a wind tunnel experiment and also dicult to simu- late with computational uid dynamics. The database is further complicated by the high dimensionality of the independent variable space, which includes the orientation of the core stage, the relative positions and orientations of the solid rocket boosters, and the thrust lev- els of the various engines. Moreover, the clearance between the core stage and the boosters during the separation event is sensitive to the aerodynamic uncertainties of the database. This paper will present the development process for Version 3 of the SLS booster separa- tion aerodynamic database and the statistics-based uncertainty quanti cation process for the database.

Methods and apparatus for constructing and implementing a universal extension module for processing objects in a database

NASA Technical Reports Server (NTRS)

Li, Chung-Sheng (Inventor); Smith, John R. (Inventor); Chang, Yuan-Chi (Inventor); Jhingran, Anant D. (Inventor); Padmanabhan, Sriram K. (Inventor); Hsiao, Hui-I (Inventor); Choy, David Mun-Hien (Inventor); Lin, Jy-Jine James (Inventor); Fuh, Gene Y. C. (Inventor); Williams, Robin (Inventor)

2004-01-01

Methods and apparatus for providing a multi-tier object-relational database architecture are disclosed. In one illustrative embodiment of the present invention, a multi-tier database architecture comprises an object-relational database engine as a top tier, one or more domain-specific extension modules as a bottom tier, and one or more universal extension modules as a middle tier. The individual extension modules of the bottom tier operationally connect with the one or more universal extension modules which, themselves, operationally connect with the database engine. The domain-specific extension modules preferably provide such functions as search, index, and retrieval services of images, video, audio, time series, web pages, text, XML, spatial data, etc. The domain-specific extension modules may include one or more IBM DB2 extenders, Oracle data cartridges and/or Informix datablades, although other domain-specific extension modules may be used.

Java Web Simulation (JWS); a web based database of kinetic models.

PubMed

Snoep, J L; Olivier, B G

2002-01-01

Software to make a database of kinetic models accessible via the internet has been developed and a core database has been set up at http://jjj.biochem.sun.ac.za/. This repository of models, available to everyone with internet access, opens a whole new way in which we can make our models public. Via the database, a user can change enzyme parameters and run time simulations or steady state analyses. The interface is user friendly and no additional software is necessary. The database currently contains 10 models, but since the generation of the program code to include new models has largely been automated the addition of new models is straightforward and people are invited to submit their models to be included in the database.

Ethanol production by engineered thermophiles.

PubMed

Olson, Daniel G; Sparling, Richard; Lynd, Lee R

2015-06-01

We compare a number of different strategies that have been pursued to engineer thermophilic microorganisms for increased ethanol production. Ethanol production from pyruvate can proceed via one of four pathways, which are named by the key pyruvate dissimilating enzyme: pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), pyruvate formate lyase (PFL), and pyruvate ferredoxin oxidoreductase (PFOR). For each of these pathways except PFL, we see examples where ethanol production has been engineered with a yield of >90% of the theoretical maximum. In each of these cases, this engineering was achieved mainly by modulating expression of native genes. We have not found an example where a thermophilic ethanol production pathway has been transferred to a non-ethanol-producing organism to produce ethanol at high yield. A key reason for the lack of transferability of ethanol production pathways is the current lack of understanding of the enzymes involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Using Internet search engines to estimate word frequency.

PubMed

Blair, Irene V; Urland, Geoffrey R; Ma, Jennifer E

2002-05-01

The present research investigated Internet search engines as a rapid, cost-effective alternative for estimating word frequencies. Frequency estimates for 382 words were obtained and compared across four methods: (1) Internet search engines, (2) the Kucera and Francis (1967) analysis of a traditional linguistic corpus, (3) the CELEX English linguistic database (Baayen, Piepenbrock, & Gulikers, 1995), and (4) participant ratings of familiarity. The results showed that Internet search engines produced frequency estimates that were highly consistent with those reported by Kucera and Francis and those calculated from CELEX, highly consistent across search engines, and very reliable over a 6-month period of time. Additional results suggested that Internet search engines are an excellent option when traditional word frequency analyses do not contain the necessary data (e.g., estimates for forenames and slang). In contrast, participants' familiarity judgments did not correspond well with the more objective estimates of word frequency. Researchers are advised to use search engines with large databases (e.g., AltaVista) to ensure the greatest representativeness of the frequency estimates.

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

PubMed Central

Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

2013-01-01

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

%22Trojan Horse%22 strategy for deconstruction of biomass for biofuels production.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, Blake Alexander; Sinclair, Michael B.; Yu, Eizadora

2011-02-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multiagency national priority (DOE/USDA/EERE). Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyzemore » the cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology-engineered plants that self-produce a suite of cellulase enzymes. Deconstruction can then be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The unique aspects of our approach are the rationally engineered enzymes which become Trojan horses during pretreatment conditions. During this study we rationally engineered Cazy enzymes and then integrated them into plant cells by multiple transformation techniques. The regenerated plants were assayed for first expression of these messages and then for the resulting proteins. The plants were then subjected to consolidated bioprocessing and characterized in detail. Our results and possible implications of this work on developing dedicated energy crops and their advantage in a consolidated bioprocessing system.« less

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

An integrated knowledge system for wind tunnel testing - Project Engineers' Intelligent Assistant

NASA Technical Reports Server (NTRS)

Lo, Ching F.; Shi, George Z.; Hoyt, W. A.; Steinle, Frank W., Jr.

1993-01-01

The Project Engineers' Intelligent Assistant (PEIA) is an integrated knowledge system developed using artificial intelligence technology, including hypertext, expert systems, and dynamic user interfaces. This system integrates documents, engineering codes, databases, and knowledge from domain experts into an enriched hypermedia environment and was designed to assist project engineers in planning and conducting wind tunnel tests. PEIA is a modular system which consists of an intelligent user-interface, seven modules and an integrated tool facility. Hypermedia technology is discussed and the seven PEIA modules are described. System maintenance and updating is very easy due to the modular structure and the integrated tool facility provides user access to commercial software shells for documentation, reporting, or database updating. PEIA is expected to provide project engineers with technical information, increase efficiency and productivity, and provide a realistic tool for personnel training.

Scale-Up of GRCop: From Laboratory to Rocket Engines

NASA Technical Reports Server (NTRS)

Ellis, David L.

2016-01-01

GRCop is a high temperature, high thermal conductivity copper-based series of alloys designed primarily for use in regeneratively cooled rocket engine liners. It began with laboratory-level production of a few grams of ribbon produced by chill block melt spinning and has grown to commercial-scale production of large-scale rocket engine liners. Along the way, a variety of methods of consolidating and working the alloy were examined, a database of properties was developed and a variety of commercial and government applications were considered. This talk will briefly address the basic material properties used for selection of compositions to scale up, the methods used to go from simple ribbon to rocket engines, the need to develop a suitable database, and the issues related to getting the alloy into a rocket engine or other application.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation.

PubMed

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-19

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Stable metal-organic frameworks containing single-molecule traps for enzyme encapsulation

NASA Astrophysics Data System (ADS)

Feng, Dawei; Liu, Tian-Fu; Su, Jie; Bosch, Mathieu; Wei, Zhangwen; Wan, Wei; Yuan, Daqiang; Chen, Ying-Pin; Wang, Xuan; Wang, Kecheng; Lian, Xizhen; Gu, Zhi-Yuan; Park, Jihye; Zou, Xiaodong; Zhou, Hong-Cai

2015-01-01

Enzymatic catalytic processes possess great potential in chemical manufacturing, including pharmaceuticals, fuel production and food processing. However, the engineering of enzymes is severely hampered due to their low operational stability and difficulty of reuse. Here, we develop a series of stable metal-organic frameworks with rationally designed ultra-large mesoporous cages as single-molecule traps (SMTs) for enzyme encapsulation. With a high concentration of mesoporous cages as SMTs, PCN-333(Al) encapsulates three enzymes with record-high loadings and recyclability. Immobilized enzymes that most likely undergo single-enzyme encapsulation (SEE) show smaller Km than free enzymes while maintaining comparable catalytic efficiency. Under harsh conditions, the enzyme in SEE exhibits better performance than free enzyme, showing the effectiveness of SEE in preventing enzyme aggregation or denaturation. With extraordinarily large pore size and excellent chemical stability, PCN-333 may be of interest not only for enzyme encapsulation, but also for entrapment of other nanoscaled functional moieties.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

PubMed Central

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao

2017-01-01

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam. PMID:29054871

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

PubMed

Gao, Shuaihua; Zhu, Shaozhou; Huang, Rong; Li, Hongxia; Wang, Hao; Zheng, Guojun

2018-01-01

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (-)-Vince lactam. Copyright © 2017 American Society for Microbiology.

Computer-Aided Systems Engineering for Flight Research Projects Using a Workgroup Database

NASA Technical Reports Server (NTRS)

Mizukami, Masahi

2004-01-01

An online systems engineering tool for flight research projects has been developed through the use of a workgroup database. Capabilities are implemented for typical flight research systems engineering needs in document library, configuration control, hazard analysis, hardware database, requirements management, action item tracking, project team information, and technical performance metrics. Repetitive tasks are automated to reduce workload and errors. Current data and documents are instantly available online and can be worked on collaboratively. Existing forms and conventional processes are used, rather than inventing or changing processes to fit the tool. An integrated tool set offers advantages by automatically cross-referencing data, minimizing redundant data entry, and reducing the number of programs that must be learned. With a simplified approach, significant improvements are attained over existing capabilities for minimal cost. By using a workgroup-level database platform, personnel most directly involved in the project can develop, modify, and maintain the system, thereby saving time and money. As a pilot project, the system has been used to support an in-house flight experiment. Options are proposed for developing and deploying this type of tool on a more extensive basis.

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

PubMed

Latimer, Luke N; Dueber, John E

2017-06-01

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

NASA Astrophysics Data System (ADS)

Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

2016-05-01

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

Katherine Young, P.E. | NREL

Science.gov Websites

) Water rights and resources engineering Database planning and development Research Interests Collection lean principles to streamline exploration and drilling and reduce error/risk Research, development and Groundwater modeling Quantitative methods in water resource engineering Water resource engineering and

Effect of ethyleneoxide groups of anionic surfactants on lipase activity.

PubMed

Magalhães, Solange S; Alves, Luís; Sebastião, Marco; Medronho, Bruno; Almeida, Zaida L; Faria, Tiago Q; Brito, Rui M M; Moreno, Maria J; Antunes, Filipe E

2016-09-01

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant-enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE 0 S) and of sodium lauryl ether sulfate with two EO units (SLE 2 S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE 2 S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276-1282, 2016. © 2016 American Institute of Chemical Engineers.

Engineering geological mapping in Wallonia (Belgium) : present state and recent computerized approach

NASA Astrophysics Data System (ADS)

Delvoie, S.; Radu, J.-P.; Ruthy, I.; Charlier, R.

2012-04-01

An engineering geological map can be defined as a geological map with a generalized representation of all the components of a geological environment which are strongly required for spatial planning, design, construction and maintenance of civil engineering buildings. In Wallonia (Belgium) 24 engineering geological maps have been developed between the 70s and the 90s at 1/5,000 or 1/10,000 scale covering some areas of the most industrialized and urbanized cities (Liège, Charleroi and Mons). They were based on soil and subsoil data point (boring, drilling, penetration test, geophysical test, outcrop…). Some displayed data present the depth (with isoheights) or the thickness (with isopachs) of the different subsoil layers up to about 50 m depth. Information about geomechanical properties of each subsoil layer, useful for engineers and urban planners, is also synthesized. However, these maps were built up only on paper and progressively needed to be updated with new soil and subsoil data. The Public Service of Wallonia and the University of Liège have recently initiated a study to evaluate the feasibility to develop engineering geological mapping with a computerized approach. Numerous and various data (about soil and subsoil) are stored into a georelational database (the geotechnical database - using Access, Microsoft®). All the data are geographically referenced. The database is linked to a GIS project (using ArcGIS, ESRI®). Both the database and GIS project consist of a powerful tool for spatial data management and analysis. This approach involves a methodology using interpolation methods to update the previous maps and to extent the coverage to new areas. The location (x, y, z) of each subsoil layer is then computed from data point. The geomechanical data of these layers are synthesized in an explanatory booklet joined to maps.

Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

PubMed

Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

2015-04-21

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

System for Performing Single Query Searches of Heterogeneous and Dispersed Databases

NASA Technical Reports Server (NTRS)

Maluf, David A. (Inventor); Okimura, Takeshi (Inventor); Gurram, Mohana M. (Inventor); Tran, Vu Hoang (Inventor); Knight, Christopher D. (Inventor); Trinh, Anh Ngoc (Inventor)

2017-01-01

The present invention is a distributed computer system of heterogeneous databases joined in an information grid and configured with an Application Programming Interface hardware which includes a search engine component for performing user-structured queries on multiple heterogeneous databases in real time. This invention reduces overhead associated with the impedance mismatch that commonly occurs in heterogeneous database queries.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

PubMed Central

Lambert, Abigail R.; Kuhar, Ryan; Jarjour, Jordan; Kulshina, Nadia; Parmeggiani, Fabio; Danaher, Patrick; Gano, Jacob; Baker, David; Stoddard, Barry L.; Scharenberg, Andrew M.

2012-01-01

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications. PMID:22684507

Inhibition of biofilm formation by T7 bacteriophages producing quorum-quenching enzymes.

PubMed

Pei, Ruoting; Lamas-Samanamud, Gisella R

2014-09-01

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

PubMed Central

Lamas-Samanamud, Gisella R.

2014-01-01

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. PMID:24951790

Database Access Manager for the Software Engineering Laboratory (DAMSEL) user's guide

NASA Technical Reports Server (NTRS)

1990-01-01

Operating instructions for the Database Access Manager for the Software Engineering Laboratory (DAMSEL) system are presented. Step-by-step instructions for performing various data entry and report generation activities are included. Sample sessions showing the user interface display screens are also included. Instructions for generating reports are accompanied by sample outputs for each of the reports. The document groups the available software functions by the classes of users that may access them.

FERN Ethnomedicinal Plant Database: Exploring Fern Ethnomedicinal Plants Knowledge for Computational Drug Discovery.

PubMed

Thakar, Sambhaji B; Ghorpade, Pradnya N; Kale, Manisha V; Sonawane, Kailas D

2015-01-01

Fern plants are known for their ethnomedicinal applications. Huge amount of fern medicinal plants information is scattered in the form of text. Hence, database development would be an appropriate endeavor to cope with the situation. So by looking at the importance of medicinally useful fern plants, we developed a web based database which contains information about several group of ferns, their medicinal uses, chemical constituents as well as protein/enzyme sequences isolated from different fern plants. Fern ethnomedicinal plant database is an all-embracing, content management web-based database system, used to retrieve collection of factual knowledge related to the ethnomedicinal fern species. Most of the protein/enzyme sequences have been extracted from NCBI Protein sequence database. The fern species, family name, identification, taxonomy ID from NCBI, geographical occurrence, trial for, plant parts used, ethnomedicinal importance, morphological characteristics, collected from various scientific literatures and journals available in the text form. NCBI's BLAST, InterPro, phylogeny, Clustal W web source has also been provided for the future comparative studies. So users can get information related to fern plants and their medicinal applications at one place. This Fern ethnomedicinal plant database includes information of 100 fern medicinal species. This web based database would be an advantageous to derive information specifically for computational drug discovery, botanists or botanical interested persons, pharmacologists, researchers, biochemists, plant biotechnologists, ayurvedic practitioners, doctors/pharmacists, traditional medicinal users, farmers, agricultural students and teachers from universities as well as colleges and finally fern plant lovers. This effort would be useful to provide essential knowledge for the users about the adventitious applications for drug discovery, applications, conservation of fern species around the world and finally to create social awareness.

MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.

PubMed

Konwar, Kishori M; Hanson, Niels W; Bhatia, Maya P; Kim, Dongjae; Wu, Shang-Ju; Hahn, Aria S; Morgan-Lang, Connor; Cheung, Hiu Kan; Hallam, Steven J

2015-10-15

Next-generation sequencing is producing vast amounts of sequence information from natural and engineered ecosystems. Although this data deluge has an enormous potential to transform our lives, knowledge creation and translation need software applications that scale with increasing data processing and analysis requirements. Here, we present improvements to MetaPathways, an annotation and analysis pipeline for environmental sequence information that expedites this transformation. We specifically address pathway prediction hazards through integration of a weighted taxonomic distance and enable quantitative comparison of assembled annotations through a normalized read-mapping measure. Additionally, we improve LAST homology searches through BLAST-equivalent E-values and output formats that are natively compatible with prevailing software applications. Finally, an updated graphical user interface allows for keyword annotation query and projection onto user-defined functional gene hierarchies, including the Carbohydrate-Active Enzyme database. MetaPathways v2.5 is available on GitHub: http://github.com/hallamlab/metapathways2. shallam@mail.ubc.ca Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

PubMed

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-02-28

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Applications and Methods for Continuous Monitoring of Physiological Chemistry

DTIC Science & Technology

2016-02-04

product and test platform to verify the performance characteristics of the enzymes when used in diagnostic device fabrication. 1.3 Results This...project had three primary objectives: 1. Engineer a cortisol oxidase enzyme suitable for use in diagnostic devices 2. Large scale production and...for both animal and human use , and for direct sale to other entities to manufacture biosensors and other products for human monitoring. The enzymes

Annual Report to Congress - Fiscal Year 2002, from the Strategic Environmental Research and Development Program

DTIC Science & Technology

2003-03-01

and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX . . . . . A-48 CU-1318 – Engineering Transgenic...1317 Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro-Substituted Explosives TNT, RDX, and HMX...A A-48 PROJECT SUMMARY PROJECT TITLE & ID: Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro

Computational enzyme design: transitioning from catalytic proteins to enzymes.

PubMed

Mak, Wai Shun; Siegel, Justin B

2014-08-01

The widespread interest in enzymes stem from their ability to catalyze chemical reactions under mild and ecologically friendly conditions with unparalleled catalytic proficiencies. While thousands of naturally occurring enzymes have been identified and characterized, there are still numerous important applications for which there are no biological catalysts capable of performing the desired chemical transformation. In order to engineer enzymes for which there is no natural starting point, efforts using a combination of quantum chemistry and force-field based protein molecular modeling have led to the design of novel proteins capable of catalyzing chemical reactions not catalyzed by naturally occurring enzymes. Here we discuss the current status and potential avenues to pursue as the field of computational enzyme design moves forward. Published by Elsevier Ltd.

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

PubMed Central

Oh, Doo-Byoung

2015-01-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444] PMID:25999178

A Symphony of Software.

ERIC Educational Resources Information Center

Currents, 2002

2002-01-01

Offers a descriptive table of databases that help higher education institutions orchestrate advancement operations. Information includes vendor, contact, software, price, database engine/server platform, recommended reporting tools, record capacity, and client type. (EV)

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization

PubMed Central

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-01-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified. PMID:27812109

A Parallel Relational Database Management System Approach to Relevance Feedback in Information Retrieval.

ERIC Educational Resources Information Center

Lundquist, Carol; Frieder, Ophir; Holmes, David O.; Grossman, David

1999-01-01

Describes a scalable, parallel, relational database-drive information retrieval engine. To support portability across a wide range of execution environments, all algorithms adhere to the SQL-92 standard. By incorporating relevance feedback algorithms, accuracy is enhanced over prior database-driven information retrieval efforts. Presents…

Electronic Reference Library: Silverplatter's Database Networking Solution.

ERIC Educational Resources Information Center

Millea, Megan

Silverplatter's Electronic Reference Library (ERL) provides wide area network access to its databases using TCP/IP communications and client-server architecture. ERL has two main components: The ERL clients (retrieval interface) and the ERL server (search engines). ERL clients provide patrons with seamless access to multiple databases on multiple…

Subject Specific Databases: A Powerful Research Tool

ERIC Educational Resources Information Center

Young, Terrence E., Jr.

2004-01-01

Subject specific databases, or vortals (vertical portals), are databases that provide highly detailed research information on a particular topic. They are the smallest, most focused search tools on the Internet and, in recent years, they've been on the rise. Currently, more of the so-called "mainstream" search engines, subject directories, and…

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

PubMed Central

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

2016-01-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

PubMed

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

2016-02-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

Engineering yeast for the expression and secretion of cellulase cocktails

USDA-ARS?s Scientific Manuscript database

Enzyme systems that digest the cellulose in plant cell walls have potential value in the biorefining of renewable feedstocks such as crop residues, straws, and grasses to biofuels and other bioproducts. The bacterium Clostridium cellulovorans is a useful source of biomass-degrading enzymes because ...

Usage of the Jess Engine, Rules and Ontology to Query a Relational Database

NASA Astrophysics Data System (ADS)

Bak, Jaroslaw; Jedrzejek, Czeslaw; Falkowski, Maciej

We present a prototypical implementation of a library tool, the Semantic Data Library (SDL), which integrates the Jess (Java Expert System Shell) engine, rules and ontology to query a relational database. The tool extends functionalities of previous OWL2Jess with SWRL implementations and takes full advantage of the Jess engine, by separating forward and backward reasoning. The optimization of integration of all these technologies is an advancement over previous tools. We discuss the complexity of the query algorithm. As a demonstration of capability of the SDL library, we execute queries using crime ontology which is being developed in the Polish PPBW project.

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues*

PubMed Central

Cheng, Hai-Li; Zhao, Rui-Yu; Chen, Tian-Jiao; Yu, Wen-Bo; Wang, Fen; Cheng, Ke-Di; Zhu, Ping

2013-01-01

Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1–1 and Lxyl-p1–2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1–1, G-Lxyl-p1–2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s−1mm−1 against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases. PMID:23665501

Flood trends and river engineering on the Mississippi River system

USGS Publications Warehouse

Pinter, N.; Jemberie, A.A.; Remo, J.W.F.; Heine, R.A.; Ickes, B.S.

2008-01-01

Along >4000 km of the Mississippi River system, we document that climate, land-use change, and river engineering have contributed to statistically significant increases in flooding over the past 100-150 years. Trends were tested using a database of >8 million hydrological measurements. A geospatial database of historical engineering construction was used to quantify the response of flood levels to each unit of engineering infrastructure. Significant climate- and/or land use-driven increases in flow were detected, but the largest and most pervasive contributors to increased flooding on the Mississippi River system were wing dikes and related navigational structures, followed by progressive levee construction. In the area of the 2008 Upper Mississippi flood, for example, about 2 m of the flood crest is linked to navigational and flood-control engineering. Systemwide, large increases in flood levels were documented at locations and at times of wing-dike and levee construction. Copyright 2008 by the American Geophysical Union.

SpirPep: an in silico digestion-based platform to assist bioactive peptides discovery from a genome-wide database.

PubMed

Anekthanakul, Krittima; Hongsthong, Apiradee; Senachak, Jittisak; Ruengjitchatchawalya, Marasri

2018-04-20

Bioactive peptides, including biological sources-derived peptides with different biological activities, are protein fragments that influence the functions or conditions of organisms, in particular humans and animals. Conventional methods of identifying bioactive peptides are time-consuming and costly. To quicken the processes, several bioinformatics tools are recently used to facilitate screening of the potential peptides prior their activity assessment in vitro and/or in vivo. In this study, we developed an efficient computational method, SpirPep, which offers many advantages over the currently available tools. The SpirPep web application tool is a one-stop analysis and visualization facility to assist bioactive peptide discovery. The tool is equipped with 15 customized enzymes and 1-3 miscleavage options, which allows in silico digestion of protein sequences encoded by protein-coding genes from single, multiple, or genome-wide scaling, and then directly classifies the peptides by bioactivity using an in-house database that contains bioactive peptides collected from 13 public databases. With this tool, the resulting peptides are categorized by each selected enzyme, and shown in a tabular format where the peptide sequences can be tracked back to their original proteins. The developed tool and webpages are coded in PHP and HTML with CSS/JavaScript. Moreover, the tool allows protein-peptide alignment visualization by Generic Genome Browser (GBrowse) to display the region and details of the proteins and peptides within each parameter, while considering digestion design for the desirable bioactivity. SpirPep is efficient; it takes less than 20 min to digest 3000 proteins (751,860 amino acids) with 15 enzymes and three miscleavages for each enzyme, and only a few seconds for single enzyme digestion. Obviously, the tool identified more bioactive peptides than that of the benchmarked tool; an example of validated pentapeptide (FLPIL) from LC-MS/MS was demonstrated. The web and database server are available at http://spirpepapp.sbi.kmutt.ac.th . SpirPep, a web-based bioactive peptide discovery application, is an in silico-based tool with an overview of the results. The platform is a one-stop analysis and visualization facility; and offers advantages over the currently available tools. This tool may be useful for further bioactivity analysis and the quantitative discovery of desirable peptides.

Cyanide bioremediation: the potential of engineered nitrilases.

PubMed

Park, Jason M; Trevor Sewell, B; Benedik, Michael J

2017-04-01

The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.

Engineered microorganisms capable of producing target compounds under anaerobic conditions

DOEpatents

Buelter, Thomas [Denver, CO; Meinhold, Peter [Denver, CO; Feldman, Reid M. Renny [San Francisco, CA; Hawkins, Andrew C [Parker, CO; Urano, Jun [Irvine, CA; Bastian, Sabine [Pasadena, CA; Arnold, Frances [La Canada, CA

2012-01-17

The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

PubMed

Martínez-Núñez, Mario Alberto; Rodríguez-Escamilla, Zuemy; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

2017-07-13

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

Artificial concurrent catalytic processes involving enzymes.

PubMed

Köhler, Valentin; Turner, Nicholas J

2015-01-11

The concurrent operation of multiple catalysts can lead to enhanced reaction features including (i) simultaneous linear multi-step transformations in a single reaction flask (ii) the control of intermediate equilibria (iii) stereoconvergent transformations (iv) rapid processing of labile reaction products. Enzymes occupy a prominent position for the development of such processes, due to their high potential compatibility with other biocatalysts. Genes for different enzymes can be co-expressed to reconstruct natural or construct artificial pathways and applied in the form of engineered whole cell biocatalysts to carry out complex transformations or, alternatively, the enzymes can be combined in vitro after isolation. Moreover, enzyme variants provide a wider substrate scope for a given reaction and often display altered selectivities and specificities. Man-made transition metal catalysts and engineered or artificial metalloenzymes also widen the range of reactivities and catalysed reactions that are potentially employable. Cascades for simultaneous cofactor or co-substrate regeneration or co-product removal are now firmly established. Many applications of more ambitious concurrent cascade catalysis are only just beginning to appear in the literature. The current review presents some of the most recent examples, with an emphasis on the combination of transition metal with enzymatic catalysis and aims to encourage researchers to contribute to this emerging field.

Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

PubMed

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

2015-10-30

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Design Considerations for a Web-based Database System of ELISpot Assay in Immunological Research

PubMed Central

Ma, Jingming; Mosmann, Tim; Wu, Hulin

2005-01-01

The enzyme-linked immunospot (ELISpot) assay has been a primary means in immunological researches (such as HIV-specific T cell response). Due to huge amount of data involved in ELISpot assay testing, the database system is needed for efficient data entry, easy retrieval, secure storage, and convenient data process. Besides, the NIH has recently issued a policy to promote the sharing of research data (see http://grants.nih.gov/grants/policy/data_sharing). The Web-based database system will be definitely benefit to data sharing among broad research communities. Here are some considerations for a database system of ELISpot assay (DBSEA). PMID:16779326

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

PubMed

Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

2012-07-28

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

NASA Image eXchange (NIX)

NASA Technical Reports Server (NTRS)

vonOfenheim. William H. C.; Heimerl, N. Lynn; Binkley, Robert L.; Curry, Marty A.; Slater, Richard T.; Nolan, Gerald J.; Griswold, T. Britt; Kovach, Robert D.; Corbin, Barney H.; Hewitt, Raymond W.

1998-01-01

This paper discusses the technical aspects of and the project background for the NASA Image exchange (NIX). NIX, which provides a single entry point to search selected image databases at the NASA Centers, is a meta-search engine (i.e., a search engine that communicates with other search engines). It uses these distributed digital image databases to access photographs, animations, and their associated descriptive information (meta-data). NIX is available for use at the following URL: http://nix.nasa.gov./NIX, which was sponsored by NASAs Scientific and Technical Information (STI) Program, currently serves images from seven NASA Centers. Plans are under way to link image databases from three additional NASA Centers. images and their associated meta-data, which are accessible by NIX, reside at the originating Centers, and NIX utilizes a virtual central site that communicates with each of these sites. Incorporated into the virtual central site are several protocols to support searches from a diverse collection of database engines. The searches are performed in parallel to ensure optimization of response times. To augment the search capability, browse functionality with pre-defined categories has been built into NIX, thereby ensuring dissemination of 'best-of-breed' imagery. As a final recourse, NIX offers access to a help desk via an on-line form to help locate images and information either within the scope of NIX or from available external sources.

The composite load spectra project

NASA Technical Reports Server (NTRS)

Newell, J. F.; Ho, H.; Kurth, R. E.

1990-01-01

Probabilistic methods and generic load models capable of simulating the load spectra that are induced in space propulsion system components are being developed. Four engine component types (the transfer ducts, the turbine blades, the liquid oxygen posts and the turbopump oxidizer discharge duct) were selected as representative hardware examples. The composite load spectra that simulate the probabilistic loads for these components are typically used as the input loads for a probabilistic structural analysis. The knowledge-based system approach used for the composite load spectra project provides an ideal environment for incremental development. The intelligent database paradigm employed in developing the expert system provides a smooth coupling between the numerical processing and the symbolic (information) processing. Large volumes of engine load information and engineering data are stored in database format and managed by a database management system. Numerical procedures for probabilistic load simulation and database management functions are controlled by rule modules. Rules were hard-wired as decision trees into rule modules to perform process control tasks. There are modules to retrieve load information and models. There are modules to select loads and models to carry out quick load calculations or make an input file for full duty-cycle time dependent load simulation. The composite load spectra load expert system implemented today is capable of performing intelligent rocket engine load spectra simulation. Further development of the expert system will provide tutorial capability for users to learn from it.

[Phenotypic and genotypic spectra of patients with glucose-6-phosphate dehydrogenase deficiency gene known pathogenic variants: a single-center study].

PubMed

Chen, X; Yang, L; Wang, H J; Wu, B B; Lu, Y L; Dong, X R; Zhou, W H

2018-05-02

Objective: To analyze the hotspots of known pathogenic disease-causing variants of glucose-6-phosphate dehydrogenase (G6PD) and the phenotype spectrum of neonatal patients with known pathogenic disease-causing variants of G6PD. Methods: The known pathogenic disease-causing variants of G6PD were collected from Human Gene Mutation Database. Screening was performed for these variants among the 7 966 cases (2 357 neonatal, 5 609 non-neonatal) in the database of sequencing at Molecular Diagnosis Center, Children's Hospital of Fudan University. All these samples were from patients suspected with genetic disorder. The database contained Whole Exon Sequencing data and Clinical Exon Sequencing data. We screened out the patients with known pathogenic disease-causing variants of G6PD, analyzed the hotspot of G6PD and the phenotype spectrum of neonatal patients with known pathogenic disease-causing variants of G6PD. Results: (1) Among the next generation sequencing data of the 7 966 samples, 86 samples (1.1%) were detected as positive for the known pathogenic disease-causing variants of G6PD (positive samples set). In the positive sample set, 51 patients (33 males, 18 females) were newborn babies. Forty-three patients (26 males, 17 females) had the enzyme activity data of G6PD. (2) Among the 86 samples, Arg463His, Arg459Leu, Leu342Phe, Val291Met were the leading 4 disease-causing variants found in 72 samples (84%). (3) Male neonatal patients with the same variants had the statistically significant differences in enzyme activity: among 13 patients with Arg463His, enzyme activity of 9 patients was ranked as grade Ⅲ, 1 case ranked as Ⅳ, 3 cases had no activity data;among 10 patients with Arg459Leu, enzyme activity of 4 patients was ranked as Ⅱ, 4 cases ranked as Ⅲ, 2 cases had no activity data;among 2 patients with His32Arg, enzyme activity of one patient was ranked as Ⅱ, another was Ⅲ. Male neonatal patients with the same mutation and enzyme activity also had the statistically significant differences in phenotype spectrum: among 9 patients with Arg463His and level Ⅲ enzyme activity, 6 presented hyperbilirubinemia, 2 met the criteria for exchange transfusion therapy, 2 showed hemolysis;among 4 patients with Arg459Leu and level Ⅱ enzyme activity, 3 presented hyperbilirubinemia;among 4 patients with Arg459Leu and level Ⅲ enzyme activity, 2 presented hyperbilirubinemia, 1 met the standard of exchange transfusion therapy;among 3 patients with Val291Met and level Ⅲ enzyme activity, 1 presented hyperbilirubinemia. Conclusions: Arg463His, Arg459Leu, Leu342Phe, Val291Met were the hotspots variants for the G6PD. Patients with the same G6PD variants and sex present different phenotype, patients with the same G6PD variants, sex and enzyme activity also present different phenotype .

Flight-determined engine exhaust characteristics of an F404 engine in an F-18 airplane

NASA Technical Reports Server (NTRS)

Ennix, Kimberly A.; Burcham, Frank W., Jr.; Webb, Lannie D.

1993-01-01

Personnel at the NASA Langley Research Center (NASA-Langley) and the NASA Dryden Flight Research Facility (NASA-Dryden) recently completed a joint acoustic flight test program. Several types of aircraft with high nozzle pressure ratio engines were flown to satisfy a twofold objective. First, assessments were made of subsonic climb-to-cruise noise from flights conducted at varying altitudes in a Mach 0.30 to 0.90 range. Second, using data from flights conducted at constant altitude in a Mach 0.30 to 0.95 range, engineers obtained a high quality noise database. This database was desired to validate the Aircraft Noise Prediction Program and other system noise prediction codes. NASA-Dryden personnel analyzed the engine data from several aircraft that were flown in the test program to determine the exhaust characteristics. The analysis of the exhaust characteristics from the F-18 aircraft are reported. An overview of the flight test planning, instrumentation, test procedures, data analysis, engine modeling codes, and results are presented.

Appreciating Formal Similarities in the Kinetics of Homogeneous, Heterogeneous, and Enzyme Catalysis

ERIC Educational Resources Information Center

Ashby, Michael T.

2007-01-01

Because interest in catalysts is widespread, the kinetics of catalytic reactions have been investigated by widely diverse groups of individuals, including chemists, engineers, and biologists. This has lead to redundancy in theories, particularly with regard to the topics of homogeneous, heterogeneous, and enzyme catalysis. From a pedagogical…

Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

USDA-ARS?s Scientific Manuscript database

The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...

"Mr. Database" : Jim Gray and the History of Database Technologies.

PubMed

Hanwahr, Nils C

2017-12-01

Although the widespread use of the term "Big Data" is comparatively recent, it invokes a phenomenon in the developments of database technology with distinct historical contexts. The database engineer Jim Gray, known as "Mr. Database" in Silicon Valley before his disappearance at sea in 2007, was involved in many of the crucial developments since the 1970s that constitute the foundation of exceedingly large and distributed databases. Jim Gray was involved in the development of relational database systems based on the concepts of Edgar F. Codd at IBM in the 1970s before he went on to develop principles of Transaction Processing that enable the parallel and highly distributed performance of databases today. He was also involved in creating forums for discourse between academia and industry, which influenced industry performance standards as well as database research agendas. As a co-founder of the San Francisco branch of Microsoft Research, Gray increasingly turned toward scientific applications of database technologies, e. g. leading the TerraServer project, an online database of satellite images. Inspired by Vannevar Bush's idea of the memex, Gray laid out his vision of a Personal Memex as well as a World Memex, eventually postulating a new era of data-based scientific discovery termed "Fourth Paradigm Science". This article gives an overview of Gray's contributions to the development of database technology as well as his research agendas and shows that central notions of Big Data have been occupying database engineers for much longer than the actual term has been in use.

Classroom Laboratory Report: Using an Image Database System in Engineering Education.

ERIC Educational Resources Information Center

Alam, Javed; And Others

1991-01-01

Describes an image database system assembled using separate computer components that was developed to overcome text-only computer hardware storage and retrieval limitations for a pavement design class. (JJK)

Engineering of Paraoxonases for Pre- and Post-treatment of Intoxication by a Broad Spectrum of Nerve Agents and Pesticides

DTIC Science & Technology

2014-08-01

chemical warfare nerve agents (CWNA). Enzymes identified in these screens should be capable of catalytically neutralizing the target agent under...soluble form. 4. Large-scale production of selected enzyme candidates, and their kinetic, structural and pharmacological evaluation 6...employed, with an enzyme protein concentration of 0.5-2 mM in the assay cuvette, the activity measured was indistinguishable from the rate of

Adaptation of Decoy Fusion Strategy for Existing Multi-Stage Search Workflows

NASA Astrophysics Data System (ADS)

Ivanov, Mark V.; Levitsky, Lev I.; Gorshkov, Mikhail V.

2016-09-01

A number of proteomic database search engines implement multi-stage strategies aiming at increasing the sensitivity of proteome analysis. These approaches often employ a subset of the original database for the secondary stage of analysis. However, if target-decoy approach (TDA) is used for false discovery rate (FDR) estimation, the multi-stage strategies may violate the underlying assumption of TDA that false matches are distributed uniformly across the target and decoy databases. This violation occurs if the numbers of target and decoy proteins selected for the second search are not equal. Here, we propose a method of decoy database generation based on the previously reported decoy fusion strategy. This method allows unbiased TDA-based FDR estimation in multi-stage searches and can be easily integrated into existing workflows utilizing popular search engines and post-search algorithms.

Improving sensitivity in proteome studies by analysis of false discovery rates for multiple search engines.

PubMed

Jones, Andrew R; Siepen, Jennifer A; Hubbard, Simon J; Paton, Norman W

2009-03-01

LC-MS experiments can generate large quantities of data, for which a variety of database search engines are available to make peptide and protein identifications. Decoy databases are becoming widely used to place statistical confidence in result sets, allowing the false discovery rate (FDR) to be estimated. Different search engines produce different identification sets so employing more than one search engine could result in an increased number of peptides (and proteins) being identified, if an appropriate mechanism for combining data can be defined. We have developed a search engine independent score, based on FDR, which allows peptide identifications from different search engines to be combined, called the FDR Score. The results demonstrate that the observed FDR is significantly different when analysing the set of identifications made by all three search engines, by each pair of search engines or by a single search engine. Our algorithm assigns identifications to groups according to the set of search engines that have made the identification, and re-assigns the score (combined FDR Score). The combined FDR Score can differentiate between correct and incorrect peptide identifications with high accuracy, allowing on average 35% more peptide identifications to be made at a fixed FDR than using a single search engine.

From Scarcity to Visibility: Gender Differences in the Careers of Doctoral Scientists and Engineers.

ERIC Educational Resources Information Center

Long, J. Scott, Ed.

This study documents the changes that have occurred in the representation of women in science and engineering and the characteristics of women scientists and engineers. Data from two National Science Foundation databases, the Survey of Earned Doctorates for New Ph.D.s and the Survey of Doctoral Recipients for the science & engineering doctoral…

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose.

PubMed

Zhu, Zhiguang; Zhang, Y-H Percival

2017-01-01

The direct generation of electricity from the most abundant renewable sugar, glucose, is an appealing alternative to the production of liquid biofuels and biohydrogen. However, enzyme-catalyzed bioelectricity generation from glucose suffers from low yields due to the incomplete oxidation of the six-carbon compound glucose via one or few enzymes. Here, we demonstrate a synthetic ATP- and CoA-free 12-enzyme pathway to implement the complete oxidation of glucose in vitro. This pathway is comprised of glucose phosphorylation via polyphosphate glucokinase, NADH generation catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), electron transfer from NADH to the anode, and glucose 6-phosphate regeneration via the non-oxidative pentose phosphate pathway and gluconeogenesis. The faraday efficiency from glucose to electrons via this pathway was as high as 98.8%, suggesting the generation of nearly 24 electrons per molecule of glucose. The generated current density was greatly increased from 2.8 to 6.9mAcm -2 by replacing a low-activity G6PDH with a high-activity G6PDH and introducing a new enzyme, 6-phosphogluconolactonase, between G6PDH and 6PGDH. These results suggest the great potential of high-yield bioelectricity generation through in vitro metabolic engineering. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

PubMed Central

Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

2013-01-01

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

PubMed

Der, Bryan S; Edwards, David R; Kuhlman, Brian

2012-05-08

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Engineering strategies for the fermentative production of plant alkaloids in yeast

PubMed Central

Trenchard, Isis J.; Smolke, Christina D.

2015-01-01

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 µg/L stylopine, 548 µg/L cis-N-methylstylopine, 252 µg/L protopine, and 80 µg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization. PMID:25981946

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

ELISA-BASE: An Integrated Bioinformatics Tool for Analyzing and Tracking ELISA Microarray Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.

ELISA-BASE is an open-source database for capturing, organizing and analyzing protein enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Soft-ware Environment (BASE) database system, which was developed for DNA microarrays. In order to make BASE suitable for protein microarray experiments, we developed several plugins for importing and analyzing quantitative ELISA microarray data. Most notably, our Protein Microarray Analysis Tool (ProMAT) for processing quantita-tive ELISA data is now available as a plugin to the database.

Philip T. Pienkos | NREL

Science.gov Websites

Valorization: Development of a Methane-to-Adipic Acid Bioprocess (consultant) Areas of Expertise Molecular engineering Enzyme and protein engineering Techno-economic analysis Education Ph.D., Molecular Biology Principal Research Supervisor, NREL, Aug. 2007-May 2011 Research Director, Molecular Logix, Inc., The

Research and application of microbial enzymes--India's contribution.

PubMed

Chand, Subhash; Mishra, Prashant

2003-01-01

Enzymes have attracted the attention of scientists world over due to their wide range of physiological, analytical and industrial applications. Although enzymes have been isolated, purified and studied from microbial, animal and plant sources, microorganisms represent the most common source of enzymes due to their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation. With the advent of molecular biology techniques, a number of genes of industrially important enzymes has been cloned and expressed in order to improve the production of enzymes, substrate utilization and other commercially useful properties. Special attention has been focused on enzymes isolated from thermophiles due to their inherent stability and industrial applications. In addition, a variety of methods have been employed to modify enzymes for their industrial usage including strain improvement, chemical modifications, modification of reaction environment, immobilization and protein engineering. A wide range of applications of enzymes in different bioprocess industries is discussed.

Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.

PubMed

Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M

2013-01-01

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation of an Aerothermal Data Base for the X33 Spacecraft

NASA Technical Reports Server (NTRS)

Roberts, Cathy; Huynh, Loc

1998-01-01

The X-33 experimental program is a cooperative program between industry and NASA, managed by Lockheed-Martin Skunk Works to develop an experimental vehicle to demonstrate new technologies for a single-stage-to-orbit, fully reusable launch vehicle (RLV). One of the new technologies to be demonstrated is an advanced Thermal Protection System (TPS) being designed by BF Goodrich (formerly Rohr, Inc.) with support from NASA. The calculation of an aerothermal database is crucial to identifying the critical design environment data for the TPS. The NASA Ames X-33 team has generated such a database using Computational Fluid Dynamics (CFD) analyses, engineering analysis methods and various programs to compare and interpolate the results from the CFD and the engineering analyses. This database, along with a program used to query the database, is used extensively by several X-33 team members to help them in designing the X-33. This paper will describe the methods used to generate this database, the program used to query the database, and will show some of the aerothermal analysis results for the X-33 aircraft.

An Overview and History of Glyco-Engineering in Insect Expression Systems.

PubMed

Geisler, Christoph; Mabashi-Asazuma, Hideaki; Jarvis, Donald L

2015-01-01

Insect systems, including the baculovirus-insect cell and Drosophila S2 cell systems are widely used as recombinant protein production platforms. Historically, however, no insect-based system has been able to produce glycoproteins with human-type glycans, which often influence the clinical efficacy of therapeutic glycoproteins and the overall structures and functions of other recombinant glycoprotein products. In addition, some insect cell systems produce N-glycans with immunogenic epitopes. Over the past 20 years, these problems have been addressed by efforts to glyco-engineer insect-based expression systems. These efforts have focused on introducing the capacity to produce complex-type, terminally sialylated N-glycans and eliminating the capacity to produce immunogenic N-glycans. Various glyco-engineering approaches have included genetically engineering insect cells, baculoviral vectors, and/or insects with heterologous genes encoding the enzymes required to produce various glycosyltransferases, sugars, nucleotide sugars, and nucleotide sugar transporters, as well as an enzyme that can deplete GDP-fucose. In this chapter, we present an overview and history of glyco-engineering in insect expression systems as a prelude to subsequent chapters, which will highlight various methods used for this purpose.

Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Production of Enzymes from Marine Actinobacteria.

PubMed

Zhao, X Q; Xu, X N; Chen, L Y

Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

Predicting Minimum Control Speed on the Ground (VMCG) and Minimum Control Airspeed (VMCA) of Engine Inoperative Flight Using Aerodynamic Database and Propulsion Database Generators

NASA Astrophysics Data System (ADS)

Hadder, Eric Michael

There are many computer aided engineering tools and software used by aerospace engineers to design and predict specific parameters of an airplane. These tools help a design engineer predict and calculate such parameters such as lift, drag, pitching moment, takeoff range, maximum takeoff weight, maximum flight range and much more. However, there are very limited ways to predict and calculate the minimum control speeds of an airplane in engine inoperative flight. There are simple solutions, as well as complicated solutions, yet there is neither standard technique nor consistency throughout the aerospace industry. To further complicate this subject, airplane designers have the option of using an Automatic Thrust Control System (ATCS), which directly alters the minimum control speeds of an airplane. This work addresses this issue with a tool used to predict and calculate the Minimum Control Speed on the Ground (VMCG) as well as the Minimum Control Airspeed (VMCA) of any existing or design-stage airplane. With simple line art of an airplane, a program called VORLAX is used to generate an aerodynamic database used to calculate the stability derivatives of an airplane. Using another program called Numerical Propulsion System Simulation (NPSS), a propulsion database is generated to use with the aerodynamic database to calculate both VMCG and VMCA. This tool was tested using two airplanes, the Airbus A320 and the Lockheed Martin C130J-30 Super Hercules. The A320 does not use an Automatic Thrust Control System (ATCS), whereas the C130J-30 does use an ATCS. The tool was able to properly calculate and match known values of VMCG and VMCA for both of the airplanes. The fact that this tool was able to calculate the known values of VMCG and VMCA for both airplanes means that this tool would be able to predict the VMCG and VMCA of an airplane in the preliminary stages of design. This would allow design engineers the ability to use an Automatic Thrust Control System (ATCS) as part of the design of an airplane and still have the ability to predict the VMCG and VMCA of the airplane.

Phenotip - a web-based instrument to help diagnosing fetal syndromes antenatally.

PubMed

Porat, Shay; de Rham, Maud; Giamboni, Davide; Van Mieghem, Tim; Baud, David

2014-12-10

Prenatal ultrasound can often reliably distinguish fetal anatomic anomalies, particularly in the hands of an experienced ultrasonographer. Given the large number of existing syndromes and the significant overlap in prenatal findings, antenatal differentiation for syndrome diagnosis is difficult. We constructed a hierarchic tree of 1140 sonographic markers and submarkers, organized per organ system. Subsequently, a database of prenatally diagnosable syndromes was built. An internet-based search engine was then designed to search the syndrome database based on a single or multiple sonographic markers. Future developments will include a database with magnetic resonance imaging findings as well as further refinements in the search engine to allow prioritization based on incidence of syndromes and markers.

Searches Conducted for Engineers.

ERIC Educational Resources Information Center

Lorenz, Patricia

This paper reports an industrial information specialist's experience in performing online searches for engineers and surveys the databases used. Engineers seeking assistance fall into three categories: (1) those who recognize the value of online retrieval; (2) referrals by colleagues; and (3) those who do not seek help. As more successful searches…

Multimedia explorer: image database, image proxy-server and search-engine.

PubMed Central

Frankewitsch, T.; Prokosch, U.

1999-01-01

Multimedia plays a major role in medicine. Databases containing images, movies or other types of multimedia objects are increasing in number, especially on the WWW. However, no good retrieval mechanism or search engine currently exists to efficiently track down such multimedia sources in the vast of information provided by the WWW. Secondly, the tools for searching databases are usually not adapted to the properties of images. HTML pages do not allow complex searches. Therefore establishing a more comfortable retrieval involves the use of a higher programming level like JAVA. With this platform independent language it is possible to create extensions to commonly used web browsers. These applets offer a graphical user interface for high level navigation. We implemented a database using JAVA objects as the primary storage container which are then stored by a JAVA controlled ORACLE8 database. Navigation depends on a structured vocabulary enhanced by a semantic network. With this approach multimedia objects can be encapsulated within a logical module for quick data retrieval. PMID:10566463

Multimedia explorer: image database, image proxy-server and search-engine.

PubMed

Frankewitsch, T; Prokosch, U

1999-01-01

Multimedia plays a major role in medicine. Databases containing images, movies or other types of multimedia objects are increasing in number, especially on the WWW. However, no good retrieval mechanism or search engine currently exists to efficiently track down such multimedia sources in the vast of information provided by the WWW. Secondly, the tools for searching databases are usually not adapted to the properties of images. HTML pages do not allow complex searches. Therefore establishing a more comfortable retrieval involves the use of a higher programming level like JAVA. With this platform independent language it is possible to create extensions to commonly used web browsers. These applets offer a graphical user interface for high level navigation. We implemented a database using JAVA objects as the primary storage container which are then stored by a JAVA controlled ORACLE8 database. Navigation depends on a structured vocabulary enhanced by a semantic network. With this approach multimedia objects can be encapsulated within a logical module for quick data retrieval.

Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

PubMed

Han, Lei; Liang, Bo; Song, Jianxia

2018-02-01

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

Ocean Drilling Program: Science Operator Search Engine

Science.gov Websites

and products Drilling services and tools Online Janus database Search the ODP/TAMU web site ODP's main -USIO site, plus IODP, ODP, and DSDP Publications, together or separately. ODP | Search | Database

MINEs: open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics.

PubMed

Jeffryes, James G; Colastani, Ricardo L; Elbadawi-Sidhu, Mona; Kind, Tobias; Niehaus, Thomas D; Broadbelt, Linda J; Hanson, Andrew D; Fiehn, Oliver; Tyo, Keith E J; Henry, Christopher S

2015-01-01

In spite of its great promise, metabolomics has proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography-mass spectrometry (LC-MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases. Here we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likely to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC-MS accurate mass data enabled the identity of an unknown peak to be confidently predicted. MINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose results include irrelevant synthetic compounds. Furthermore, MINEs complement and expand on previous in silico generated compound databases that focus on human metabolism. We are actively developing the database; future versions of this resource will incorporate transformation rules for spontaneous chemical reactions and more advanced filtering and prioritization of candidate structures. Graphical abstractMINE database construction and access methods. The process of constructing a MINE database from the curated source databases is depicted on the left. The methods for accessing the database are shown on the right.

Military, Charter, Unreported Domestic Traffic and General Aviation 1976, 1984, 1992, and 2015 Emission Scenarios

NASA Technical Reports Server (NTRS)

Mortlock, Alan; VanAlstyne, Richard

1998-01-01

The report describes development of databases estimating aircraft engine exhaust emissions for the years 1976 and 1984 from global operations of Military, Charter, historic Soviet and Chinese, Unreported Domestic traffic, and General Aviation (GA). These databases were developed under the National Aeronautics and Space Administration's (NASA) Advanced Subsonic Assessment (AST). McDonnell Douglas Corporation's (MDC), now part of the Boeing Company has previously estimated engine exhaust emissions' databases for the baseline year of 1992 and a 2015 forecast year scenario. Since their original creation, (Ward, 1994 and Metwally, 1995) revised technology algorithms have been developed. Additionally, GA databases have been created and all past NIDC emission inventories have been updated to reflect the new technology algorithms. Revised data (Baughcum, 1996 and Baughcum, 1997) for the scheduled inventories have been used in this report to provide a comparison of the total aviation emission forecasts from various components. Global results of two historic years (1976 and 1984), a baseline year (1992) and a forecast year (2015) are presented. Since engine emissions are directly related to fuel usage, an overview of individual aviation annual global fuel use for each inventory component is also given in this report.

TWRS technical baseline database manager definition document

DOE Office of Scientific and Technical Information (OSTI.GOV)

Acree, C.D.

1997-08-13

This document serves as a guide for using the TWRS Technical Baseline Database Management Systems Engineering (SE) support tool in performing SE activities for the Tank Waste Remediation System (TWRS). This document will provide a consistent interpretation of the relationships between the TWRS Technical Baseline Database Management software and the present TWRS SE practices. The Database Manager currently utilized is the RDD-1000 System manufactured by the Ascent Logic Corporation. In other documents, the term RDD-1000 may be used interchangeably with TWRS Technical Baseline Database Manager.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli

DOE PAGES

Whitaker, W. Brian; Jones, J. Andrew; Bennett, R. Kyle; ...

2016-11-01

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less

Emerging engineering principles for yield improvement in microbial cell design.

PubMed

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling.

Emerging engineering principles for yield improvement in microbial cell design

PubMed Central

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling. PMID:24688676

Nanobiocatalyst advancements and bioprocessing applications

PubMed Central

Misson, Mailin; Zhang, Hu; Jin, Bo

2015-01-01

The nanobiocatalyst (NBC) is an emerging innovation that synergistically integrates advanced nanotechnology with biotechnology and promises exciting advantages for improving enzyme activity, stability, capability and engineering performances in bioprocessing applications. NBCs are fabricated by immobilizing enzymes with functional nanomaterials as enzyme carriers or containers. In this paper, we review the recent developments of novel nanocarriers/nanocontainers with advanced hierarchical porous structures for retaining enzymes, such as nanofibres (NFs), mesoporous nanocarriers and nanocages. Strategies for immobilizing enzymes onto nanocarriers made from polymers, silicas, carbons and metals by physical adsorption, covalent binding, cross-linking or specific ligand spacers are discussed. The resulting NBCs are critically evaluated in terms of their bioprocessing performances. Excellent performances are demonstrated through enhanced NBC catalytic activity and stability due to conformational changes upon immobilization and localized nanoenvironments, and NBC reutilization by assembling magnetic nanoparticles into NBCs to defray the high operational costs associated with enzyme production and nanocarrier synthesis. We also highlight several challenges associated with the NBC-driven bioprocess applications, including the maturation of large-scale nanocarrier synthesis, design and development of bioreactors to accommodate NBCs, and long-term operations of NBCs. We suggest these challenges are to be addressed through joint collaboration of chemists, engineers and material scientists. Finally, we have demonstrated the great potential of NBCs in manufacturing bioprocesses in the near future through successful laboratory trials of NBCs in carbohydrate hydrolysis, biofuel production and biotransformation. PMID:25392397

Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates.

PubMed

Aldor, Ilana S; Keasling, Jay D

2003-10-01

Implementing several metabolic engineering strategies, either individually or in combination, it is possible to construct microbial plastic factories to produce a variety of polyhydroxyalkanoate (PHA) biopolymers with desirable structures and material properties. Approaches include external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation and, most recently, protein engineering of PHA biosynthetic enzymes. In addition, mathematical models and molecular methods can be used to elucidate metabolically engineered systems and to identify targets for performance improvement.

Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.

PubMed

Budnik, Lygia T; Scheer, Edwin; Burge, P Sherwood; Baur, Xaver

2017-01-01

The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Link, P.K.

A total of 48 papers were presented at the Engineering Geology and Geotechnical Engineering 30th Symposium. These papers are presented in this proceedings under the following headings: site characterization--Pocatello area; site characterization--Boise Area; site assessment; Idaho National Engineering Laboratory; geophysical methods; remediation; geotechnical engineering; and hydrogeology, northern and western Idaho. Individual papers have been processed separately for inclusion in the Energy Science and Technology Database.

Probabilistic simulation of concurrent engineering of propulsion systems

NASA Technical Reports Server (NTRS)

Chamis, C. C.; Singhal, S. N.

1993-01-01

Technology readiness and the available infrastructure is assessed for timely computational simulation of concurrent engineering for propulsion systems. Results for initial coupled multidisciplinary, fabrication-process, and system simulators are presented including uncertainties inherent in various facets of engineering processes. An approach is outlined for computationally formalizing the concurrent engineering process from cradle-to-grave via discipline dedicated workstations linked with a common database.

ATLAS of Biochemistry: A Repository of All Possible Biochemical Reactions for Synthetic Biology and Metabolic Engineering Studies.

PubMed

Hadadi, Noushin; Hafner, Jasmin; Shajkofci, Adrian; Zisaki, Aikaterini; Hatzimanikatis, Vassily

2016-10-21

Because the complexity of metabolism cannot be intuitively understood or analyzed, computational methods are indispensable for studying biochemistry and deepening our understanding of cellular metabolism to promote new discoveries. We used the computational framework BNICE.ch along with cheminformatic tools to assemble the whole theoretical reactome from the known metabolome through expansion of the known biochemistry presented in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We constructed the ATLAS of Biochemistry, a database of all theoretical biochemical reactions based on known biochemical principles and compounds. ATLAS includes more than 130 000 hypothetical enzymatic reactions that connect two or more KEGG metabolites through novel enzymatic reactions that have never been reported to occur in living organisms. Moreover, ATLAS reactions integrate 42% of KEGG metabolites that are not currently present in any KEGG reaction into one or more novel enzymatic reactions. The generated repository of information is organized in a Web-based database ( http://lcsb-databases.epfl.ch/atlas/ ) that allows the user to search for all possible routes from any substrate compound to any product. The resulting pathways involve known and novel enzymatic steps that may indicate unidentified enzymatic activities and provide potential targets for protein engineering. Our approach of introducing novel biochemistry into pathway design and associated databases will be important for synthetic biology and metabolic engineering.

Retrieving high-resolution images over the Internet from an anatomical image database

NASA Astrophysics Data System (ADS)

Strupp-Adams, Annette; Henderson, Earl

1999-12-01

The Visible Human Data set is an important contribution to the national collection of anatomical images. To enhance the availability of these images, the National Library of Medicine has supported the design and development of a prototype object-oriented image database which imports, stores, and distributes high resolution anatomical images in both pixel and voxel formats. One of the key database modules is its client-server Internet interface. This Web interface provides a query engine with retrieval access to high-resolution anatomical images that range in size from 100KB for browser viewable rendered images, to 1GB for anatomical structures in voxel file formats. The Web query and retrieval client-server system is composed of applet GUIs, servlets, and RMI application modules which communicate with each other to allow users to query for specific anatomical structures, and retrieve image data as well as associated anatomical images from the database. Selected images can be downloaded individually as single files via HTTP or downloaded in batch-mode over the Internet to the user's machine through an applet that uses Netscape's Object Signing mechanism. The image database uses ObjectDesign's object-oriented DBMS, ObjectStore that has a Java interface. The query and retrieval systems has been tested with a Java-CDE window system, and on the x86 architecture using Windows NT 4.0. This paper describes the Java applet client search engine that queries the database; the Java client module that enables users to view anatomical images online; the Java application server interface to the database which organizes data returned to the user, and its distribution engine that allow users to download image files individually and/or in batch-mode.

Engineering Lipases: walking the fine line between activity and stability

NASA Astrophysics Data System (ADS)

Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

2017-11-01

Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

Modules for in vitro metabolic engineering: Pathway assembly for bio-based production of value-added chemicals.

PubMed

Taniguchi, Hironori; Okano, Kenji; Honda, Kohsuke

2017-06-01

Bio-based chemical production has drawn attention regarding the realization of a sustainable society. In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals. This method involves the reconstitution of natural or artificial metabolic pathways by assembling purified/semi-purified enzymes in vitro . Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel, enabling easier pathway design with high modularity. Multiple modules have been designed, built, tested, and improved by different groups for different purpose. In this review, we focus on these in vitro metabolic engineering modules, especially focusing on the carbon metabolism, and present an overview of input modules, output modules, and other modules related to cofactor management.

Combining chemoinformatics with bioinformatics: in silico prediction of bacterial flavor-forming pathways by a chemical systems biology approach "reverse pathway engineering".

PubMed

Liu, Mengjin; Bienfait, Bruno; Sacher, Oliver; Gasteiger, Johann; Siezen, Roland J; Nauta, Arjen; Geurts, Jan M W

2014-01-01

The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the "missing links" between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology.

The limits to biocatalysis: pushing the envelope.

PubMed

Sheldon, Roger A; Brady, Dean

2018-06-12

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol

PubMed Central

Favaro, Lorenzo; Jooste, Tania; Basaglia, Marina; Rose, Shaunita H.; Saayman, Maryna; Görgens, Johann F.; Casella, Sergio; van Zyl, Willem H.

2013-01-01

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one step process, is a promising strategy for the effective ethanol production from cheap lignocellulosic and starchy materials. CBP requires a highly engineered microbial strain able to both hydrolyze biomass with enzymes produced on its own and convert the resulting simple sugars into high-titer ethanol. Recently, heterologous production of cellulose and starch-degrading enzymes has been achieved in yeast hosts, which has realized direct processing of biomass to ethanol. However, essentially all efforts aimed at the efficient heterologous expression of saccharolytic enzymes in yeast have involved laboratory strains and much of this work has to be transferred to industrial yeasts that provide the fermentation capacity and robustness desired for large scale bioethanol production. Specifically, the development of an industrial CBP amylolytic yeast would allow the one-step processing of low-cost starchy substrates into ethanol. This article gives insight in the current knowledge and achievements on bioethanol production from starchy materials with industrial engineered S. cerevisiae strains. PMID:22989992

Thermostable microbial xylanases for pulp and paper industries: trends, applications and further perspectives.

PubMed

Kumar, Vishal; Marín-Navarro, Julia; Shukla, Pratyoosh

2016-02-01

Xylanases are enzymes with biotechnological relevance in a number of fields, including food, feed, biofuel, and textile industries. Their most significant application is in the paper and pulp industry, where they are used as a biobleaching agent, showing clear economic and environmental advantages over chemical alternatives. Since this process requires high temperatures and alkali media, the identification of thermostable and alkali stable xylanases represents a major biotechnological goal in this field. Moreover, thermostability is a desirable property for many other applications of xylanases. The review makes an overview of xylanase producing microorganisms and their current implementation in paper biobleaching. Future perspectives are analyzed focusing in the efforts carried out to generate thermostable enzymes by means of modern biotechnological tools, including metagenomic analysis, enzyme molecular engineering and nanotechnology. Furthermore, structural and mutagenesis studies have revealed critical sites for stability of xylanases from glycoside hydrolase families GH10 and GH11, which constitute the main classes of these enzymes. The overall conclusions of these works are summarized here and provide relevant information about putative weak spots within xylanase structures to be targeted in future protein engineering approaches.

The production, properties, and applications of thermostable steryl glucosidases.

PubMed

Aguirre, Andres; Eberhardt, Florencia; Hails, Guillermo; Cerminati, Sebastian; Castelli, María Eugenia; Rasia, Rodolfo M; Paoletti, Luciana; Menzella, Hugo G; Peiru, Salvador

2018-02-21

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable β-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

Sandia Engineering Analysis Code Access System v. 2.0.1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sjaardema, Gregory D.

The Sandia Engineering Analysis Code Access System (SEACAS) is a suite of preprocessing, post processing, translation, visualization, and utility applications supporting finite element analysis software using the Exodus database file format.

An Improved Forensic Science Information Search.

PubMed

Teitelbaum, J

2015-01-01

Although thousands of search engines and databases are available online, finding answers to specific forensic science questions can be a challenge even to experienced Internet users. Because there is no central repository for forensic science information, and because of the sheer number of disciplines under the forensic science umbrella, forensic scientists are often unable to locate material that is relevant to their needs. The author contends that using six publicly accessible search engines and databases can produce high-quality search results. The six resources are Google, PubMed, Google Scholar, Google Books, WorldCat, and the National Criminal Justice Reference Service. Carefully selected keywords and keyword combinations, designating a keyword phrase so that the search engine will search on the phrase and not individual keywords, and prompting search engines to retrieve PDF files are among the techniques discussed. Copyright © 2015 Central Police University.

A Practical Engineering Approach to Predicting Fatigue Crack Growth in Riveted Lap Joints

NASA Technical Reports Server (NTRS)

Harris, Charles E.; Piascik, Robert S.; Newman, James C., Jr.

1999-01-01

An extensive experimental database has been assembled from very detailed teardown examinations of fatigue cracks found in rivet holes of fuselage structural components. Based on this experimental database, a comprehensive analysis methodology was developed to predict the onset of widespread fatigue damage in lap joints of fuselage structure. Several computer codes were developed with specialized capabilities to conduct the various analyses that make up the comprehensive methodology. Over the past several years, the authors have interrogated various aspects of the analysis methods to determine the degree of computational rigor required to produce numerical predictions with acceptable engineering accuracy. This study led to the formulation of a practical engineering approach to predicting fatigue crack growth in riveted lap joints. This paper describes the practical engineering approach and compares predictions with the results from several experimental studies.

A Practical Engineering Approach to Predicting Fatigue Crack Growth in Riveted Lap Joints

NASA Technical Reports Server (NTRS)

Harris, C. E.; Piascik, R. S.; Newman, J. C., Jr.

2000-01-01

An extensive experimental database has been assembled from very detailed teardown examinations of fatigue cracks found in rivet holes of fuselage structural components. Based on this experimental database, a comprehensive analysis methodology was developed to predict the onset of widespread fatigue damage in lap joints of fuselage structure. Several computer codes were developed with specialized capabilities to conduct the various analyses that make up the comprehensive methodology. Over the past several years, the authors have interrogated various aspects of the analysis methods to determine the degree of computational rigor required to produce numerical predictions with acceptable engineering accuracy. This study led to the formulation of a practical engineering approach to predicting fatigue crack growth in riveted lap joints. This paper describes the practical engineering approach and compares predictions with the results from several experimental studies.

[Advances of consolidated bioprocessing based on recombinant strategy].

PubMed

Zheng, Zongbao; Zhao, Meina; Chen, Tao; Zhao, Xueming

2013-10-01

Lignocellulosic biomass represents an abundant, low-cost and renewable source of potentially fermentable sugars. It is acandidate besides petroleum as feedstock for fuel and chemical production. Recent researches on utilizing lignocellulosicsas feedstock boost development of numerous-promising processes for a variety of fuels and chemicals, such as biodiesel, biohydrogen and ethanol. However, high cost in depolymerization is a primary obstacle preventing the use of lignocellulosic biomass as feedstock. Consolidated bioprocessing (CBP), refers to the bioprocess without any exogenous cellulolyotic enzymes added, converting the lignocellulosic material into biochemicals directly, which could potentially avoid the cost of the dedicated enzyme generation step by incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single organism through genetic engineering. There are two CBP strategies, native strategy and recombinant strategy. We mainly introduce the recombinant strategy, including its principle, the two responding styles, the contributions of synthetic biology and metabolic engineering and the future challenges.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

PubMed

Jacques, Philippe; Béchet, Max; Bigan, Muriel; Caly, Delphine; Chataigné, Gabrielle; Coutte, François; Flahaut, Christophe; Heuson, Egon; Leclère, Valérie; Lecouturier, Didier; Phalip, Vincent; Ravallec, Rozenn; Dhulster, Pascal; Froidevaux, Rénato

2017-02-01

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols

PubMed Central

Avalos, José L.; Fink, Gerald R.; Stephanopoulos, Gregory

2013-01-01

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeted expression of metabolic pathways to mitochondria can increase production levels compared with expression of the same pathways in the cytoplasm. Compartmentalisation of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overexpressing only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains reveals that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves higher local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion, and reducing the loss of intermediates to competing pathways. PMID:23417095

Engineering the intracellular metabolism of Escherichia coli to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes.

PubMed

Pham, Van Dung; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

2016-02-01

To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli. Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l(-1) was produced from 10 g glucose l(-1) while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l(-1) when 20 g glucose l(-1) was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l(-1)). The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, Fanny; Peck, Ariana; Salzman, Julia

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

WaveNet: A Web-Based Metocean Data Access, Processing and Analysis Tool. Part 4 - GLOS/GLCFS Database

DTIC Science & Technology

2014-06-01

and Coastal Data Information Program ( CDIP ). This User’s Guide includes step-by-step instructions for accessing the GLOS/GLCFS database via WaveNet...access, processing and analysis tool; part 3 – CDIP database. ERDC/CHL CHETN-xx-14. Vicksburg, MS: U.S. Army Engineer Research and Development Center

The Wannabee Culture: Why No-One Does What They Used To.

ERIC Educational Resources Information Center

Dixon, Anne

1998-01-01

Electronic publishing has been an agent for change in not just how one publishes but in what one publishes. Describes HyperCite, a joint project with the Institution of Electrical Engineers (IEE) to create INSPEC database. Highlights include the database; the research phase (cross database searching and new interface); and what and how much was…

The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.

PubMed

Shilov, Ignat V; Seymour, Sean L; Patel, Alpesh A; Loboda, Alex; Tang, Wilfred H; Keating, Sean P; Hunter, Christie L; Nuwaysir, Lydia M; Schaeffer, Daniel A

2007-09-01

The Paragon Algorithm, a novel database search engine for the identification of peptides from tandem mass spectrometry data, is presented. Sequence Temperature Values are computed using a sequence tag algorithm, allowing the degree of implication by an MS/MS spectrum of each region of a database to be determined on a continuum. Counter to conventional approaches, features such as modifications, substitutions, and cleavage events are modeled with probabilities rather than by discrete user-controlled settings to consider or not consider a feature. The use of feature probabilities in conjunction with Sequence Temperature Values allows for a very large increase in the effective search space with only a very small increase in the actual number of hypotheses that must be scored. The algorithm has a new kind of user interface that removes the user expertise requirement, presenting control settings in the language of the laboratory that are translated to optimal algorithmic settings. To validate this new algorithm, a comparison with Mascot is presented for a series of analogous searches to explore the relative impact of increasing search space probed with Mascot by relaxing the tryptic digestion conformance requirements from trypsin to semitrypsin to no enzyme and with the Paragon Algorithm using its Rapid mode and Thorough mode with and without tryptic specificity. Although they performed similarly for small search space, dramatic differences were observed in large search space. With the Paragon Algorithm, hundreds of biological and artifact modifications, all possible substitutions, and all levels of conformance to the expected digestion pattern can be searched in a single search step, yet the typical cost in search time is only 2-5 times that of conventional small search space. Despite this large increase in effective search space, there is no drastic loss of discrimination that typically accompanies the exploration of large search space.

MINEs: Open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics

DOE PAGES

Jeffryes, James G.; Colastani, Ricardo L.; Elbadawi-Sidhu, Mona; ...

2015-08-28

Metabolomics have proven difficult to execute in an untargeted and generalizable manner. Liquid chromatography–mass spectrometry (LC–MS) has made it possible to gather data on thousands of cellular metabolites. However, matching metabolites to their spectral features continues to be a bottleneck, meaning that much of the collected information remains uninterpreted and that new metabolites are seldom discovered in untargeted studies. These challenges require new approaches that consider compounds beyond those available in curated biochemistry databases. Here we present Metabolic In silico Network Expansions (MINEs), an extension of known metabolite databases to include molecules that have not been observed, but are likelymore » to occur based on known metabolites and common biochemical reactions. We utilize an algorithm called the Biochemical Network Integrated Computational Explorer (BNICE) and expert-curated reaction rules based on the Enzyme Commission classification system to propose the novel chemical structures and reactions that comprise MINE databases. Starting from the Kyoto Encyclopedia of Genes and Genomes (KEGG) COMPOUND database, the MINE contains over 571,000 compounds, of which 93% are not present in the PubChem database. However, these MINE compounds have on average higher structural similarity to natural products than compounds from KEGG or PubChem. MINE databases were able to propose annotations for 98.6% of a set of 667 MassBank spectra, 14% more than KEGG alone and equivalent to PubChem while returning far fewer candidates per spectra than PubChem (46 vs. 1715 median candidates). Application of MINEs to LC–MS accurate mass data enabled the identity of an unknown peak to be confidently predicted. MINE databases are freely accessible for non-commercial use via user-friendly web-tools at http://minedatabase.mcs.anl.gov and developer-friendly APIs. MINEs improve metabolomics peak identification as compared to general chemical databases whose results include irrelevant synthetic compounds. MINEs complement and expand on previous in silico generated compound databases that focus on human metabolism. We are actively developing the database; future versions of this resource will incorporate transformation rules for spontaneous chemical reactions and more advanced filtering and prioritization of candidate structures.« less

Remote online monitoring and measuring system for civil engineering structures

NASA Astrophysics Data System (ADS)

Kujawińska, Malgorzata; Sitnik, Robert; Dymny, Grzegorz; Karaszewski, Maciej; Michoński, Kuba; Krzesłowski, Jakub; Mularczyk, Krzysztof; Bolewicki, Paweł

2009-06-01

In this paper a distributed intelligent system for civil engineering structures on-line measurement, remote monitoring, and data archiving is presented. The system consists of a set of optical, full-field displacement sensors connected to a controlling server. The server conducts measurements according to a list of scheduled tasks and stores the primary data or initial results in a remote centralized database. Simultaneously the server performs checks, ordered by the operator, which may in turn result with an alert or a specific action. The structure of whole system is analyzed along with the discussion on possible fields of application and the ways to provide a relevant security during data transport. Finally, a working implementation consisting of a fringe projection, geometrical moiré, digital image correlation and grating interferometry sensors and Oracle XE database is presented. The results from database utilized for on-line monitoring of a threshold value of strain for an exemplary area of interest at the engineering structure are presented and discussed.

Hydra: a scalable proteomic search engine which utilizes the Hadoop distributed computing framework

PubMed Central

2012-01-01

Background For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. Results We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. Conclusion The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources. PMID:23216909

Hydra: a scalable proteomic search engine which utilizes the Hadoop distributed computing framework.

PubMed

Lewis, Steven; Csordas, Attila; Killcoyne, Sarah; Hermjakob, Henning; Hoopmann, Michael R; Moritz, Robert L; Deutsch, Eric W; Boyle, John

2012-12-05

For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources.

Production of extremophilic bacterial cellulase enzymes in aspergillus niger.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gladden, John Michael

2013-09-01

Enzymes can be used to catalyze a myriad of chemical reactions and are a cornerstone in the biotechnology industry. Enzymes have a wide range of uses, ranging from medicine with the production of pharmaceuticals to energy were they are applied to biofuel production. However, it is difficult to produce large quantities of enzymes, especially if they are non-native to the production host. Fortunately, filamentous fungi, such as Aspergillus niger, are broadly used in industry and show great potential for use a heterologous enzyme production hosts. Here, we present work outlining an effort to engineer A. niger to produce thermophilic bacterialmore » cellulases relevant to lignocellulosic biofuel production.« less

Sensitivity and Antioxidant Response of Chlorella sp. MM3 to Used Engine Oil and Its Water Accommodated Fraction.

PubMed

Ramadass, Kavitha; Megharaj, Mallavarapu; Venkateswarlu, Kadiyala; Naidu, Ravi

2016-07-01

We exposed the microalgal strain, Chlorella sp. MM3, to unused or used engine oil, or their water accommodated fractions (WAFs) to determine growth inhibition and response of antioxidant enzymes. Oil type and oil concentration greatly affected the microalgal growth. Used oil at 0.04 % (0.4 g L(-1)) resulted in 50 % inhibition in algal growth, measured in terms of chlorophyll-a, while the corresponding concentration of unused oil was nontoxic. Similarly, used oil WAF showed significant toxicity to the algal growth at 10 % level, whereas WAF from unused oil was nontoxic even at 100 % concentration. Peroxidase enzyme in the microalga significantly increased with used oil at concentrations above 0.04 g L(-1) whereas the induction of superoxide dismutase and catalase was apparent only at 0.06 g L(-1). Activities of the antioxidant enzymes increased significantly when the microalga was exposed to 75 and 100 % WAF obtained from used oil. The used oil toxicity on microalga could be due to the presence of toxic soluble mono- and polyaromatic compounds, heavy metals, and other compounds attained by the oil during its use in the motor engines.

Min Zhang | NREL

Science.gov Websites

Min Zhang Photo of Min Zhang Min Zhang Researcher V-Molecular Biology Min.Zhang@nrel.gov | 303-384 -7753 Research Interests Using a systems biology approach to identify, analyze, and engineer pathways Metabolic engineering Molecular biology Microbial physiology Systems biology Fermentation development Enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spreitzer, Robert Joseph

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO 2 fixation in photosynthesis. However, it is a slow enzyme, and O 2 competes with CO 2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO 2. If carboxylation could be increased or oxygenation decreased, an increase in net CO 2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants,more » and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO 2/O 2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO 2/O 2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.« less

Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

PubMed

Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

2016-12-01

The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

Lynx: a database and knowledge extraction engine for integrative medicine.

PubMed

Sulakhe, Dinanath; Balasubramanian, Sandhya; Xie, Bingqing; Feng, Bo; Taylor, Andrew; Wang, Sheng; Berrocal, Eduardo; Dave, Utpal; Xu, Jinbo; Börnigen, Daniela; Gilliam, T Conrad; Maltsev, Natalia

2014-01-01

We have developed Lynx (http://lynx.ci.uchicago.edu)--a web-based database and a knowledge extraction engine, supporting annotation and analysis of experimental data and generation of weighted hypotheses on molecular mechanisms contributing to human phenotypes and disorders of interest. Its underlying knowledge base (LynxKB) integrates various classes of information from >35 public databases and private collections, as well as manually curated data from our group and collaborators. Lynx provides advanced search capabilities and a variety of algorithms for enrichment analysis and network-based gene prioritization to assist the user in extracting meaningful knowledge from LynxKB and experimental data, whereas its service-oriented architecture provides public access to LynxKB and its analytical tools via user-friendly web services and interfaces.

Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering.

PubMed

Teixeira, Liliana S Moreira; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

2012-02-01

State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

PubMed Central

Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2012-01-01

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

An ontology-based search engine for protein-protein interactions

PubMed Central

2010-01-01

Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

An ontology-based search engine for protein-protein interactions.

PubMed

Park, Byungkyu; Han, Kyungsook

2010-01-18

Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology.

Microgravity

NASA Image and Video Library

1987-02-01

Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

Engineering Ultrastable Protein Filaments into 2D and 3D Templates for Advanced Nanomaterials: A New Dimension in Materials Design

DTIC Science & Technology

2013-08-16

Littlechild,   Thermophilic  archaeal   enzymes  and  applications  in  biocatalysis,  Biochem  Soc  Trans  39   (2011...spatially arrange thermostable enzymes for high- temperature biocatalysis[7][8] and as biotemplates for fabrication and patterning of inorganic...upon which to position hyperthermostable enzymes that have optimal activities at or above 100°C  [8], which are temperatures beyond the stability of

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol.

PubMed

Han, Gui Hwan; Seong, Wonjae; Fu, Yaoyao; Yoon, Paul K; Kim, Seong Keun; Yeom, Soo-Jin; Lee, Dae-Hee; Lee, Seung-Goo

2017-03-01

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Decision making in family medicine

PubMed Central

Labrecque, Michel; Ratté, Stéphane; Frémont, Pierre; Cauchon, Michel; Ouellet, Jérôme; Hogg, William; McGowan, Jessie; Gagnon, Marie-Pierre; Njoya, Merlin; Légaré, France

2013-01-01

Abstract Objective To compare the ability of users of 2 medical search engines, InfoClinique and the Trip database, to provide correct answers to clinical questions and to explore the perceived effects of the tools on the clinical decision-making process. Design Randomized trial. Setting Three family medicine units of the family medicine program of the Faculty of Medicine at Laval University in Quebec city, Que. Participants Fifteen second-year family medicine residents. Intervention Residents generated 30 structured questions about therapy or preventive treatment (2 questions per resident) based on clinical encounters. Using an Internet platform designed for the trial, each resident answered 20 of these questions (their own 2, plus 18 of the questions formulated by other residents, selected randomly) before and after searching for information with 1 of the 2 search engines. For each question, 5 residents were randomly assigned to begin their search with InfoClinique and 5 with the Trip database. Main outcome measures The ability of residents to provide correct answers to clinical questions using the search engines, as determined by third-party evaluation. After answering each question, participants completed a questionnaire to assess their perception of the engine’s effect on the decision-making process in clinical practice. Results Of 300 possible pairs of answers (1 answer before and 1 after the initial search), 254 (85%) were produced by 14 residents. Of these, 132 (52%) and 122 (48%) pairs of answers concerned questions that had been assigned an initial search with InfoClinique and the Trip database, respectively. Both engines produced an important and similar absolute increase in the proportion of correct answers after searching (26% to 62% for InfoClinique, for an increase of 36%; 24% to 63% for the Trip database, for an increase of 39%; P = .68). For all 30 clinical questions, at least 1 resident produced the correct answer after searching with either search engine. The mean (SD) time of the initial search for each question was 23.5 (7.6) minutes with InfoClinique and 22.3 (7.8) minutes with the Trip database (P = .30). Participants’ perceptions of each engine’s effect on the decision-making process were very positive and similar for both search engines. Conclusion Family medicine residents’ ability to provide correct answers to clinical questions increased dramatically and similarly with the use of both InfoClinique and the Trip database. These tools have strong potential to increase the quality of medical care. PMID:24130286

Tags Extarction from Spatial Documents in Search Engines

NASA Astrophysics Data System (ADS)

Borhaninejad, S.; Hakimpour, F.; Hamzei, E.

2015-12-01

Nowadays the selective access to information on the Web is provided by search engines, but in the cases which the data includes spatial information the search task becomes more complex and search engines require special capabilities. The purpose of this study is to extract the information which lies in spatial documents. To that end, we implement and evaluate information extraction from GML documents and a retrieval method in an integrated approach. Our proposed system consists of three components: crawler, database and user interface. In crawler component, GML documents are discovered and their text is parsed for information extraction; storage. The database component is responsible for indexing of information which is collected by crawlers. Finally the user interface component provides the interaction between system and user. We have implemented this system as a pilot system on an Application Server as a simulation of Web. Our system as a spatial search engine provided searching capability throughout the GML documents and thus an important step to improve the efficiency of search engines has been taken.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

PubMed

Vickery, Christopher R; Cardenas, Javier; Bowman, Marianne E; Burkart, Michael D; Da Silva, Nancy A; Noel, Joseph P

2018-06-01

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts. © 2018 Wiley Periodicals, Inc.

Impact of Commercial Search Engines and International Databases on Engineering Teaching and Research

ERIC Educational Resources Information Center

Chanson, Hubert

2007-01-01

For the last three decades, the engineering higher education and professional environments have been completely transformed by the "electronic/digital information revolution" that has included the introduction of personal computer, the development of email and world wide web, and broadband Internet connections at home. Herein the writer compares…

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

USDA-ARS?s Scientific Manuscript database

The seeds of many non-domesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered ...

DataBase on Demand

NASA Astrophysics Data System (ADS)

Gaspar Aparicio, R.; Gomez, D.; Coterillo Coz, I.; Wojcik, D.

2012-12-01

At CERN a number of key database applications are running on user-managed MySQL database services. The database on demand project was born out of an idea to provide the CERN user community with an environment to develop and run database services outside of the actual centralised Oracle based database services. The Database on Demand (DBoD) empowers the user to perform certain actions that had been traditionally done by database administrators, DBA's, providing an enterprise platform for database applications. It also allows the CERN user community to run different database engines, e.g. presently open community version of MySQL and single instance Oracle database server. This article describes a technology approach to face this challenge, a service level agreement, the SLA that the project provides, and an evolution of possible scenarios.

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlin, DA; Bertolani, SJ; Siegel, JB

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase.

PubMed

Carlin, D A; Bertolani, S J; Siegel, J B

2015-02-11

Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

PubMed

Romero, Philip A; Tran, Tuan M; Abate, Adam R

2015-06-09

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass.

PubMed

Manisha; Yadav, Sudesh Kumar

2017-12-01

Hydrolytic enzymes are indispensable tools in the production of various foodstuffs, drugs, and consumables owing to their applications in almost every industrial process nowadays. One of the foremost areas of interest involving the use of hydrolytic enzymes is in the transformation of lignocellulosic biomass into value added products. However, limitations of the processes due to inadequate enzyme activity and stability with a narrow range of pH and temperature optima often limit their effective usage. The innovative technologies, involving manipulation of enzyme activity and stability through mutagenesis, genetic engineering and metagenomics lead to a major leap in all the fields using hydrolytic enzymes. This article provides recent advancement towards the isolation and use of microbes for lignocellulosic biomass utilisation, microbes producing the hydrolytic enzymes, the modern age technologies used to manipulate and enhance the hydrolytic enzyme activity and the applications of such enzymes in value added products development from lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

MSblender: A probabilistic approach for integrating peptide identifications from multiple database search engines.

PubMed

Kwon, Taejoon; Choi, Hyungwon; Vogel, Christine; Nesvizhskii, Alexey I; Marcotte, Edward M

2011-07-01

Shotgun proteomics using mass spectrometry is a powerful method for protein identification but suffers limited sensitivity in complex samples. Integrating peptide identifications from multiple database search engines is a promising strategy to increase the number of peptide identifications and reduce the volume of unassigned tandem mass spectra. Existing methods pool statistical significance scores such as p-values or posterior probabilities of peptide-spectrum matches (PSMs) from multiple search engines after high scoring peptides have been assigned to spectra, but these methods lack reliable control of identification error rates as data are integrated from different search engines. We developed a statistically coherent method for integrative analysis, termed MSblender. MSblender converts raw search scores from search engines into a probability score for every possible PSM and properly accounts for the correlation between search scores. The method reliably estimates false discovery rates and identifies more PSMs than any single search engine at the same false discovery rate. Increased identifications increment spectral counts for most proteins and allow quantification of proteins that would not have been quantified by individual search engines. We also demonstrate that enhanced quantification contributes to improve sensitivity in differential expression analyses.

MSblender: a probabilistic approach for integrating peptide identifications from multiple database search engines

PubMed Central

Kwon, Taejoon; Choi, Hyungwon; Vogel, Christine; Nesvizhskii, Alexey I.; Marcotte, Edward M.

2011-01-01

Shotgun proteomics using mass spectrometry is a powerful method for protein identification but suffers limited sensitivity in complex samples. Integrating peptide identifications from multiple database search engines is a promising strategy to increase the number of peptide identifications and reduce the volume of unassigned tandem mass spectra. Existing methods pool statistical significance scores such as p-values or posterior probabilities of peptide-spectrum matches (PSMs) from multiple search engines after high scoring peptides have been assigned to spectra, but these methods lack reliable control of identification error rates as data are integrated from different search engines. We developed a statistically coherent method for integrative analysis, termed MSblender. MSblender converts raw search scores from search engines into a probability score for all possible PSMs and properly accounts for the correlation between search scores. The method reliably estimates false discovery rates and identifies more PSMs than any single search engine at the same false discovery rate. Increased identifications increment spectral counts for all detected proteins and allow quantification of proteins that would not have been quantified by individual search engines. We also demonstrate that enhanced quantification contributes to improve sensitivity in differential expression analyses. PMID:21488652

WaveNet: A Web-Based Metocean Data Access, Processing, and Analysis Tool. Part 3 - CDIP Database

DTIC Science & Technology

2014-06-01

and Analysis Tool; Part 3 – CDIP Database by Zeki Demirbilek, Lihwa Lin, and Derek Wilson PURPOSE: This Coastal and Hydraulics Engineering...Technical Note (CHETN) describes coupling of the Coastal Data Information Program ( CDIP ) database to WaveNet, the first module of MetOcnDat (Meteorological...provides a step-by-step procedure to access, process, and analyze wave and wind data from the CDIP database. BACKGROUND: WaveNet addresses a basic

Thermal Protection System Imagery Inspection Management System -TIIMS

NASA Technical Reports Server (NTRS)

Goza, Sharon; Melendrez, David L.; Henningan, Marsha; LaBasse, Daniel; Smith, Daniel J.

2011-01-01

TIIMS is used during the inspection phases of every mission to provide quick visual feedback, detailed inspection data, and determination to the mission management team. This system consists of a visual Web page interface, an SQL database, and a graphical image generator. These combine to allow a user to ascertain quickly the status of the inspection process, and current determination of any problem zones. The TIIMS system allows inspection engineers to enter their determinations into a database and to link pertinent images and video to those database entries. The database then assigns criteria to each zone and tile, and via query, sends the information to a graphical image generation program. Using the official TIPS database tile positions and sizes, the graphical image generation program creates images of the current status of the orbiter, coloring zones, and tiles based on a predefined key code. These images are then displayed on a Web page using customized JAVA scripts to display the appropriate zone of the orbiter based on the location of the user's cursor. The close-up graphic and database entry for that particular zone can then be seen by selecting the zone. This page contains links into the database to access the images used by the inspection engineer when they make the determination entered into the database. Status for the inspection zones changes as determinations are refined and shown by the appropriate color code.

Teaching English Engineering Terminology in a Hypermedia Environment.

ERIC Educational Resources Information Center

Stamison-Atmatzidi, M.; And Others

1995-01-01

Discusses a hypermedia prototype system constituting a hypermedia dictionary environment and a database of field-specific reading passages with related exercises, for utilization in the teaching of English engineering terminology in foreign language environments. (eight references) (CK)

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C.

PubMed

Gaßmeyer, Sarah Katharina; Yoshikawa, Hiroyuki; Enoki, Junichi; Hülsemann, Nadine; Stoll, Raphael; Miyamoto, Kenji; Kourist, Robert

2015-06-23

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functionalized nanocompartments (Synthosomes): limitations and prospective applications in industrial biotechnology.

PubMed

Onaca, Ozana; Nallani, Madhavan; Ihle, Saskia; Schenk, Alexander; Schwaneberg, Ulrich

2006-01-01

Synthosomes are mechanically stable vesicles with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. The polymer vesicles are nanometer-sized (50-1000 nm) and functionalized by loading them with enzymes for bioconversions or encapsulating charged macromolecules for selective compound recovery/release. The Synthosome system might become a novel technology platform for biocatalysis and selective product recovery. Progress in Synthosome research comprises employed block copolymers, transmembrane channel engineering, and functionalizations, which are discussed here in detail. The challenges in transmembrane protein engineering, as well as cost-effective production, in block copolymer design and the state of the art in Synthosome characterization comprising quantification of encapsulated protein, translocation efficiency, number of transmembrane channels per vesicle, and enzyme kinetics are also presented and discussed. An assessment of the Synthosome technology platform for prospective applications in industrial (white) biotechnology concludes this review.

THE DRINKING WATER TREATABILITY DATABASE (Slides)

EPA Science Inventory

The Drinking Water Treatability Database (TDB) assembles referenced data on the control of contaminants in drinking water, housed on an interactive, publicly-available, USEPA web site (www.epa.gov/tdb). The TDB is of use to drinking water utilities, treatment process design engin...

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

PubMed

Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, Veeranki V

2017-01-01

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping.

PubMed

Chang, Hsueh-Wei; Cheng, Yu-Huei; Chuang, Li-Yeh; Yang, Cheng-Hong

2010-04-08

PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2. The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system. The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

OrChem - An open source chemistry search engine for Oracle(R).

PubMed

Rijnbeek, Mark; Steinbeck, Christoph

2009-10-22

Registration, indexing and searching of chemical structures in relational databases is one of the core areas of cheminformatics. However, little detail has been published on the inner workings of search engines and their development has been mostly closed-source. We decided to develop an open source chemistry extension for Oracle, the de facto database platform in the commercial world. Here we present OrChem, an extension for the Oracle 11G database that adds registration and indexing of chemical structures to support fast substructure and similarity searching. The cheminformatics functionality is provided by the Chemistry Development Kit. OrChem provides similarity searching with response times in the order of seconds for databases with millions of compounds, depending on a given similarity cut-off. For substructure searching, it can make use of multiple processor cores on today's powerful database servers to provide fast response times in equally large data sets. OrChem is free software and can be redistributed and/or modified under the terms of the GNU Lesser General Public License as published by the Free Software Foundation. All software is available via http://orchem.sourceforge.net.

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

PubMed

Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

2016-09-22

Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

Industrial Fungal Enzymes: An Occupational Allergen Perspective

PubMed Central

Green, Brett J.; Beezhold, Donald H.

2011-01-01

Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation.

PubMed

Sinha, Rajeshwari; Khare, Sunil K

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

PubMed Central

Sinha, Rajeshwari; Khare, Sunil K.

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

Thermostable Carbonic Anhydrases in Biotechnological Applications

PubMed Central

Di Fiore, Anna; Alterio, Vincenzo; Monti, Simona M.; De Simone, Giuseppina; D’Ambrosio, Katia

2015-01-01

Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application. PMID:26184158

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Positive dermal hypersensitivity and specific antibodies in workers exposed to bio-engineered enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biagini, R.E.; Henningsen, G.M.; Driscoll, R.

1991-03-15

Thirty-six employees who produced industrial enzymes from bio-engineered strains of bacteria and fungi were evaluated by skin prick testing and enzyme linked immunosorbent assays for specific IgE and IgG antibodies. The workers complained of asthma- and flu-like' symptoms which generally lessened away from work. The enzymes evaluated were {alpha}-amylase from A. niger (ind-AAN), B. licheniformis (ind-AAL) and B. subtilis (ind-AAS); purified {alpha}-amylase from B. subtilis (AAS) and A. niger (AAN); alkaline protease from B. licheniformis (ind-APL) and purified alkaline protease (APL); amylase glucosidase from A. niger (ind-AGN) and purified amylase glucosidase (AGN). Significantly positive skin tests were found for APL,more » AGN and ind-AAN. Significantly elevated specific IgE results were observed for AAN, AGN, and ind-AAN; elevated specific IgGs were observed for AAN, ind-AAN, ind-AAS, ind-AAL and ind-AGN. Radioimmunoassays of air filter samples (using sera with high Ab titers) for 4 of the ind-enzymes showed only ind-AAN at extremely high environmental levels. These results indicate that occupational exposure to some ind-enzymes causes immediate onset dermal hypersensitivity reactions. The results are equivocal as to whether these reactions are IgE mediated, as IgE titers were low. Contrary to this, IgG titers were extremely high and suggest that these biomarkers can be used as indicators of both individual exposure and environmental analyses.« less

The structure of Plasmodium falciparum serine hydroxymethyltransferase reveals a novel redox switch that regulates its activities

PubMed Central

Chitnumsub, Penchit; Ittarat, Wanwipa; Jaruwat, Aritsara; Noytanom, Krittikar; Amornwatcharapong, Watcharee; Pornthanakasem, Wichai; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

2014-01-01

Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT), an enzyme in the dTMP synthesis cycle, is an antimalarial target because inhibition of its expression or function has been shown to be lethal to the parasite. As the wild-type enzyme could not be crystallized, protein engineering of residues on the surface was carried out. The surface-engineered mutant PfSHMT-F292E was successfully crystallized and its structure was determined at 3 Å resolution. The PfSHMT-F292E structure is a good representation of PfSHMT as this variant revealed biochemical properties similar to those of the wild type. Although the overall structure of PfSHMT is similar to those of other SHMTs, unique features including the presence of two loops and a distinctive cysteine pair formed by Cys125 and Cys364 in the tetrahydrofolate (THF) substrate binding pocket were identified. These structural characteristics have never been reported in other SHMTs. Biochemical characterization and mutation analysis of these two residues confirm that they act as a disulfide/sulfhydryl switch to regulate the THF-dependent catalytic function of the enzyme. This redox switch is not present in the human enzyme, in which the cysteine pair is absent. The data reported here can be further exploited as a new strategy to specifically disrupt the activity of the parasite enzyme without interfering with the function of the human enzyme. PMID:24914963