Science.gov

Sample records for alpha-glucan acting enzymes

  1. Safety evaluation of highly-branched cyclic dextrin and a 1,4-alpha-glucan branching enzyme from Bacillus stearothermophilus.

    PubMed

    Choi, Sharon S H; Danielewska-Nikiel, Barbara; Ohdan, Kohji; Kojima, Iwao; Takata, Hiroki; Kuriki, Takashi

    2009-12-01

    Highly-branched cyclic dextrin (HBCD), a dextrin food ingredient presently only used in Japan, was investigated for digestibility and potential toxicity. HBCD was readily hydrolyzed in vitro to maltose and maltotriose by human salivary and porcine pancreatic alpha-amylases. Incubation of HBCD with a rat intestinal homogenate, containing digestive enzymes, resulted in the formation of maltose, maltotriose, and maltotetraose, and with longer incubation times, resulted in the formation of glucose. In an acute toxicity study, Wistar rats orally administered a single-dose of 2000mg/kg body weight of HBCD did not display mortality or any signs or symptoms of toxicity or abnormalities upon necropsy. Transient loose stools were observed, but were resolved within 24h of HBCD administration, and therefore, were not considered as compound-specific adverse effects. In the Ames assay, HBCD was non-mutagenic with or without metabolic activation. Toxicity testing of the branching enzyme (BE) involved in the synthesis of HBCD showed that the BE also was not acutely toxic when orally administered to rats and was non-mutagenic in the mouse lymphoma assay. The results of this study demonstrate that HBCD is digested to normal and safe products of carbohydrate digestion, and therefore, support the safety of HBCD for human consumption. PMID:19651182

  2. Self-poisoning of Mycobacterium tuberculosis by targeting GlgE in an alpha-glucan pathway.

    PubMed

    Kalscheuer, Rainer; Syson, Karl; Veeraraghavan, Usha; Weinrick, Brian; Biermann, Karolin E; Liu, Zhen; Sacchettini, James C; Besra, Gurdyal; Bornemann, Stephen; Jacobs, William R

    2010-05-01

    New chemotherapeutics are urgently required to control the tuberculosis pandemic. We describe a new pathway from trehalose to alpha-glucan in Mycobacterium tuberculosis comprising four enzymatic steps mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Using traditional and chemical reverse genetics, we show that GlgE inactivation causes rapid death of M. tuberculosis in vitro and in mice through a self-poisoning accumulation of maltose 1-phosphate. Poisoning elicits pleiotropic phosphosugar-induced stress responses promoted by a self-amplifying feedback loop where trehalose-forming enzymes are upregulated. Moreover, the pathway from trehalose to alpha-glucan exhibited a synthetic lethal interaction with the glucosyltransferase Rv3032, which is involved in biosynthesis of polymethylated alpha-glucans, because key enzymes in each pathway could not be simultaneously inactivated. The unique combination of maltose 1-phosphate toxicity and gene essentiality within a synthetic lethal pathway validates GlgE as a distinct potential drug target that exploits new synergistic mechanisms to induce death in M. tuberculosis. PMID:20305657

  3. Variation in storage alpha-glucans of the Porphyridiales (Rhodophyta).

    PubMed

    Shimonaga, Takahiro; Konishi, Mai; Oyama, Yasunori; Fujiwara, Shoko; Satoh, Aya; Fujita, Naoko; Colleoni, Christophe; Buléon, Alain; Putaux, Jean-Luc; Ball, Steven G; Yokoyama, Akiko; Hara, Yoshiaki; Nakamura, Yasunori; Tsuzuki, Mikio

    2008-01-01

    Storage glucans were analyzed in the Porphyridiales which include the most primitive and phylogenetically diverged species in the Rhodophyta, to understand early evolution of the glucan structure in the Rhodophyta. The storage glucans of both Galdieria sulphuraria and Cyanidium caldarium consisted of glycogen, while those of Rhodosorus marinus, Porphyridium purpureum, P. sordidum and Rhodella violacea could be defined as semi-amylopectin. X-ray diffraction analysis of the glucans demonstrated variation in the crystalline structure: the patterns in P. purpureum and R. violacea were of A- and B-types, respectively, while alpha-glucans of R. marinus and P. sordidum displayed structures with lower crystallinity. Electron microscopic observations indicated that the alpha-glucans of P. sordidum consisted of two kinds of granules; a minor component of more dense granules with crystalline leaflets and a major component of softer ones without crystalline structure. Gel permeation chromatography showed that all the species containing the semi-amylopectin-type glucans also contained amylose, although the relative amounts of this fraction were different depending on the species. Our results are consistent with two distinct evolution scenarios defined either by the independent acquisition of semi-crystalline starch-like structures in the different plant lineages or more probably by the loss of starch and reversion to glycogen synthesis in cyanidian algae growing in hot and acid environments. PMID:18079144

  4. The Structural Basis of Alpha-Glucan Recognition by a Family 41 Carbohydrate-Binding Module from Therotoga Maritima

    SciTech Connect

    van Bueren,A.; Boraston, A.

    2006-01-01

    Starch recognition by carbohydrate-binding modules (CBMs) is important for the activity of starch-degrading enzymes. The N-terminal family 41 CBM, TmCBM41 (from pullulanase PulA secreted by Thermotoga maritima) was shown to have {alpha}-glucan binding activity with specificity for {alpha}-1, 4-glucans but was able to tolerate the {alpha}-1, 6-linkages found roughly every three or four glucose units in pullulan. Using X-ray crystallography, the structures were solved for TmCBM41 in an uncomplexed form and in complex with maltotetraose and 63-{alpha}-d-glucosyl-maltotriose (GM3). Ligand binding was facilitated by stacking interactions between the {alpha}-faces of the glucose residues and two tryptophan side-chains in the two main subsites of the carbohydrate-binding site. Overall, this mode of starch binding is quite well conserved by other starch-binding modules. The structure in complex with GM3 revealed a third binding subsite with the flexibility to accommodate an {alpha}-1, 4- or an {alpha}-1, 6-linked glucose.

  5. Enzymes acting at strand interruptions in DNA.

    PubMed

    Lindahl, T; Satoh, M S; Dianov, G

    1995-01-30

    Endogenous and environmental DNA-damaging agents often generate single-strand interruptions in DNA. The lesions trigger a complex set of cellular reactions. In most eukaryotic cells, cellular poly(ADP-ribose) formation is the most acute response to such damage. Recently, such events have been amenable to study with soluble cell-free extracts of human cells. These investigations clarify the modulating role on DNA repair by poly (ADP-ribose), and suggest that the primary function of this unusual polymer is to act as an antirecombinant agent. Similar biochemical studies of subsequent repair events have revealed a branched pathway for the ubiquitous DNA base excision-repair process. The alternative pathway provides the cell with back-up functions for individual steps in this essential form of DNA repair. PMID:7746855

  6. Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

    PubMed Central

    Bosello, Mattia; Berti, Francesco; Mariani, Massimo; Telford, John L.; Grandi, Guido; Soriani, Marco

    2008-01-01

    Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade ?-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of ?-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of ?-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections. PMID:19023424

  7. Water and a protic ionic liquid acted as refolding additives for chemically denatured enzymes.

    PubMed

    Attri, Pankaj; Venkatesu, P; Kumar, Anil

    2012-10-01

    In this communication, we present the ability of water and a protic ionic liquid, triethyl ammonium phosphate (TEAP) to act as refolding additives for the urea-induced chemical denaturated state of the two enzymes, ?-chymotrypsin and succinylated Con A. We show that the enzymatic activity is regained and in certain circumstances enhanced. PMID:22814381

  8. Enzyme

    MedlinePLUS

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  9. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule

    PubMed Central

    Hu, Ming Chang; Shi, Mingjun; Zhang, Jianning; Pastor, Johanne; Nakatani, Teruyo; Lanske, Beate; Razzaque, M. Shawkat; Rosenblatt, Kevin P.; Baum, Michel G.; Kuro-o, Makoto; Moe, Orson W.

    2010-01-01

    Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho’s known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant ?-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by ?-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.—Hu, M. C., Shi, M., Zhang, J., Pastor, J., Nakatani, T., Lanske, B., Shawkat Razzaque, M., Rosenblatt, K. P., Baum, M. G., Kuro-o, M., Moe, O. W. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. PMID:20466874

  10. A Natural Vanishing Act: The Enzyme-Catalyzed Degradation of Carbon Nanomaterials

    PubMed Central

    Kotchey, Gregg P.; Hasan, Saad A.; Kapralov, Alexander A.; Ha, Seung Han; Kim, Kang; Shvedova, Anna A.; Kagan, Valerian E.; Star, Alexander

    2012-01-01

    CONSPECTUS Over the past three decades, revolutionary research in nanotechnology by the scientific, medical, and engineering communities has yielded a treasure trove of discoveries with diverse applications that promise to benefit humanity. With their unique electronic and mechanical properties, carbon nanomaterials (CNMs) represent a prime example of the promise of nanotechnology with applications in areas that include electronics, fuel cells, composites, and nanomedicine. Because of toxicological issues associated with CNMs, however, their full commercial potential may not be achieved. The ex vitro, in vitro, and in vivo data presented in this Account provide fundamental insights into the biopersistence of CNMs, such as carbon nanotubes and graphene, and their oxidation/biodegradation processes as catalyzed by peroxidase enzymes. We also communicate our current understanding of the mechanism for the enzymatic oxidation/biodegradation. Finally, we outline potential future directions that could enhance our mechanistic understanding of the CNM oxidation/biodegradation and could yield benefits in terms of human health and environmental safety. The conclusions presented in this Account may catalyze a rational rethinking of CNM incorporation in diverse applications. For example, armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation/biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. In nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. On the other hand, in the construction of aircraft, a CNM composite material should be stable to oxidizing conditions in the environment. Therefore, pristine, inert CNMs would be ideal for this application. Finally, the incorporation of CNMs with defect sites in consumer goods could provide a facile mechanism that promotes the degradation of these materials once these products reach landfills. PMID:22824066

  11. The catecholamine biosynthetic enzyme dopamine ?-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter

    PubMed Central

    Mustapic, Maja; Maihofer, Adam X.; Mahata, Manjula; Chen, Yuqing; Baker, Dewleen G.; O'Connor, Daniel T.; Nievergelt, Caroline M.

    2014-01-01

    Dopamine beta-hydroxylase (DBH) is the biosynthetic enzyme catalyzing formation of norepinephrine. Changes in DBH expression or activity have been implicated in the pathogenesis of cardiovascular and neuropsychiatric disorders. Genetic determination of DBH enzymatic activity and its secretion are only incompletely understood. We began with a genome-wide association search for loci contributing to DBH activity in human plasma. Initially, in a population sample of European ancestry, we identified the proximal DBH promoter as a region harboring three common trait-determining variants (top hit rs1611115, P = 7.2 × 10?51). We confirmed their effects on transcription and showed that the three variants each acted additively on gene expression. Results were replicated in a population sample of Native American descent (top hit rs1611115, P = 4.1 × 10?15). Jointly, DBH variants accounted for 57% of DBH trait variation. We further identified a genome-wide significant SNP at the LOC338797 locus on chromosome 12 as trans-quantitative trait locus (QTL) (rs4255618, P = 4.62 × 10?8). Conditional analyses on DBH identified a third genomic region contributing to DBH variation: a likely cis-QTL adjacent to DBH in SARDH (rs7040170, P = 1.31 × 10?14) on chromosome 9q. We conclude that three common SNPs in the DBH promoter act additively to control phenotypic variation in DBH levels, and that two additional novel loci (SARDH and LOC338797) may also contribute to the expression of this catecholamine biosynthetic trait. Identification of DBH variants with strong effects makes it possible to take advantage of Mendelian randomization approaches to test causal effects of this intermediate trait on disease. PMID:24986918

  12. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  13. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A. (Los Alamos, NM); Wilson, Mahlon S. (Los Alamos, NM); Rishpon, Judith (Ramat-Aviv, IL); Gottesfeld, Shimshon (Los Alamos, NM)

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  14. Identification of cis-Acting Elements Important for Expression of the Starch-Branching Enzyme I Gene in Maize Endosperm1

    PubMed Central

    Kim, Kyung-Nam; Guiltinan, Mark J.

    1999-01-01

    The genes encoding the starch-branching enzymes (SBE) SBEI, SBEIIa, and SBEIIb in maize (Zea mays) are differentially regulated in tissue specificity and during kernel development. To gain insight into the regulatory mechanisms controlling their expression, we analyzed the 5?-flanking sequences of Sbe1 using a transient gene expression system. Although the 2.2-kb 5?-flanking sequence between ?2,190 and +27 relative to the transcription initiation site was sufficient to promote transcription, the addition of the transcribed region between +28 and +228 containing the first exon and intron resulted in high-level expression in suspension-cultured maize endosperm cells. A series of 5? deletion and linker-substitution mutants identified two critical positive cis elements, ?314 to ?295 and ?284 to ?255. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from maize kernels interact with the 60-bp fragment containing these two elements. Expression of the Sbe1 gene is regulated by sugar concentration in suspension-cultured maize endosperm cells, and the region ?314 to ?145 is essential for this effect. Interestingly, the expression of mEmBP-1, a bZIP transcription activator, in suspension-cultured maize endosperm cells resulted in a 5-fold decrease in Sbe1 promoter activity, suggesting a possible regulatory role of the G-box present in the Sbe1 promoter from ?227 to ?220. PMID:10482678

  15. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  16. Pancreatic Enzymes

    MedlinePLUS

    ... adequate nutrition and prevent weight loss. MCT (Medium Chain Triglyceride) oil may help control weight loss in ... enzyme products are regulated by the United States Food and Drug Administration (FDA) to ensure their effectiveness, ...

  17. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  18. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  19. Extracellular Enzymes Lab Biochemistry

    E-print Network

    Vallino, Joseph J.

    of the enzyme · The 3D enzyme structure and catalytic activity can be lost by exposing the enzyme to high temperatures, salinity, pH, and other extremes. These extremes "denature" the enzyme. · Many enzymes have) enzymes are affected by other compounds (modulators) that can either inhibit or activate an enzyme

  20. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  1. Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

    SciTech Connect

    Maltz, Lauren

    2015-08-25

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D ?-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.

  2. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  3. Enzyme responsive acetaminophen hydrogels

    E-print Network

    Vemula, Praveen

    Utilization of enzyme catalysis as a tool to disassemble self-assembled hydrogels to control the release encapsulated drug provides an opportunity to design a wide range of enzyme-specific low-molecular-weight hydrogelators ...

  4. Self-powered enzyme micropumps

    NASA Astrophysics Data System (ADS)

    Sengupta, Samudra; Patra, Debabrata; Ortiz-Rivera, Isamar; Agrawal, Arjun; Shklyaev, Sergey; Dey, Krishna K.; Córdova-Figueroa, Ubaldo; Mallouk, Thomas E.; Sen, Ayusman

    2014-05-01

    Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

  5. One Enzyme, Two Functions

    PubMed Central

    Altenhöfer, Sebastian; Witte, Ines; Teiber, John F.; Wilgenbus, Petra; Pautz, Andrea; Li, Huige; Daiber, Andreas; Witan, Heidrun; Clement, Albrecht M.; Förstermann, Ulrich; Horke, Sven

    2010-01-01

    The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O2?? dismutase activities and cytochrome c expression unaltered, and it did not oxidize O2?? but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His114 and His133 are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys311, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer. PMID:20530481

  6. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  7. Rational enzyme redesign

    SciTech Connect

    Ornstein, R.L.

    1994-05-01

    Protein engineering is first a means of elucidating structure-function relations in an enzyme, and second, a means of changing a protein to make it serve a different, but generally related, purpose. In principle, one may change the functional characteristics of an enzyme by altering its substrate specificity, kinetics, optimum range of activity, and chemical mechanism. Obviously one cannot make all possible combinations of amino acid changes for even the smallest enzyme, so the essential question is which changes to make. The intent of rational protein/enzyme redesign is to alter a protein/enzyme in a timely and premeditated fashion. This article provides an outline of the process of rational enzyme redesign.

  8. REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY

    E-print Network

    REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY ACT REGULATORY FLEXBILITY REGULATORY

  9. Nanostructures for enzyme stabilization

    SciTech Connect

    Kim, Jungbae; Grate, Jay W.; Wang, Ping

    2006-02-02

    The last decade has witnessed notable breakthroughs in nanotechnology with development of various nanostructured materials such as mesoporous materials and nanoparticles. These nanostructures have been used as a host for enzyme immobilization via various approaches, such as enzyme adsorption, covalent attachment, enzyme encapsulation, and sophisticated combinations of methods. This review discusses the stabilization mechanisms behind these diverse approaches; such as confinement, pore size and volume, charge interaction, hydrophobic interaction, and multipoint attachment. In addition, we will introduce recent rigorous approaches to improve the enzyme stability in these nanostructures or develop new nanostructures for the enzyme stabilization. Especially, we will introduce our recent invention of a nanostructure, called single enzyme nanoparticles (SENs). In the form of SENs, each enzyme molecule is surrounded with a nanometer scale network, resulting in stabilization of enzyme activity without any serious limitation for the substrate transfer from solution to the active site. SENs can be further immobilized into mesoporous silica with a large surface area, providing a hierarchical approach for stable, immobilized enzyme systems for various applications, such as bioconversion, bioremediation, and biosensors.

  10. Food and feed enzymes.

    PubMed

    Fraatz, Marco Alexander; Rühl, Martin; Zorn, Holger

    2014-01-01

    Humans have benefited from the unique catalytic properties of enzymes, in particular for food production, for thousands of years. Prominent examples include the production of fermented alcoholic beverages, such as beer and wine, as well as bakery and dairy products. The chapter reviews the historic background of the development of modern enzyme technology and provides an overview of the industrial food and feed enzymes currently available on the world market. The chapter highlights enzyme applications for the improvement of resource efficiency, the biopreservation of food, and the treatment of food intolerances. Further topics address the improvement of food safety and food quality. PMID:23873095

  11. Enzymes on material surfaces.

    PubMed

    Talbert, Joey N; Goddard, Julie M

    2012-05-01

    Enzyme interactions with material surfaces are of interest for industrial food and pharmaceutical transformations, biosensors, artificial cells, cell free reactions, drug and nutrition delivery technologies, and imaging. When in contact with a material surface, an enzyme may lose or appear to lose activity due to the nature of the enzyme, the nature of the material, and/or the nature of the interface between the enzyme, material, and substrate environment. The purpose of this review is to survey recent advances that have been made towards the preservation, optimization, and enhancement of enzyme activity on material surfaces within the context of well-known concepts that describe the loss of activity after immobilization. This review breaks down the immobilized enzyme system to look at the individual components of the system-namely the enzyme, the material, and the interface. For each piece, possible causes for the loss of enzyme activity are described as well as strategies that have been applied to limit the affect. At the conclusion we identify areas of future research needed to overcome limitations in the current state-of-the art for immobilized enzyme systems. PMID:22269888

  12. Extracting enzyme processivity from kinetic assays.

    PubMed

    Barel, Itay; Reich, Norbert O; Brown, Frank L H

    2015-12-14

    A steady-state analysis for the catalytic turnover of molecules containing two substrate sites is presented. A broad class of Markovian dynamic models, motivated by the action of DNA modifying enzymes and the rich variety of translocation mechanisms associated with these systems (e.g., sliding, hopping, intersegmental transfer, etc.), is considered. The modeling suggests an elementary and general method of data analysis, which enables the extraction of the enzyme's processivity directly and unambiguously from experimental data. This analysis is not limited to the initial velocity regime. The predictions are validated both against detailed numerical models and by revisiting published experimental data for EcoRI endonuclease acting on DNA. PMID:26671366

  13. Analysis of Hydrogen Tunneling in an Enzyme Active Site Using von Neumann Measurements

    E-print Network

    Iyengar, Srinivasan S.

    Analysis of Hydrogen Tunneling in an Enzyme Active Site Using von Neumann Measurements Isaiah treat the enzyme active site as a measurement device which acts on the tunneling hydrogen nucleus via earlier quantum wavepacket study of hydrogen transfer in the biological enzyme soybean lipoxygenase-1

  14. Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation of Crystalline Cellulose Fibers

    E-print Network

    Dutcher, John

    Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation types of T. reesei cellulolytic enzymes TrCel6A, TrCel7A, and TrCel7Band their mixtures. TrCel6A and Tr. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant

  15. A Hands-On Classroom Simulation to Demonstrate Concepts in Enzyme Kinetics

    ERIC Educational Resources Information Center

    Junker, Matthew

    2010-01-01

    A classroom exercise is described to introduce enzyme kinetics in an undergraduate biochemistry or chemistry course. The exercise is a simulation in which a student acts as an enzyme that "catalyzes" the unscrewing of a nut from a bolt. With other students assisting, the student enzyme carries out reactions with bolt-nut substrates under different…

  16. Commercial production of microbial enzymes

    SciTech Connect

    Munro, I.G.

    1985-01-01

    The advantages and uses of industrially produced microbial enzymes are described. The processes involved in the production of these enzymes, cultivation techniques, enzyme extraction, enzyme purification and immobilization are outlined. Both the history of enzyme technology and its future development are discussed.

  17. Basic models for differential inhibition of enzymes.

    PubMed

    Cappiello, Mario; Moschini, Roberta; Balestri, Francesco; Mura, Umberto; Del-Corso, Antonella

    2014-03-14

    The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as "intra-site differential inhibition" and the molecules able to exert this action as "differential inhibitors". This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate. PMID:24530393

  18. Balancing Acts

    MedlinePLUS

    ... Current Issue Past Issues Special Section: Focus on Communication Balancing Acts Past Issues / Fall 2008 Table of ... from the National Institute on Deafness and Other Communication Disorders (NIDCD). It involves simulated trips down the ...

  19. Acting Atoms.

    ERIC Educational Resources Information Center

    Farin, Susan Archie

    1997-01-01

    Describes a fun game in which students act as electrons, protons, and neutrons. This activity is designed to help students develop a concrete understanding of the abstract concept of atomic structure. (DKM)

  20. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  1. Overproduction of ligninolytic enzymes

    SciTech Connect

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  2. Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The four mucosal alpha-glucosidases, which differ in their digestive roles, generate glucose from glycemic carbohydrates and accordingly can be viewed as a control point for rate of glucose delivery to the body. In this study, individual recombinant enzymes were used to understand how alpha-glucan o...

  3. Random-walk enzymes

    PubMed Central

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-01-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C ? U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics. PMID:26465508

  4. Lignin-degrading enzymes.

    PubMed

    Pollegioni, Loredano; Tonin, Fabio; Rosini, Elena

    2015-04-01

    A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by ?-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies. PMID:25649492

  5. Random-walk enzymes

    NASA Astrophysics Data System (ADS)

    Mak, Chi H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

    2015-09-01

    Enzymes that rely on random walk to search for substrate targets in a heterogeneously dispersed medium can leave behind complex spatial profiles of their catalyzed conversions. The catalytic signatures of these random-walk enzymes are the result of two coupled stochastic processes: scanning and catalysis. Here we develop analytical models to understand the conversion profiles produced by these enzymes, comparing an intrusive model, in which scanning and catalysis are tightly coupled, against a loosely coupled passive model. Diagrammatic theory and path-integral solutions of these models revealed clearly distinct predictions. Comparison to experimental data from catalyzed deaminations deposited on single-stranded DNA by the enzyme activation-induced deoxycytidine deaminase (AID) demonstrates that catalysis and diffusion are strongly intertwined, where the chemical conversions give rise to new stochastic trajectories that were absent if the substrate DNA was homogeneous. The C ?U deamination profiles in both analytical predictions and experiments exhibit a strong contextual dependence, where the conversion rate of each target site is strongly contingent on the identities of other surrounding targets, with the intrusive model showing an excellent fit to the data. These methods can be applied to deduce sequence-dependent catalytic signatures of other DNA modification enzymes, with potential applications to cancer, gene regulation, and epigenetics.

  6. Aminoglycoside Modifying Enzymes

    PubMed Central

    Ramirez, Maria S.; Tolmasky, Marcelo E.

    2010-01-01

    Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different ?OH or ?NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltranferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes. PMID:20833577

  7. The Moderately Efficient Enzyme: Futile Encounters and Enzyme Floppiness.

    PubMed

    Bar-Even, Arren; Milo, Ron; Noor, Elad; Tawfik, Dan S

    2015-08-18

    The pioneering model of Henri, Michaelis, and Menten was based on the fast equilibrium assumption: the substrate binds its enzyme reversibly, and substrate dissociation is much faster than product formation. Here, we examine this assumption from a somewhat different point of view, asking what fraction of enzyme-substrate complexes are futile, i.e., result in dissociation rather than product formation. In Knowles' notion of a "perfect" enzyme, all encounters of the enzyme with its substrate result in conversion to product. Thus, the perfect enzyme's catalytic efficiency, kcat/KM, is constrained by only the diffusion on-rate, and the fraction of futile encounters (defined as ?) approaches zero. The available data on >1000 different enzymes suggest that for ?90% of enzymes ? > 0.99 and for the "average enzyme" ? ? 0.9999; namely, <1 of 10(4) encounters is productive. Thus, the "fast equilibrium" assumption holds for the vast majority of enzymes. We discuss possible molecular origins for the dominance of futile encounters, including the coexistence of multiple sub-states of an enzyme's active site (enzyme floppiness) and/or its substrate. Floppiness relates to the inherent flexibility of proteins, but also to conflicting demands, or trade-offs, between rate acceleration (the rate-determining chemical step) and catalytic turnover, or between turnover rate and accuracy. The study of futile encounters and active-site floppiness may contribute to a better understanding of enzyme catalysis, enzyme evolution, and improved enzyme design. PMID:26219075

  8. ENZYMES IN ONTOGENESIS (ORTHOPTERA)

    PubMed Central

    Bodine, Joseph Hall; Carlson, Loren D.

    1941-01-01

    1. Proteins, when added to activators (sodium oleate. Aerosol) of protyrosinase, greatly decrease the degree of activation. 2. Certain proteins adsorbed on activator micelles are markedly affected by temperature and are rendered more sensitive by ultraviolet light. 3. Ideas are expressed as to the possible nature of activating and inhibiting phenomena especially as they relate to the enzyme tyrosinase. PMID:19873225

  9. The Enzyme Function Initiative†

    PubMed Central

    Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

    2011-01-01

    The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID:21999478

  10. Affordable Care Act (Medicaid)

    MedlinePLUS

    ... Affordable Care Act Affordable Care Act Provisions Affordable Care Act See Affordable Care Act Federal Policy Guidance. ... related policy guidance can be found below. AFFORDABLE CARE ACT PROVISIONS DESCRIPTION Eligibility Fills in current gaps ...

  11. Enzyme synthesis in the regulation of hepatic "malic" enzyme activity.

    PubMed

    Murphy, G; Walker, D G

    1974-10-01

    A homogeneous preparation of ;malic' enzyme (EC 1.1.1.40) from livers of thyroxine-treated rats was used to prepare in rabbits an antiserum to the enzyme that reacts monospecifically with the ;malic' enzyme in livers of rats in several physiological states. Changes in enzyme activity resulting from modification of the state of the animal are hence due to an altered amount of enzyme protein. The antiserum has been used to precipitate out ;malic' enzyme from heat-treated supernatant preparations of livers from both adult and neonatal rats, in a number of physiological conditions, that had been injected 30min earlier with l-[4,5-(3)H]leucine. The low incorporations of radioactivity into the immunoprecipitable enzyme have permitted the qualitative conclusion that changed enzyme activity in adult rats arises mainly from alterations in the rate of enzyme synthesis. The marked increase in ;malic' enzyme activity that occurs naturally or as a result of thyroxine treatment of the weanling rat is likewise due to a marked increase in the rate of enzyme synthesis possibly associated with a concurrent diminished rate of enzyme degradation. PMID:4462568

  12. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  13. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R. (Columbus, OH); Bednarski, Mark D. (Berkeley, CA); Gruber, Patrick R. (St. Paul, MN)

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  14. Enzyme Molar Fractions: A Powerful Tool for Understanding Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Serra, Juan L.; And Others

    1986-01-01

    Deduces the relationship between reduced velocity and molar fractions for productive enzyme complexes; obtains the mathematical expression of molar fractions for an enzyme with two specific binding sites per molecule; and proposes a useful plot to follow the dependence of enzyme molar fractions with the concentration of one of its ligands. (JN)

  15. Quorum quenching enzymes.

    PubMed

    Fetzner, Susanne

    2015-05-10

    Bacteria use cell-to-cell communication systems based on chemical signal molecules to coordinate their behavior within the population. These quorum sensing systems are potential targets for antivirulence therapies, because many bacterial pathogens control the expression of virulence factors via quorum sensing networks. Since biofilm maturation is also usually influenced by quorum sensing, quenching these systems may contribute to combat biofouling. One possibility to interfere with quorum sensing is signal inactivation by enzymatic degradation or modification. Such quorum quenching enzymes are wide-spread in the bacterial world and have also been found in eukaryotes. Lactonases and acylases that hydrolyze N-acyl homoserine lactone (AHL) signaling molecules have been investigated most intensively, however, different oxidoreductases active toward AHLs or 2-alkyl-4(1H)-quinolone signals as well as other signal-converting enzymes have been described. Several approaches have been assessed which aim at alleviating virulence, or biofilm formation, by reducing the signal concentration in the bacterial environment. These involve the application or stimulation of signal-degrading bacteria as biocontrol agents in the protection of crop plants against soft-rot disease, the use of signal-degrading bacteria as probiotics in aquaculture, and the immobilization or entrapment of quorum quenching enzymes or bacteria to control biofouling in membrane bioreactors. While most approaches to use quorum quenching as antivirulence strategy are still in the research phase, the growing number of organisms and enzymes known to interfere with quorum sensing opens up new perspectives for the development of innovative antibacterial strategies. PMID:25220028

  16. Engineering Cellulase Enzymes for Bioenergy

    E-print Network

    Atreya, Meera Elizabeth

    2015-01-01

    measuring enzyme specific activities, pH and temperatureenzyme to understand its temperature optimum and stability range, pH optimum, activityActivity assay at 70?C is consistent with a ?-glucosidase enzyme that is inactive at this temperature.

  17. Treating Wastewater With Immobilized Enzymes

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.

    1991-01-01

    Experiments show enzymes are immobilized on supporting materials to make biocatalyst beds for treatment of wastewater. With suitable combination of enzymes, concentrations of various inorganic and organic contaminants, including ammonia and urea, reduced significantly.

  18. The Catalytic Function of Enzymes.

    ERIC Educational Resources Information Center

    Splittgerber, Allan G.

    1985-01-01

    Discusses: structure of the enzyme molecule; active site; reaction mechanism; transition state; factors affecting enzyme reaction rates, concentration of enzyme; concentration of substrate; product concentration; temperature effects and pH effects; factors causing a lowering of activation energy; proximity and orientation effects; substrate strain…

  19. Enzyme-Coated Carbon Nanotubes as Single-Molecule Biosensors

    E-print Network

    Dekker, Cees

    chemical sensor for the detection of NO2 and NH3 gas.4 Semiconducting SWNTs [or semiconducting silicon) on degenerately doped silicon wafers with a 200 nm thermally grown oxide. Electrodes consisting of a 30 nm gold. The enzyme-coated tube is found to act as a pH sensor with large and reversible changes in conductance upon

  20. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  1. Act resilient.

    PubMed

    Joseph, Genie; Bice-Stephens, Wynona

    2014-01-01

    Attendees have reported changing from being fearful to serene, from listless to energized, from disengaged to connected, and becoming markedly less anxious in a few weeks. Anecdotally, self-reported stress levels have been reduced by over 50% after just one class. Attendees learn not to be afraid of their feelings by working with emotions in a playful manner. When a person can act angry, but separate himself from his personal story, the emotional energy exists in a separate form that is not attached to specific events, and can be more easily dealt with and neutralized. Attendees are taught to "take out the emotional trash" through expressive comedy. They become less intimated by their own emotional intensity and triggers as they learn how even metaphorical buckets of anger, shame, guilt and hurt can be emotionally emptied. The added benefit is that this is accomplished without the disclosure of personal information of the requirement to reexperience past pain which can trigger its own cascade of stress. PMID:24706248

  2. Enzymes, embryos, and ancestors.

    PubMed

    Gerhart, John

    2010-01-01

    In the 1950s, cellular regulatory mechanisms were newly recognized; with Arthur Pardee I investigated the initial enzyme of pyrimidine biosynthesis, which he discovered is controlled by feedback inhibition. The protein proved unusual in having separate but interacting sites for substrates and regulators. Howard Schachman and I dissociated the protein into different subunits, one binding regulators and one substrates. The enzyme became an early prime example of allostery. In developmental biology I studied the egg of the frog, Xenopus laevis, characterizing early processes of axis formation. My excellent students and I described cortical rotation, a 30° movement of the egg's cortex over tracks of parallel microtubules anchored to the underlying cytoplasmic core, and we perturbed it to alter Spemann's organizer and effect spectacular phenotypes. The entire sequence of events has been elucidated by others at the molecular level, making Xenopus a prime example of vertebrate axis formation. Marc Kirschner, Christopher Lowe, and I then compared hemichordate (half-chordate) and chordate early development. Despite anatomical-physiological differences, these groups share numerous steps of axis formation, ones that were probably already in use in their pre-Cambrian ancestor. I've thoroughly enjoyed exploring these areas during a 50-year period of great advances in biological sciences by the worldwide research community. PMID:20929311

  3. Microbial Enzymes with Special Characteristics for Biotechnological Applications

    PubMed Central

    Nigam, Poonam Singh

    2013-01-01

    This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations. PMID:24970183

  4. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...2014-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture ...58.436 Rennet, pepsin, other milk clotting enzymes and flavor enzymes. Enzyme preparations used...

  5. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...2013-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture ...58.436 Rennet, pepsin, other milk clotting enzymes and flavor enzymes. Enzyme preparations used...

  6. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture ...58.436 Rennet, pepsin, other milk clotting enzymes and flavor enzymes. Enzyme preparations used...

  7. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...2012-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture ...58.436 Rennet, pepsin, other milk clotting enzymes and flavor enzymes. Enzyme preparations used...

  8. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...2011-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture ...58.436 Rennet, pepsin, other milk clotting enzymes and flavor enzymes. Enzyme preparations used...

  9. DGAT enzymes and triacylglycerol biosynthesis

    PubMed Central

    Yen, Chi-Liang Eric; Stone, Scot J.; Koliwad, Suneil; Harris, Charles; Farese, Robert V.

    2008-01-01

    Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases. PMID:18757836

  10. Immobilized Cell and Enzyme Technology

    NASA Astrophysics Data System (ADS)

    Dunnill, P.

    1980-08-01

    The development of immobilized enzyme and cell technology is summarized. Industrial processes for sucrose inversion, penicillin deacylation and glucose isomerization using immobilized enzymes are described. An alternative process for glucose isomerization using immobilized cells, and some other industrial applications of immobilized cells are indicated. Recent developments in immobilized enzyme and cell technology are assessed and the relative merits of the different biochemical catalyst forms are considered.

  11. Making the Rate: Enzyme Dynamics

    ERIC Educational Resources Information Center

    Ragsdale, Frances R.

    2004-01-01

    An enzyme exercise to address the problem of students inability to visualize chemical reaction at the molecular level is described. This exercise is designed as a dry lab exercise but can be modified into a classroom activity then can be augmented by a wet lab procedure, thereby providing students with a practical exposure to enzyme function.

  12. An investigation into keratinolytic enzymes to enhance ungual drug delivery.

    PubMed

    Mohorcic, M; Torkar, A; Friedrich, J; Kristl, J; Murdan, S

    2007-03-01

    The topical therapy of nail diseases is limited by the low permeability of drugs through the nail plate. To increase drug penetration, the integrity of the nail plate must be compromised to a certain extent. We hypothesised that keratinolytic enzymes might decrease the barrier properties of the nail plate by hydrolysing the nail keratins, and thereby enhance ungual drug permeation. To determine enzyme action on nail plates, nail clippings were incubated at 35 degrees C, in the presence of keratinase at optimal pH for 48h, after which the nail plates were examined using scanning electron microscopy. It was found that the enzyme acted on the intercellular matrix which holds nail cells together, such that corneocytes on the dorsal surface separated from one another and 'lifted off' the nail plate. In addition, the surface of the corneocytes was corroded. Permeation studies using modified Franz diffusion cells and bovine hoof membranes as a model for the nail plate showed that the enzyme enhanced drug permeation through the hoof membrane. The permeability and partition coefficients, and the drug flux were found to be significantly increased in the presence of the enzyme. We can conclude that the enzyme, via its hydrolytic action on nail plate proteins, could increase ungual drug delivery. PMID:17097244

  13. Integrated microdroplet-based system for enzyme synthesis and sampling

    NASA Astrophysics Data System (ADS)

    Lapierre, Florian; Best, Michel; Stewart, Robert; Oakeshott, John; Peat, Thomas; Zhu, Yonggang

    2013-12-01

    Microdroplet-based microfluidic devices are emerging as powerful tools for a wide range of biochemical screenings and analyses. Monodispersed aqueous microdroplets from picoliters to nanoliters in volume are generated inside microfluidic channels within an immiscible oil phase. This results in the formation of emulsions which can contain various reagents for chemical reactions and can be considered as discrete bioreactors. In this paper an integrated microfluidic platform for the synthesis, screening and sorting of libraries of an organophosphate degrading enzyme is presented. The variants of the selected enzyme are synthesized from a DNA source using in-vitro transcription and translation method. The synthesis occurs inside water-in-oil emulsion droplets, acting as bioreactors. Through a fluorescence based detection system, only the most efficient enzymes are selected. All the necessary steps from the enzyme synthesis to selection of the best genes (producing the highest enzyme activity) are thus integrated inside a single and unique device. In the second part of the paper, an innovative design of the microfluidic platform is presented, integrating an electronic prototyping board for ensuring the communication between the various components of the platform (camera, syringe pumps and high voltage power supply), resulting in a future handheld, user-friendly, fully automated device for enzyme synthesis, screening and selection. An overview on the capabilities as well as future perspectives of this new microfluidic platform is provided.

  14. ENZYMES IN ONTOGENESIS (ORTHOPTERA)

    PubMed Central

    Allen, Thomas Hunter; Bodine, Joseph Hall

    1940-01-01

    1. Protyrosinase from the egg of the grasshopper, Melanoplus differentialis, can be activated by excess sodium oleate or Aerosol. 2. The 3:4 quinone products of the reaction of activated protyrosinase with tyramine or tyrosine will oxidize ascorbic acid to dehydroascorbic acid. 3. The velocity of this latter oxidation of ascorbic acid increases with the amount of tyramine or tyrosine. 4. The oxidation of ascorbic acid by the tyramine-tyrosinase reaction delays the time of appearance of a red color associated with an indole quinone intermediary product in the formation of melanin. 5. Protyrosinase, in itself, and in the presence of tyrosinase substrates does not bring about the oxidation of ascorbic acid. 6. A naturally occurring substrate in a preparation of protyrosinase, sufficient to cause the oxidation of ascorbic acid, can be removed by dialysis against a 0.9 per cent sodium chloride solution. 7. Dialysis against such a solution does not change the properties of protyrosinase; the inactive enzyme must still be activated before it will catalyze the oxidation of tyramine or tyrosine. 8. When the natural substrate, tyrosine, or tyramine is absent, activation of protyrosinase does not result in the oxidation of ascorbic acid. PMID:19873203

  15. Deubiquitinating enzymes as oncotargets

    PubMed Central

    McClurg, Urszula L.; Robson, Craig N.

    2015-01-01

    Carcinogenesis is a complex process tightly regulated at multiple levels by post-translational modifications. Epigenetics plays a major role in cancer development, all stable changes to the gene expression process that are not a result of a direct change in the DNA code are described as epigenetics. Epigenetic processes are regulated by post-translational modifications including ubiquitination which can directly affect either histones or transcription factors or may target their co-factors and interacting partners exerting an indirect effect. Deubiquitination of these target proteins is equally important and alterations in this pathway can also lead to cancer development, progression and metastasis. Only the correct, unaltered balance between ubiquitination and deubiquitination ensures healthy cellular homeostasis. In this review we focus on the role of deubiquitinating (DUB) enzymes in various aspects of epigenetics including the regulation of transcription factors, histone modifications, DNA damage repair pathways and cell cycle regulation. We discuss the impact of those processes on tumourigenesis and potential therapeutic applications of DUBs for cancer treatment. PMID:25962961

  16. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  17. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S. (Bellport, NY); MacGregor, Robert R. (Sag Harbor, NY); Wolf, Alfred P. (Setauket, NY); Langstrom, Bengt (Upsala, SE)

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  18. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  19. Enzyme activity determination using ultrasound

    NASA Astrophysics Data System (ADS)

    Holmes, M. J.; Southworth, T.; Watson, N. G.; Povey, M. J. W.

    2014-04-01

    Here are presented the results of a novel approach to the measurement of enzyme reaction rates in which ultrasound velocity measurement is used. Our results show enzyme activity is observable, in the acoustic context, and that furthermore this offers the potential to estimate the rate of reaction over different substrate concentrations and temperatures. Findings are corroborated with optical microscopy and rheological measurements. Ultrasound velocity measurement can be performed without the need for aliquot extraction and offers an efficient, non-invasive and dynamic method to monitor enzyme activity.

  20. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864...Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are...

  1. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 2014-04-01 2014-04-01 false Enzyme preparations. 864.4400 Section 864...Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are...

  2. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 2011-04-01 2011-04-01 false Enzyme preparations. 864.4400 Section 864...Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are...

  3. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 2012-04-01 2012-04-01 false Enzyme preparations. 864.4400 Section 864...Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are...

  4. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 2013-04-01 2013-04-01 false Enzyme preparations. 864.4400 Section 864...Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are...

  5. Enzyme immobilisation in permselective microcapsules.

    PubMed

    Pachariyanon, Pavadee; Barth, Ekkehard; Agar, David W

    2011-01-01

    The objective of this investigation was to study the permselective behaviour of calcium alginate membranes, including the modifying effects of silica additives, which were subsequently used as microcapsule shells. Diffusion experiments and HPLC were carried out to ascertain the size-exclusion property of the membranes for a mixed molecular-weight dextran solution. Hollow microcapsules containing the enzyme dextranase were prepared using double concentric nozzles and the encapsulation performance was evaluated based on an analysis of the enzyme reactivity and stability. To improve mass transport within the microcapsules, magnetic nanoparticles were introduced into the liquid core and agitated using an alternating external magnetic field. The modified membranes exhibited better size-exclusion behaviour than the unmodified membranes. The magnetic nanoparticles slightly improved mass transport inside the microcapsule. The encapsulated enzyme yielded nearly 80% of the free enzyme activity and retained about 80% of the initial catalytic activity even after being used for eight reaction cycles. PMID:21736522

  6. Molybdenum enzymes in higher organisms

    PubMed Central

    Hille, Russ; Nishino, Takeshi; Bittner, Florian

    2010-01-01

    Recent progress in our understanding of the structural and catalytic properties of molybdenum-containing enzymes in eukaryotes is reviewed, along with aspects of the biosynthesis of the cofactor and its insertion into apoprotein. PMID:21516203

  7. Immobilized enzymes affect biofilm formation.

    PubMed

    Cordeiro, Ana L; Hippius, Catharina; Werner, Carsten

    2011-09-01

    The effect of the activity of immobilized enzymes on the initial attachment of pathogenic bacteria commonly associated with nosocomial infections (Pseudomonas aeruginosa and Staphylococcus epidermidis) was investigated. The proteolytic enzymes, subtilisin A and the glycoside hydrolase cellulose, were covalently attached onto poly(ethylene-alt-maleic) anhydride copolymer films. A comparison between active and heat-inactivated surfaces showed that while the activity of immobilized cellulase reduced the attachment of S. epidermidis by 67%, it had no effect on the attachment of P. aeruginosa. Immobilized subtilisin A had opposite effects: the active enzyme had no effect on the attachment of S. epidermidis but reduced the attachment of P. aeruginosa by 44%. The results suggest that different biomolecules are involved in the initial steps of attachment of different bacteria, and that the development of broad-spectrum antifouling enzymatic coatings will need to involve the co-immobilization of enzymes. PMID:21618024

  8. Heavy metals and enzyme induction.

    PubMed

    Stoytchev, T; Kadiiska, M

    1983-01-01

    In experiments on male albino rats it was established that single toxic doses of heavy metal salts inhibited to a different extent the enzyme-inducing action of phenobarbital and methylcholanthrene as determined by ethylmorphine-N-demethylase, respectively aniline hydroxylase activity. The majority of salts (of Cu, Co, Cd, Pb, Ni, Zn and Hg) inhibited more strongly the enzyme induction produced by methylcholanthrene as compared with that caused by phenobarbital. Subtoxic doses of Co, Cd, Zn and Ni salts given daily with the drinking water for 30 days shortened the hexobarbital sleeping time and increased the ethylmorphine-N-demethylase activity and cytochrome P-450 content in the liver microsomes. This suggests an enzyme-inducing action of these heavy metal salts at oral administration in subtoxic doses. Cu, Bi, Sn, Pb did not produce enzyme induction. The changes in the three indices of the As and Hg action were not consistent in our experiments. PMID:6624490

  9. Enzymes: principles and biotechnological applications.

    PubMed

    Robinson, Peter K

    2015-11-15

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  10. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    SciTech Connect

    Maltz, Lauren

    2015-08-27

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D ?- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes

  11. Taking the Mystery Out of Enzymes.

    ERIC Educational Resources Information Center

    DeYoung, H. Garrett

    1984-01-01

    Discusses structure and function of enzymes, design of new enzymes and enzyme substitutes, and enzyme uses in industry, medicine, and wastewater treatment. The latter is a low-cost method which can remove as much as 99 percent of toxic substances found in many industrial wastewater streams. (JN)

  12. Micromotors Powered by Enzyme Catalysis.

    PubMed

    Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

    2015-12-01

    Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture. PMID:26587897

  13. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  14. Enzyme-based listericidal nanocomposites.

    PubMed

    Solanki, Kusum; Grover, Navdeep; Downs, Patrick; Paskaleva, Elena E; Mehta, Krunal K; Lee, Lillian; Schadler, Linda S; Kane, Ravi S; Dordick, Jonathan S

    2013-01-01

    Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion. These Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) at challenges up to 10(5)?CFU/ml both in non-growth sustaining PBS as well as under growth conditions on lettuce. This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure. PMID:23545700

  15. Enzyme-Based Listericidal Nanocomposites

    PubMed Central

    Solanki, Kusum; Grover, Navdeep; Downs, Patrick; Paskaleva, Elena E.; Mehta, Krunal K.; Lee, Lillian; Schadler, Linda S.; Kane, Ravi S.; Dordick, Jonathan S.

    2013-01-01

    Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion. These Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) at challenges up to 105?CFU/ml both in non-growth sustaining PBS as well as under growth conditions on lettuce. This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure. PMID:23545700

  16. Recovery Act Milestones

    SciTech Connect

    Rogers, Matt

    2009-01-01

    Every 100 days, the Department of Energy is held accountable for a progress report on the American Recovery and Reinvestment Act. Update at 200 days, hosted by Matt Rogers, Senior Advisor to Secretary Steven Chu for Recovery Act Implementation.

  17. Recovery Act Milestones

    ScienceCinema

    Rogers, Matt

    2013-05-29

    Every 100 days, the Department of Energy is held accountable for a progress report on the American Recovery and Reinvestment Act. Update at 200 days, hosted by Matt Rogers, Senior Advisor to Secretary Steven Chu for Recovery Act Implementation.

  18. Public Health Act, 1961 

    E-print Network

    Her Majesty's Stationary Office

    1961-01-01

    An Act to amend the provisions of the Public Health Act, 1936, relating to building byelaws, to make such amendments of the law relating to public health and the functions of county councils and other local authorities as ...

  19. Characteristics necessary for an interconvertible enzyme cascade to generate a highly sensitive response to an effector.

    PubMed

    Cárdenas, M L; Cornish-Bowden, A

    1989-01-15

    A monocyclic interconvertible enzyme cascade, in which active and inactive states of an enzyme are interconverted by two opposing enzyme-catalysed reactions, does not necessarily produce a greater degree of sensitivity to an effector than one could expect from direct interaction between effector and target reaction. On the contrary, a cascade in which an effector acts on one of the enzymes catalysing the interconversion reactions by altering the apparent value of its specificity constant will always generate a less sensitive response than direct interaction would give. Nonetheless, even if both interconversion reactions obey Michaelis-Menten kinetics with the ordinary types of inhibition and activation, one can easily generate an enormous sensitivity in which a 0.5% change in concentration can increase the proportion of target enzyme in the active state from 10% to 90%: this corresponds approximately to a Hill coefficient of 800. To maximize the sensitivity, the following conditions must be satisfied: (1) both modifier enzymes must act under conditions of near saturation; (2) the effector must act on both of them in opposite directions; (3) it must alter the apparent values of their catalytic constants; (4) the enzyme subject to inhibition by the effector must respond at much lower effector concentrations than the enzyme subject to activation. As the last of these conditions appears to be counter-intuitive, it suggests that feeble activation of modifier enzymes in real systems may have passed unnoticed, or been dismissed as physiologically insignificant, although in reality crucial to the effective response of the system. PMID:2930453

  20. Lithuanian biochemist builds enzyme empire

    SciTech Connect

    Dickman, S.

    1992-09-11

    Vidas Janulaitis is professor of biochemistry at the University of Vilnius, head of the Institute of Applied Enzymology - and creator of one of the world's largest collections of restriction enzymes, with more than 100 on offer. He also appears to be the first successful biotechnology entrepreneur to emerge from the former Soviet Union. This paper shows how Janulaitis managed to rise above the chaos that has accompanied the dismantlement of the Soviet Union to become one of the world's top suppliers of new restriction enzymes - especially given that the venture capitalists who rushed off to make deals with Moscow labs in the early days of perestroika mostly came back disappointed.

  1. 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.

  2. Forgetting ACT UP

    ERIC Educational Resources Information Center

    Juhasz, Alexandra

    2012-01-01

    When ACT UP is remembered as the pinnacle of postmodern activism, other forms and forums of activism that were taking place during that time--practices that were linked, related, just modern, in dialogue or even opposition to ACT UP's "confrontational activism"--are forgotten. In its time, ACT UP was embedded in New York City, and a larger world,…

  3. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  4. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  5. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  6. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  7. 7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be...

  8. Reproducible EnzymeReproducible Enzyme Assembly and CatalyticAssembly and Catalytic

    E-print Network

    Rubloff, Gary W.

    Reproducible EnzymeReproducible Enzyme Assembly and CatalyticAssembly and Catalytic Activity Accomplishments #12;Reproducible Enzyme Assembly and CatalyticReproducible Enzyme Assembly and Catalytic Activity in Reusable BioMEMSActivity in Reusable BioMEMS Accomplishment Pro-tagged Pfs enzymes are spatially assembled

  9. Medicinal Chemistry and Enzyme Kinetics

    E-print Network

    Truhlar, Donald G

    of bioavailability and ADMETox in drug design See a separate research highlight on solvation for further medicinalMedicinal Chemistry and Enzyme Kinetics Elizabeth Amin and C. R. Wagner, Medicinal Chemistry Jiali and parameters for MM, QM/MM work Prof. Elizabeth A. Amin, Department of Medicinal Chemistry, College of Pharmacy

  10. Rennin--a Neglected Enzyme?

    ERIC Educational Resources Information Center

    Gill, John; Saunders, Terry

    1987-01-01

    Presents investigations to explore the substrate specificity, pH, concentration, and temperature relations of an enzyme with only inexpensive commercial rennet and basic laboratory equipment. Describes how the activities were carried out with a group of 15-year-old students. (CW)

  11. August 25, 2015 Enzyme detective

    E-print Network

    with producing the initial enzyme itself with the help of the bacterium Escherichia coli, or E. coli for short. Despite other strains of E. coli getting a bad rap for causing disease, the laboratory strains Bacik uses are quite benign. Bacik provides the E. coli with external genetic material and a "switch" that tells them

  12. Rapid-Equilibrium Enzyme Kinetics

    ERIC Educational Resources Information Center

    Alberty, Robert A.

    2008-01-01

    Rapid-equilibrium rate equations for enzyme-catalyzed reactions are especially useful because if experimental data can be fit by these simpler rate equations, the Michaelis constants can be interpreted as equilibrium constants. However, for some reactions it is necessary to use the more complicated steady-state rate equations. Thermodynamics is…

  13. Insolubilized enzymes for food synthesis

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1972-01-01

    Cellulose matrix with numerous enzyme-coated silica particles of colloidal size permanently bound at various sites within matrix was produced that has high activity and possesses requisite physical characteristics for filtration or column operations. Product also allows coupling step in synthesis of edible food to proceed under mild conditions.

  14. The enzymes associated with denitrification

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  15. ORGANOPHOSPHATE DEGRADING ENZYMES - PHASE I

    EPA Science Inventory

    Agave BioSystems in collaboration with Carl A. Batt proposes to develop decon-nanoparticles, which will leverage ongoing opportunities in enzyme engineering and the fabrication of functionalized magnetic nanoparticles. Enhanced performance will be engineered into the system t...

  16. Direct-acting mutagens in automobile exhaust.

    PubMed

    Wang, Y Y; Rappaport, S M; Sawyer, R F; Talcott, R E; Wei, E T

    1978-07-01

    Particulate matter in city air contains chemicals which are mutagenic in the Ames Salmonella typhimurium assay. In residential urban areas, the principal mutagens in air do not require liver enzymes to be activated. The source of these liver independent (direct-acting) mutagens may be automobile exhaust because (1) the mutagenic activities were correlated to the lead content or air, (2) the mutagens were found exhaust samples from automobiles and from an experimental CFR single-cylinder gasoline engine, and (3) these mutagens were not found in fuel or unused motor oil, but were found in used motor oil. The strain specificity and the fact that liver enzymes were not required for activation indicated that the exhaust and airborne mutagens were not unsubstituted polycyclic aromatic hydrocarbons (PAH), aromatic amines, alkylnitrosamines or aliphatic epoxides, peroxides and hydroepoxides. A number of nitro-substituted aromatic compounds are direct-acting mutagens in the Ames test, and it is possible that nitration of PAH in exhaust may form the compounds observed here. We synthesized 6-nitrobenzo [a] pyrene and found it to be a potent, direct-acting mutagen with activity comparable to that of benzo-[a] pyrene. PMID:80258

  17. EzCatDB: the enzyme reaction database, 2015 update

    PubMed Central

    Nagano, Nozomi; Nakayama, Naoko; Ikeda, Kazuyoshi; Fukuie, Masaru; Yokota, Kiyonobu; Doi, Takuo; Kato, Tsuyoshi; Tomii, Kentaro

    2015-01-01

    The EzCatDB database (http://ezcatdb.cbrc.jp/EzCatDB/) has emphasized manual classification of enzyme reactions from the viewpoints of enzyme active-site structures and their catalytic mechanisms based on literature information, amino acid sequences of enzymes (UniProtKB) and the corresponding tertiary structures from the Protein Data Bank (PDB). Reaction types such as hydrolysis, transfer, addition, elimination, isomerization, hydride transfer and electron transfer have been included in the reaction classification, RLCP. This database includes information related to ligand molecules on the enzyme structures in the PDB data, classified in terms of cofactors, substrates, products and intermediates, which are also necessary to elucidate the catalytic mechanisms. Recently, the database system was updated. The 3D structures of active sites for each PDB entry can be viewed using Jmol or Rasmol software. Moreover, sequence search systems of two types were developed for the EzCatDB database: EzCat-BLAST and EzCat-FORTE. EzCat-BLAST is suitable for quick searches, adopting the BLAST algorithm, whereas EzCat-FORTE is more suitable for detecting remote homologues, adopting the algorithm for FORTE protein structure prediction software. Another system, EzMetAct, is also available to searching for major active-site structures in EzCatDB, for which PDB-formatted queries can be searched. PMID:25324316

  18. Immobilization of enzymes masks their active site.

    PubMed

    Kumakura, M; Kaetsu, I

    1984-03-01

    We studied the effect of immobilizing cellulase to carboxycellulose sodium by radiation polymerization on the masking of the active site of the enzyme. Masking of the enzyme during the preparation of immobilized enzyme was assayed at low temperature. The activity of immobilized enzyme was retained during repeated batch reactions, indicating that the enzyme was firmly trapped in the polymer matrix. Various compounds (designated monomers) were used to dissolve the carboxymethylcellulose; enzyme activity was affected by the nature of the monomer, by the monomer concentration, and by the solubility of the substrate in monomer. PMID:6722287

  19. Metagenomics for the discovery of pollutant degrading enzymes.

    PubMed

    Ufarté, Lisa; Laville, Élisabeth; Duquesne, Sophie; Potocki-Veronese, Gabrielle

    2015-12-01

    Organic pollutants, including xenobiotics, are often persistent and toxic organic compounds resulting from human activities and released in large amounts into terrestrial, fluvial and marine environments. However, some microbial species which are naturally exposed to these compounds in their own habitat are capable of degrading a large range of pollutants, especially poly-aromatic, halogenated and polyester molecules. These microbes constitute a huge reservoir of enzymes for the diagnosis of pollution and for bioremediation. Most are found in highly complex ecosystems like soils, activated sludge, compost or polluted water, and more than 99% have never been cultured. Meta-omic approaches are thus well suited to retrieve biocatalysts from these environmental samples. In this review, we report the latest advances in functional metagenomics aimed at the discovery of enzymes capable of acting on different kinds of polluting molecules. PMID:26526541

  20. Enzyme-catalyzed protein crosslinking.

    PubMed

    Heck, Tobias; Faccio, Greta; Richter, Michael; Thöny-Meyer, Linda

    2013-01-01

    The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications. PMID:23179622

  1. Improvements of biomass deconstruction enzymes

    SciTech Connect

    Sale, K. L.

    2012-03-01

    Sandia National Laboratories and DSM Innovation, Inc. collaborated on the investigation of the structure and function of cellulases from thermophilic fungi. Sandia's role was to use its expertise in protein structure determination and X-ray crystallography to solve the structure of these enzymes in their native state and in their substrate and product bound states. Sandia was also tasked to work with DSM to use the newly solved structure to, using computational approaches, analyze enzyme interactions with both bound substrate and bound product; the goal being to develop approaches for rationally designing improved cellulases for biomass deconstruction. We solved the structures of five cellulases from thermophilic fungi. Several of these were also solved with bound substrate/product, which allowed us to predict mutations that might enhance activity and stability.

  2. NOX Enzymes and Pulmonary Disease

    PubMed Central

    Griffith, Brian; Pendyala, Srikanth; Hecker, Louise; Lee, Patty J.; Natarajan, Viswanathan

    2009-01-01

    Abstract The primary function of the lung is to facilitate the transfer of molecular oxygen (O2; dioxygen) from the atmosphere to the systemic circulation. In addition to its essential role in aerobic metabolism, O2 serves as the physiologic terminal acceptor of electron transfer catalyzed by the NADPH oxidase (NOX) family of oxidoreductases. The evolution of the lungs and circulatory systems in vertebrates was accompanied by increasing diversification of NOX family enzymes, suggesting adaptive roles for NOX-derived reactive oxygen species in normal physiology. However, this adaptation may paradoxically carry detrimental consequences in the setting of overwhelming/persistent environmental stressors, both infectious and noninfectious, and during the process of aging. Here, we review current understanding of NOX enzymes in normal lung physiology and their pathophysiologic roles in a number of pulmonary diseases, including lung infections, acute lung injury, pulmonary arterial hypertension, obstructive lung disorders, fibrotic lung disease, and lung cancer. Antioxid. Redox Signal. 11, 2505–2516. PMID:19331546

  3. Enzymes of respiratory iron oxidation

    SciTech Connect

    Blake, R. II.

    1991-01-01

    This report focuses on the progress made in three areas of research concerned with enzymes involved in respiratory iron oxidation. The three areas are as follows: development of an improved procedure for the routine large scale culture of iron oxidizing chemolithotrophs based on the in-situ electrolysis of the soluble iron in the growth medium; to perform iron oxidation kinetic studies on whole cells using the oxygen electrode; and to identify, separate, purify, and characterize the individual cellular components.

  4. Molecular Biology Basics Planning Restriction Enzyme Digests

    E-print Network

    Aris, John P.

    (see below), and digest with second enzyme. E. Inactivation: chelate Mg++ heat inactivation phenol in order: sterile dH2O, 10X buffer, BSA, plasmid DNA, enzyme. · For multiple digestions, set up a "Master

  5. Crystallization studies of 5'-deoxyadenosyl radical enzymes

    E-print Network

    Phillips, Laura (Laura Anne)

    2007-01-01

    Both adenosylcobalamin- and S-adenosylmethionine-dependent radical enzymes use a 5'-deoxyadenosyl radical intermediate to abstract a hydrogen atom from their substrates. In the case of adenosylcobalamin-dependent enzymes, ...

  6. Extracellular enzyme kinetics scale with resource availability

    EPA Science Inventory

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...

  7. ENZYME DEGRADATION OF CHIRAL ORGANIC PHOSPHORUS INSECTICIDES

    EPA Science Inventory

    Chiral organic phosphorus pesticides (OPs) are expected to be biologically degraded enantioselectively by endogenous enzymes. Various chiral Ops were treated with the enzyme phosphotriesterase (PTE) obtained from partially purified extracts of Escherichia coli strain DH-5- carryi...

  8. Lignin-degrading enzyme activities.

    PubMed

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

    2012-01-01

    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays. PMID:22843404

  9. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    NASA Astrophysics Data System (ADS)

    Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

  10. The Amborella vacuolar processing enzyme family

    PubMed Central

    Poncet, Valérie; Scutt, Charlie; Tournebize, Rémi; Villegente, Matthieu; Cueff, Gwendal; Rajjou, Loïc; Balliau, Thierry; Zivy, Michel; Fogliani, Bruno; Job, Claudette; de Kochko, Alexandre; Sarramegna-Burtet, Valérie; Job, Dominique

    2015-01-01

    Most vacuolar proteins are synthesized on rough endoplasmic reticulum as proprotein precursors and then transported to the vacuoles, where they are converted into their respective mature forms by vacuolar processing enzymes (VPEs). In the case of the seed storage proteins, this process is of major importance, as it conditions the establishment of vigorous seedlings. Toward the goal of identifying proteome signatures that could be associated with the origin and early diversification of angiosperms, we previously characterized the 11S-legumin-type seed storage proteins from Amborella trichopoda, a rainforest shrub endemic to New Caledonia that is also the probable sister to all other angiosperms (Amborella Genome Project, 2013). In the present study, proteomic and genomic approaches were used to characterize the VPE family in this species. Three genes were found to encode VPEs in the Amborella's genome. Phylogenetic analyses showed that the Amborella sequences grouped within two major clades of angiosperm VPEs, indicating that the duplication that generated the ancestors of these clades occurred before the most recent common ancestor of living angiosperms. A further important duplication within the VPE family appears to have occurred in common ancestor of the core eudicots, while many more recent duplications have also occurred in specific taxa, including both Arabidopsis thaliana and Amborella. An analysis of natural genetic variation for each of the three Amborella VPE genes revealed the absence of selective forces acting on intronic and exonic single-nucleotide polymorphisms among several natural Amborella populations in New Caledonia. PMID:26347753

  11. Enzymes toughen up for chemical processing

    SciTech Connect

    Hairston, D.

    1995-05-01

    While enzymes have been making tremendous inroads into detergent formulation and food processing, the penetration of these protein-based catalysts into other chemical-process manufacture and hazardous waste treatment--where they are slated to replace heavy metal catalysts and other processing aids--has been relatively slow. Recently, however, enhancements in the enzyme`s properties are opening the door wider for such broadened usage. Some of these non-traditional uses of enzymes are described.

  12. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  13. Microorganisms detected by enzyme-catalyzed reaction

    NASA Technical Reports Server (NTRS)

    Vango, S. P.; Weetall, H. H.; Weliky, N.

    1966-01-01

    Enzymes detect the presence of microorganisms in soils. The enzyme lysozymi is used to release the enzyme catalase from the microorganisms in a soil sample. The catalase catalyzes the decomposition of added hydrogen peroxide to produce oxygen which is detected manometrically. The partial pressure of the oxygen serves as an index of the samples bacteria content.

  14. High-Activity Enzyme-Polyurethane Coatings

    E-print Network

    Federspiel, William J.

    High-Activity Enzyme- Polyurethane Coatings GeÂraldine F. Drevon,2 Karsten Danielmeier,3 William of the enzyme to the polymeric matrix. The distribution of immobilized DFPase as well as activity retention deactivation of DFPase-ECC leads to the formation of a hyperstable and active form of enzyme. ã 2002 Wiley

  15. A Structural Perspective on Enzymes Activated by

    E-print Network

    Di Cera, Enrico

    A Structural Perspective on Enzymes Activated by Monovalent Cations* Published, JBC Papers in Press and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 Enzymes activated resolution crystal structures of M -activated enzymes, which have become available only over the last decade

  16. Investigating Degradation Enzymes for Pentachlorophenols (PCPs)

    E-print Network

    Collins, Gary S.

    Investigating Degradation Enzymes for Pentachlorophenols (PCPs) Emily White1, Mark Nissen2, Chul chlorophenolicum that breaks down PCP has been studied extensively but a complete picture of how the enzymes work is still lacking. This study aims to solve the crystal structure of PcpC, which is an enzyme in the pathway

  17. Better Enzymes for Biofuels and Green Chemistry

    E-print Network

    Better Enzymes for Biofuels and Green Chemistry: Solving the Cofactor Imbalance Problem Imbalances-engineered the cofactor specificity of individual enzymes, this process has historically been arduous and prone to failure. Testing this procedure on a structurally and functionally diverse set of industrially relevant enzymes has

  18. Immobilization of Enzymes in Polymer Supports.

    ERIC Educational Resources Information Center

    Conlon, Hugh D.; Walt, David R.

    1986-01-01

    Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

  19. Determining Enzyme Activity by Radial Diffusion

    ERIC Educational Resources Information Center

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  20. Ma12 Mathematics of Enzyme Spring 2015

    E-print Network

    Simon, Barry

    Notes Ma12 Mathematics of Enzyme Kinetics Spring 2015 D. Ramakrishnan and R. Tanner 1 #12;Lecture 1 Dinakar Ramakrishnan 0.1 The terms Enzyme: Catalyst; one aiding (or initiating) the action without being changed by it (like Mercutio in Romeo and Juliet) from the German word Enzym, derived from the Greek word

  1. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  2. Regeneration of Cofactors for Enzyme Biocatalysis

    E-print Network

    Zhao, Huimin

    Regeneration of Cofactors for Enzyme Biocatalysis Ryan D. Woodyer, Tyler W. Johannes and Huimin with the need for more selective, safer, and cleaner reactions. Enzymes as catalysts meet many of the needs-independent enzymes such as hydrolases, which perform relatively simple chemistry (Faber 2000). In contrast, cofactor

  3. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  4. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enzyme preparations. 864.4400 Section 864.4400...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  5. Restricting SBH Ambiguity via Restriction Enzymes

    E-print Network

    Snir, Sagi

    Restricting SBH Ambiguity via Restriction Enzymes Steven Skiena1 and Sagi Snir2 1 Department, we build on [20] to enhance the resolving power of restriction enzymes. We give a hardness result­417, 2002. c Springer-Verlag Berlin Heidelberg 2002 #12;Restricting SBH Ambiguity via Restriction Enzymes

  6. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  7. Greenhouse Gas Reduction Act

    E-print Network

    Hilderbrand, Robert H.

    Maryland's Greenhouse Gas Reduction Act Plan October 2013 #12;The Maryland Department of the Environment is the agency responsible for preparing and submitting this 2013 Greenhouse Gas Reduction Act Plan Chapter 2: Update on climate change science Chapter 3: Inventory and forecast Chapter 4: Climate change

  8. ACT and College Success

    ERIC Educational Resources Information Center

    Bleyaert, Barbara

    2010-01-01

    What is the relationship between ACT scores and success in college? For decades, admissions policies in colleges and universities across the country have required applicants to submit scores from a college entrance exam, most typically the ACT (American College Testing) or SAT (Scholastic Aptitude Test). This requirement suggests that high school…

  9. Characterization of fish-skin gelatin gels and films containing the antomicrobial enzyme lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish skins are rich in collagen and can be used to produce food-grade gelatin. Films cast from fish-skin gelatins are stable at room temperature and can act as a barrier when applied to foods. Lysozyme is a food-safe, antimicrobial enzyme that can also produce gels and films. When cold-water, fish-s...

  10. The Clean Water Act

    SciTech Connect

    Piatt, J.

    1995-12-31

    The Federal Water Pollution Control Act, commonly called the Clean Water Act (CWA), was adopted on 18 October 1972. Since then it has been amended 18 times, the last amendments were adopted on 4 February 1987. As established, its objective is: to restore and maintain the chemical, physical, and biological integrity of the Nation`s waters. And has, as an interim goal: water quality which provides for the protection and propagation of fish, shellfish, and wildlife and provides for recreation in and on the water. It should be noted that Congress established as the Act`s ultimate goal: the discharge of pollutants into the navigable waters be eliminated. The Act set out to meet this lofty objective and goal through the development and implementation of controls on the point source discharges and the nonpoint source release of pollutants. The regulation of point and nonpoint sources as well as future requirements are discussed.

  11. Controls on the Temperature Sensitivity of Soil Enzymes: A Key Driver of In Situ Enzyme

    E-print Network

    Allison, Steven D.

    few studies. In theory, the temperature sensitivity of enzyme activities can be described from first the activation energy of biochemical reactions. There are two aspects to the temperature sensitivity of enzymesChapter 13 Controls on the Temperature Sensitivity of Soil Enzymes: A Key Driver of In Situ Enzyme

  12. Identification of a dehydrogenase acting on D-2-hydroxyglutarate.

    PubMed

    Achouri, Younes; Noël, Gaëtane; Vertommen, Didier; Rider, Mark H; Veiga-Da-Cunha, Maria; Van Schaftingen, Emile

    2004-07-01

    Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme. PMID:15070399

  13. Virulence-Associated Enzymes of Cryptococcus neoformans

    PubMed Central

    Almeida, Fausto; Wolf, Julie M.

    2015-01-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

  14. Virulence-Associated Enzymes of Cryptococcus neoformans.

    PubMed

    Almeida, Fausto; Wolf, Julie M; Casadevall, Arturo

    2015-12-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

  15. [Muscle enzyme activity and exercise].

    PubMed

    Gojanovic, B; Feihl, F; Gremion, G; Waeber, B

    2009-02-01

    Exercise is classically associated with muscular soreness, presenting one to two days later, delayed onset muscular soreness. Blood muscle enzymes and protein elevations are characteristic, and may cause renal failure. Creatin phosphokinase peak appears on the fourth day and depends on exercise type and individual parameters. This effect is attenuated with repeated bouts, by habituation. Metabolic complications are rare. The knowledge of this reaction, even with common exercises, allows to postpone investigations for a complex metabolic disorder, or to avoid stopping a medication for fear of a side effect, as with statins. Indeed, it is necessary to wait for seven days without any exercise before interpreting an elevated CK result. PMID:19180440

  16. E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

    PubMed Central

    St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

    2014-01-01

    Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

  17. Activity of native hydrolytic enzymes and their association with the cell wall of three ectomycorrhizal fungi.

    PubMed

    Pérez-de-Mora, Alfredo; Reuter, Bianca; Lucio, Marianna; Ahne, Alfred; Schloter, Michael; Pritsch, Karin

    2013-04-01

    The ecological and biogeochemical relevance of hydrolytic enzymes associated with the fungal cell wall has been poorly studied in ectomycorrhizal (ECM) fungi. We used a modified sequential extraction procedure to investigate the activity of various hydrolytic enzymes (?-glucosidase, acid-phosphatase, leucine-aminopeptidase, chitinase, xylanase and glucuronidase) and their association with the cell wall of three ECM fungi (Rhizopogon roseolus, Paxillus involutus and Piloderma croceum). Fungi were grown on C-rich solid medium under three different P concentrations (3.7, 0.37 and 0.037 mM). The sequential extraction procedure classifies enzymes as: (a) cytosolic, (b) loosely bound, (c) hydrophobically bound, (d) ionically bound and (e) covalently bound. Results showed that for the same fungus absolute enzymatic activity was affected by P concentration, whilst enzymatic compartmentalization among the cytosol and the cell wall fractions was not. The association of enzymes with the cell wall was fungus- and enzyme-specific. Our data indicate also that enzymes best known for being either extracellular or cytosolic or both, do act in muro as well. The ecological implications of cell wall-bound enzymes and the potential applications and limitations of sequential extractions are further discussed. PMID:23053575

  18. Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

    PubMed

    Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

    2012-10-01

    Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. PMID:22743221

  19. Assertive Community Treatment (ACT)

    MedlinePLUS

    ... be very effective in the treatment of severe mental illness. ACT is aimed at providing comprehensive multidisciplinary care ... strives to decrease the debilitating symptoms of severe mental illness that affect each individual in different ways. By ...

  20. ACTS mobile SATCOM experiments

    NASA Technical Reports Server (NTRS)

    Abbe, Brian S.; Frye, Robert E.; Jedrey, Thomas C.

    1993-01-01

    Over the last decade, the demand for reliable mobile satellite communications (satcom) for voice, data, and video applications has increased dramatically. As consumer demand grows, the current spectrum allocation at L-band could become saturated. For this reason, NASA and the Jet Propulsion Laboratory are developing the Advanced Communications Technology Satellites (ACTS) mobile terminal (AMT) and are evaluating the feasibility of K/Ka-band (20/30 GHz) mobile satcom to meet these growing needs. U.S. industry and government, acting as co-partners, will evaluate K/Ka-band mobile satcom and develop new technologies by conducting a series of applications-oriented experiments. The ACTS and the AMT testbed will be used to conduct these mobile satcom experiments. The goals of the ACTS Mobile Experiments Program and the individual experiment configurations and objectives are further presented.

  1. Public Health Service Act

    Cancer.gov

    The Public Health Service was established by act of July 16, 1798 (ch. 77, 1 Stat. 605), authorizing marine hospitals for the care of American merchant seamen. Subsequent legislation has vastly broadened the scope of its activities.

  2. Peptidoglycan Remodeling by the Coordinated Action of Multispecific Enzymes

    PubMed Central

    Alvarez, Laura; Espaillat, Akbar; Hermoso, Juan A.; de Pedro, Miguel A.

    2014-01-01

    The peptidoglycan (PG) cell wall constitutes the main defense barrier of bacteria against environmental insults and acts as communication interface. The biochemistry of this macromolecule has been well characterized throughout the years but recent discoveries have unveiled its chemical plasticity under environmental stresses. Non-canonical D-amino acids (NCDAA) are produced and released to the extracellular media by diverse bacteria. Such molecules govern cell wall adaptation to challenging environments through their incorporation into the polymer, a widespread capability among bacteria that reveals the inherent catalytic plasticity of the enzymes involved in the cell wall metabolism. Here, we analyze the recent structural and biochemical characterization of Bsr, a new family of broad spectrum racemases able to generate a wide range of NCDAA. We also discuss the necessity of a coordinated action of PG multispecific enzymes to generate adequate levels of modification in the murein sacculus. Finally, we also highlight how this catalytic plasticity of NCDAA-incorporating enzymes has allowed the development of new revolutionary methodologies for the study of PG modes of growth and in vivo dynamics. PMID:24799190

  3. Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies

    PubMed Central

    Kitatani, Kazuyuki; Taniguchi, Makoto; Okazaki, Toshiro

    2015-01-01

    Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma. PMID:25997737

  4. Enzymes of ?,?-Trehalose Metabolism in Soybean Nodules 1

    PubMed Central

    Salminen, Seppo O.; Streeter, John G.

    1986-01-01

    Metabolism of trehalose, ?,d-glucopyranosyl-?,d-glucopyranoside, was studied in nodules of Bradyrhizobium japonicum-Glycine max [L.] Merr. cv Beeson 80 symbiosis. The nodule extract was divided into three fractions: bacteroid soluble protein, bacteroid fragments, and cytosol. The bacteroid soluble protein and cytosol fractions were gel-filtered. The key biosynthetic enzyme, trehalose-6-phosphate synthetase, was consistently found only in the bacteroids. Trehalose-6-phosphate phosphatase activity was present both in the bacteroid soluble protein and cytosol fractions. Trehalase, the most abundant catabolic enzyme was present in all three fractions and showed two pH optima: pH 3.8 and 6.6. Two other degradative enzymes, phosphotrehalase, acting on trehalose-6-phosphate forming glucose and glucose-6-phosphate, and trehalose phosphorylase, forming glucose and ?-glucose-1-phosphate, were also detected in the bacteroid soluble protein and cytosol fractions. Trehalase was present in large excess over trehalose-6-phosphate synthetase. Trehalose accumulation in the nodules would appear to be predicated on spatial separation of trehalose and trehalase. PMID:16664852

  5. Microbial community composition shapes enzyme patterns in topsoil and subsoil horizons along a latitudinal transect in Western Siberia

    PubMed Central

    Schnecker, Jörg; Wild, Birgit; Takriti, Mounir; Eloy Alves, Ricardo J.; Gentsch, Norman; Gittel, Antje; Hofer, Angelika; Klaus, Karoline; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Richter, Andreas

    2015-01-01

    Soil horizons below 30 cm depth contain about 60% of the organic carbon stored in soils. Although insight into the physical and chemical stabilization of soil organic matter (SOM) and into microbial community composition in these horizons is being gained, information on microbial functions of subsoil microbial communities and on associated microbially-mediated processes remains sparse. To identify possible controls on enzyme patterns, we correlated enzyme patterns with biotic and abiotic soil parameters, as well as with microbial community composition, estimated using phospholipid fatty acid profiles. Enzyme patterns (i.e. distance-matrixes calculated from these enzyme activities) were calculated from the activities of six extracellular enzymes (cellobiohydrolase, leucine-amino-peptidase, N-acetylglucosaminidase, chitotriosidase, phosphatase and phenoloxidase), which had been measured in soil samples from organic topsoil horizons, mineral topsoil horizons, and mineral subsoil horizons from seven ecosystems along a 1500 km latitudinal transect in Western Siberia. We found that hydrolytic enzyme activities decreased rapidly with depth, whereas oxidative enzyme activities in mineral horizons were as high as, or higher than in organic topsoil horizons. Enzyme patterns varied more strongly between ecosystems in mineral subsoil horizons than in organic topsoils. The enzyme patterns in topsoil horizons were correlated with SOM content (i.e., C and N content) and microbial community composition. In contrast, the enzyme patterns in mineral subsoil horizons were related to water content, soil pH and microbial community composition. The lack of correlation between enzyme patterns and SOM quantity in the mineral subsoils suggests that SOM chemistry, spatial separation or physical stabilization of SOM rather than SOM content might determine substrate availability for enzymatic breakdown. The correlation of microbial community composition and enzyme patterns in all horizons, suggests that microbial community composition shapes enzyme patterns and might act as a modifier for the usual dependency of decomposition rates on SOM content or C/N ratios. PMID:25859057

  6. 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

  7. Engineering Enzymes in Energy Crops: Conditionally Activated Enzymes Expressed in Cellulosic Energy Crops

    SciTech Connect

    2010-01-15

    Broad Funding Opportunity Announcement Project: Enzymes are required to break plant biomass down into the fermentable sugars that are used to create biofuel. Currently, costly enzymes must be added to the biofuel production process. Engineering crops to already contain these enzymes will reduce costs and produce biomass that is more easily digested. In fact, enzyme costs alone account for $0.50-$0.75/gallon of the cost of a biomass-derived biofuel like ethanol. Agrivida is genetically engineering plants to contain high concentrations of enzymes that break down cell walls. These enzymes can be “switched on” after harvest so they won’t damage the plant while it’s growing.

  8. Enzyme analysis of Schistosoma haematobium*

    PubMed Central

    Wright, C. A.; Ross, G. C.

    1983-01-01

    Results are reported of enzyme analyses, by isoelectric focusing in polyacrylamide gels, of adult Schistosoma haematobium worms derived from 22 isolates originating from 13 countries. Polymorphisms have been identified in the glucose-6-phosphate dehydrogenase (G6PD) and phosphoglucomutase (PGM) systems. Certain forms appear to be restricted in their geographical distribution and their occurrence outside their usual areas suggests human population movements resulting in mixing of parasite strains. The possible implications of minor variations in some PGM patterns and the apparent absence of heteropolymer fractions in presumed G6PD heterozygotes are discussed. The use of the technique to detect natural multiple miracidial infections in snails is reported and discussed. ImagesFig. 1 PMID:6222843

  9. Ethanologenic Enzymes of Zymomonas mobilis

    SciTech Connect

    Ingram, Lonnie O'Neal

    1999-03-01

    Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.

  10. Monobody-mediated alteration of enzyme specificity.

    PubMed

    Tanaka, Shun-Ichi; Takahashi, Tetsuya; Koide, Akiko; Ishihara, Satoru; Koikeda, Satoshi; Koide, Shohei

    2015-10-01

    Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a ?-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics. PMID:26322825

  11. Inhibiting the Function of an Enzyme

    SciTech Connect

    2015-06-17

    In order to stop bacteria from reproducing and causing a disease like tuberculosis, researchers must first block its enzymes' ability to bind with certain molecules. A research team from Brandeis University worked with the Advanced Protein Characterization Facility at Argonne National Laboratory to define 13 different bacterial structures and uncover the mechanism by which their enzymes form and break bonds with molecules. This animation depicts how an enzyme may be inhibited using this knowledge.

  12. Advanced development of immobilized enzyme reactors

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.; Schussel, Leonard J.; Carter, Layne

    1991-01-01

    Fixed-bed reactors have been used at NASA-Marshall to purify wastewater generated by an end-use equipment facility, on the basis of a combination of multifiltration unibeds and enzyme unibeds. The enzyme beds were found to effectively remove such targeted organics as urea, alcohols, and aldehydes, down to levels lying below detection limits. The enzyme beds were also found to remove organic contaminants not specifically targeted.

  13. Modified kinetics of enzymes interacting with nanoparticles

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Breger, Joyce C.; Malanoski, Anthony; Claussen, Jonathan C.; Walper, Scott A.; Ancona, Mario G.; Brown, Carl W.; Stewart, Michael H.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2015-08-01

    Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

  14. ACTS broadband aeronautical terminal

    NASA Technical Reports Server (NTRS)

    Agan, M. J.; Densmore, A. C.

    1995-01-01

    This paper discusses the design of, and experiments with, the ACTS Broadband Aeronautical Terminal. As part of the ongoing effort to investigate commercial applications of ACTS technologies, NASA's Jet Propulsion Laboratory and various industry/government partners are developing a broadband mobile terminal for aeronautical applications. The ACTS Broadband Aeronautical Terminal is designed to explore the use of K/Ka-band for high data rate aeronautical satellite communications. Currently available commercial aeronautical satellite communications systems are only capable of achieving data rates on the order of tens of kilobits per second. The broadband terminal used in conjunction with the ACTS mechanically steerable antenna, can achieve data rates of 384 kilobits per second, while use of an ACTS spot beam antenna with this terminal will allow up to T1 data rates (1.544 megabits per second). The aeronautical terminal will be utilized to test a variety of applications that require a high data rate communications link. The use of the K/Ka-band for wideband aeronautical communications has the advantages of spectrum availability and smaller antennas, while eliminating the one major drawback of this frequency band, rain attenuation, by flying above the clouds the majority of the time.

  15. Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

    PubMed Central

    Pallottini, Valentina; Canuti, Lorena; De Canio, Michele; Urbani, Andrea; Marzano, Valeria; Cornetta, Tommaso; Stano, Pasquale; Giovanetti, Anna; Stella, Lorenzo; Canini, Antonella; Federici, Giorgio; Ricci, Giorgio

    2010-01-01

    Background In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions. Methodology/Principal Findings Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization. Conclusions/Significance The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes. PMID:21170318

  16. On Acting from Duty 

    E-print Network

    Fossee, Jordan Michael

    2013-09-24

    moral worth, the action must be motivated solely by duty. In the first section of the Groundwork, Kant provides examples of moral agents of varying character to illustrate the distinction between acting merely in accordance with duty and acting from duty... sympathetic a temper that, without any further motive of vanity or self-interest, they find an inner pleasure in spreading happiness around them and can take delight in the contentment of others as their own work. Yet I maintain that in such a case an action...

  17. Affordable Care Act.

    PubMed

    Rak, Sofija; Coffin, Janis

    2013-01-01

    The Patient Protection and Affordable Care Act of 2010 (PPACA), although a subject of much debate in the Unites States, was enacted on March 23, 2010, and upheld by the Supreme Court on June 28, 2012. This act advocates that "healthcare is a right, not a privilege." The main goals of PPACA are to minimize the number of uninsured Americans and make healthcare available to everyone at an affordable price. The Congressional Budget Office has determined that 94% of Americans will have healthcare coverage while staying under the $900 billion limit that President Barack Obama established by bending the healthcare cost curve and reducing the deficit over the next 10 years. PMID:23767130

  18. AN ENZYME-IMMOBILIZATION PROCEDURE FOR THE ANALYSIS OF ENZYME-INHIBITING CHEMICALS IN WATER

    EPA Science Inventory

    The enzymes cholinesterase and urease were mixed individually with gelatin and immobilized onto the inside surface of glass capillary tubes. After the gelatin-enzyme mixture had dried, water samples containing various enzyme inhibiting test chemicals were pumped through the tubes...

  19. Cross-linked polymer nanofibers for hyperthermophilic enzyme immobilization: approaches to improve enzyme performance.

    PubMed

    Tang, Christina; Saquing, Carl D; Morton, Stephen W; Glatz, Brittany N; Kelly, Robert M; Khan, Saad A

    2014-08-13

    We report an enzyme immobilization method effective at elevated temperatures (up to 105 °C) and sufficiently robust for hyperthermophilic enzymes. Using a model hyperthermophilic enzyme, ?-galactosidase from Thermotoga maritima, immobilization within chemically cross-linked poly(vinyl alcohol) (PVA) nanofibers to provide high specific surface area is achieved by (1) electrospinning a blend of a PVA and enzyme and (2) chemically cross-linking the polymer to entrap the enzyme within a water insoluble PVA fiber. The resulting enzyme-loaded nanofibers are water-insoluble at elevated temperatures, and enzyme leaching is not observed, indicating that the cross-linking effectively immobilizes the enzyme within the fibers. Upon immobilization, the enzyme retains its hyperthermophilic nature and shows improved thermal stability indicated by a 5.5-fold increase in apparent half-life at 90 °C, but with a significant decrease in apparent activity. The loss in apparent activity is attributed to enzyme deactivation and mass transfer limitations. Improvements in the apparent activity can be achieved by incorporating a cryoprotectant during immobilization to prevent enzyme deactivation. For example, immobilization in the presence of trehalose improved the apparent activity by 10-fold. Minimizing the mat thickness to reduce interfiber diffusion was a simple and effective method to further improve the performance of the immobilized enzyme. PMID:25058141

  20. DIGESTIVE ENZYMES IN POIKILOTHERMAL VERTEBRATES. AN INVESTIGATION OF ENZYMES IN FISHES, WITH COM-

    E-print Network

    DIGESTIVE ENZYMES IN POIKILOTHERMAL VERTEBRATES. AN INVESTIGATION OF ENZYMES IN FISHES, WITH COM in the digestive tract_un Peptic digestionn __ Esophagus _ Stomach _ Page 181 Results and discussion-Continued. 182 Tryptic digestion uu u_ 182 Ereptic digestion u n _ 186 Carbohydrate-splitting enzymes _ 184 Amylase _ 184

  1. Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme*S

    E-print Network

    Kowalczykowski, Stephen C.

    Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme*SBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity the exceptionally high rate and processivity of DNA unwinding cata- lyzed by the RecBCD enzyme. Using a stopped

  2. Enzyme Reactions and Acceptability of Plant Foods.

    ERIC Educational Resources Information Center

    Palmer, James K.

    1984-01-01

    Provides an overview of enzyme reactions which contribute to the character and acceptability of plant foods. A detailed discussion of polyphenoloxidase is also provided as an example of an enzyme which can markedly affect the character and acceptability of such foods. (JN)

  3. A toy quantum analog of enzymes

    E-print Network

    Svetlichny, George

    2015-01-01

    We present a quantum system incorporating qualitative aspects of enzyme action in which the possibility of quantum superposition of several conformations of the enzyme-substrate complex is investigated. We present numerical results showing quantum effects that transcend the case of a statistical mixture of conformations.

  4. A toy quantum analog of enzymes

    E-print Network

    George Svetlichny

    2015-10-19

    We present a quantum system incorporating qualitative aspects of enzyme action in which the possibility of quantum superposition of several conformations of the enzyme-substrate complex is investigated. We present numerical results showing quantum effects that transcend the case of a statistical mixture of conformations.

  5. Developing enzyme systems for biomass deconstruction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The conversion of agricultural crops and residues to fermentable feedstock for the production of bioethanol represents a major source of renewable energy. The key to economically viable and effective biomass conversion includes the development of novel enzymes and enzyme systems to achieve total de...

  6. A DNA enzyme that cleaves RNA

    NASA Technical Reports Server (NTRS)

    Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

    1994-01-01

    BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

  7. Enzyme Activity Experiments Using a Simple Spectrophotometer

    ERIC Educational Resources Information Center

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  8. Post-production modification of industrial enzymes.

    PubMed

    Minten, Inge J; Abello, Nicolas; Schooneveld-Bergmans, Margot E F; van den Berg, Marco A

    2014-01-01

    Industry has an increasing interest in the use of enzymes as environmentally friendly, highly efficient, and specific bio-catalysts. Enzymes have primarily evolved to function in aqueous environments at ambient temperature and pressure. These conditions however do not always correspond with industrial processes or applications, and only a small portion of all known enzymes are therefore suitable for industrial use. Protein engineering can sometimes be applied to convey more desirable properties to enzymes, such as increased stability, but is limited to the 20 naturally occurring amino acids or homologs thereof. Using post-production modification, which has the potential to combine desirable properties from the enzyme and the conjugated compounds, enzymes can be modified with both natural and synthetic molecules. This offers access to a myriad of possibilities for tuning the properties of enzymes. At this moment, however, the effects of post-production modification cannot yet be reliably predicted. The increasing number of applications will improve this so that the potential of this technology can be fully exploited. This review will focus on post-production modification of enzymes and its use and opportunities in industry. PMID:24903809

  9. Restriction Enzyme Mapping: A Simple Student Practical.

    ERIC Educational Resources Information Center

    Higgins, Stephen J.; And Others

    1990-01-01

    An experiment that uses the recombinant plasmid pX1108 to illustrate restriction mapping is described. The experiment involves three restriction enzymes and employs single and double restriction enzyme digestions. A list of needed materials, procedures, safety precautions, results, and discussion are included. (KR)

  10. Enzymes: A Workshop for Secondary School Students.

    ERIC Educational Resources Information Center

    Bering, C. Larry

    1994-01-01

    Describes the weekend science workshop on enzymes and includes several projects that involve students directly, parts of which can be incorporated into a traditional chemistry, biology, or physical science course at the secondary level. Subjects include catalysts and catalytic converters in cars, enzymes as consumer products and in industrial…

  11. Enzyme Catalysis and the Gibbs Energy

    ERIC Educational Resources Information Center

    Ault, Addison

    2009-01-01

    Gibbs-energy profiles are often introduced during the first semester of organic chemistry, but are less often presented in connection with enzyme-catalyzed reactions. In this article I show how the Gibbs-energy profile corresponds to the characteristic kinetics of a simple enzyme-catalyzed reaction. (Contains 1 figure and 1 note.)

  12. Illustrating Enzyme Inhibition Using Gibbs Energy Profiles

    ERIC Educational Resources Information Center

    Bearne, Stephen L.

    2012-01-01

    Gibbs energy profiles have great utility as teaching and learning tools because they present students with a visual representation of the energy changes that occur during enzyme catalysis. Unfortunately, most textbooks divorce discussions of traditional kinetic topics, such as enzyme inhibition, from discussions of these same topics in terms of…

  13. Biocatalytic material comprising multilayer enzyme coated fiber

    DOEpatents

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    The present invention relates generally to high stability, high activity biocatalytic materials and processes for using the same. The materials comprise enzyme aggregate coatings having high biocatalytic activity and stability useful in heterogeneous environment. These new materials provide a new biocatalytic immobilized enzyme system with applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  14. Nocardiopsis species as potential sources of diverse and novel extracellular enzymes.

    PubMed

    Bennur, Tahsin; Kumar, Ameeta Ravi; Zinjarde, Smita; Javdekar, Vaishali

    2014-11-01

    Members of the genus Nocardiopsis are generally encountered in locations that are inherently extreme. They are present in frozen soils, desert sand, compost, saline or hypersaline habitats (marine systems, salterns and soils) and alkaline places (slag dumps, lake soils and sediments). In order to survive under these severe conditions, they produce novel and diverse enzymes that allow them to utilize the available nutrients and to thrive. The members of this genus are multifaceted and release an assortment of extracellular hydrolytic enzymes. They produce enzymes that are cold-adapted (?-amylases), thermotolerant (?-amylases and xylanases), thermoalkalotolerant (cellulases, ?-1,3-glucanases), alkali-tolerant thermostable (inulinases), acid-stable (keratinase) and alkalophilic (serine proteases). Some of the enzymes derived from Nocardiopsis species act on insoluble polymers such as glucans (pachyman and curdlan), keratin (feathers and prion proteins) and polyhydroxyalkanoates. Extreme tolerance exhibited by proteases has been attributed to the presence of some amino acids (Asn and Pro) in loop structures, relocation of multiple salt bridges to outer regions of the protein or the presence of a distinct polyproline II helix. The range of novel enzymes is projected to increase in the forthcoming years, as new isolates are being continually reported, and the development of processes involving such enzymes is envisaged in the future. PMID:25269602

  15. ACTS Mobile Terminals

    NASA Technical Reports Server (NTRS)

    Abbe, Brian S.; Agan, Martin J.; Jedrey, Thomas C.

    1997-01-01

    The development of the Advanced Communications Technology Satellite (ACTS) Mobile Terminal (AMT) and its follow-on, the Broadband Aeronautical Terminal (BAT), have provided an excellent testbed for the evaluation of K- and Ka-band mobile satellite communications systems. An overview of both of these terminals is presented in this paper.

  16. Acts of Endearment

    PubMed Central

    Stephens, G. Gayle

    1992-01-01

    Legitimate and clinically useful affection between physicians and patients can be nurtured by attending to duties enjoined by traditional codes of ethics. Three acts of endearment have special importance for today's family physicians: smoothing the bed of death; keeping patients' secrets; and not abandoning patients on account of incurability. PMID:20469528

  17. The USA PATRIOT Act.

    ERIC Educational Resources Information Center

    Minow, Mary; Coyle, Karen; Kaufman, Paula

    2002-01-01

    Explains the USA PATRIOT (Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct Terrorism) Act, passed after the September 11 terrorist attacks, and its implications for libraries and patron records. Considers past dealings with the FBI; court orders; search warrants; wiretaps; and subpoenas. Includes:…

  18. Respect for Acting.

    ERIC Educational Resources Information Center

    Hagen, Uta

    This book, based on the author's experience as a professional actress, is divided into three sections. The first part, "The Actor," deals with techniques the actor uses to function physically, verbally, and emotionally and discusses the actor's concept of himself and the art of acting. The second part, "The Object Exercises," consists of a series…

  19. Acting like a Pro

    ERIC Educational Resources Information Center

    Walker, Marlon A.

    2012-01-01

    The Saturday morning acting class in the Pearson Hall auditorium at Miles College boasts the school's highest attendance all year. The teacher, actress Robin Givens, was a lure few students--and others from surrounding areas--could resist. Some came to learn about their prospective field from a professional. Others were there for pointers to…

  20. Improving America's Schools Act

    NASA Technical Reports Server (NTRS)

    Cradler, John; Bridgforth, Elizabeth

    1995-01-01

    The Improving America's Schools ACT (IASA) emphasizes coherent systemic education reform, with Goals 2000 setting common standards for IASA and the recently authorized School-to-Work Program. IASA addresses the need to raise academic achievement, increase opportunities to learn, improve professional development, increase community involvement, utilize instructional applications of technology, and improve assessment, and allow more local flexibility in the use of funds.

  1. Family Educational Rights & Privacy Act

    E-print Network

    Spirtes, Peter

    or handicap in violation of Title VI of the Civil Rights Act of 1964, Title IX of the Educational AmendmentsFamily Educational Rights & Privacy Act (FERPA) Statement of Assurance Carnegie Mellon University Educational Rights and Privacy Act (FERPA). The act guarantees a student's right to access their educational

  2. Substrate analogues for isoprenoid enzymes

    SciTech Connect

    Stremler, K.E.

    1987-01-01

    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  3. Biotechnological uses of enzymes from psychrophiles

    PubMed Central

    Cavicchioli, R.; Charlton, T.; Ertan, H.; Omar, S. Mohd; Siddiqui, K. S.; Williams, T. J.

    2011-01-01

    Summary The bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ??5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold?adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning. PMID:21733127

  4. Highly Efficient Self-Replicating RNA Enzymes

    PubMed Central

    Robertson, Michael P.; Joyce, Gerald F.

    2014-01-01

    SUMMARY An RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form additional copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme. Directed evolution was used to improve the efficiency of the enzyme and increase its exponential growth rate to 0.14 min?1, corresponding to a doubling time of 5 min. This is close to the limit of 0.21 min?1 imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day. PMID:24388759

  5. Lignocellulolytic enzyme profiles of edible mushroom fungi.

    PubMed

    Buswell, J A; Cai, Y J; Chang, S T; Peberdy, J F; Fu, S Y; Yu, H S

    1996-09-01

    One of the most economically-viable processes for the bioconversion of many types of lignocellulosic wastes is represented by edible mushroom cultivation. Lentinula edodes, Volvariella volvacea and Pleurotus sajor-caju are three important commercially cultivated mushrooms which exhibit varying abilities to utilise different lignocellulosics as growth substrate. Examination of the lignocellulolytic enzyme profiles of the three species show this diversity to be reflected in qualitative variations in the major enzymic determinants (i.e. cellulases, ligninases) required for substrate bioconversion. For example, L. edodes, which is cultivated on highly lignified substrates such as wood or sawdust, produces two extracellular enzymes which have been associated with lignin depolymerisation in other fungi, (manganese peroxidase and laccase). Conversely, V. volvacea, which prefers high cellulose-, low lignin-containing substrates produces a family of cellulolytic enzymes including at least five endoglucanases, five cellobiohydrolases and two ?-glucosidases, but none of the recognised lignin-degrading enzymes. PMID:24415386

  6. Process for preparing multilayer enzyme coating on a fiber

    DOEpatents

    Kim, Jungbae (Richland, WA); Kwak, Ja Hun (Richland, WA); Grate, Jay W. (West Richland, WA)

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  7. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    PubMed Central

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  8. Characterization of the Streptococcus mutans Pyruvate Formate-Lyase (PFL)-Activating Enzyme Gene by Complementary Reconstitution of the In Vitro PFL-Reactivating System

    PubMed Central

    Yamamoto, Yasuhito; Sato, Yutaka; Takahashi-Abbe, Shoko; Takahashi, Nobuhiro; Kizaki, Harutoshi

    2000-01-01

    The act gene was identified and an act mutant as well as the pfl mutant was constructed in Streptococcus mutans. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The S. mutans act gene encoded the sole enzyme able to activate the PFL protein in this organism. PMID:10899886

  9. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  10. Potato Peroxidase for the Study of Enzyme Properties.

    ERIC Educational Resources Information Center

    Shamaefsky, Brian R.

    1993-01-01

    Explains how the surface of a freshly sliced potato can be used for a variety of enzyme action experiments including the influence of pH on enzyme action, the enzyme denaturation potential of boiling water, the inhibition of enzymes by heavy metals, and the effects of salt concentration on enzyme effectiveness. (PR)

  11. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared from refined beef fat; butterfat...

  12. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are...

  13. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are...

  14. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are...

  15. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are...

  16. Immobilized enzymes in organic media: Determinants of water dependence. Progress statement

    SciTech Connect

    Nandi, S.; DeFilippi, I.; Bedwell, B.; Zemel, H.

    1994-08-01

    The overall goals of this project are to investigate the critical factors that limit commercial scale applications of enzymes in organic solvents, and to scale-up a process for the production of a precursor to a specialty polymer. The overall performance of an immobilized enzyme can be influenced by its intrinsic structure and by external factors such as water content, support, pH, etc.. We have investigated the interrelation between support morphology and water content, and its effect on overall enzyme performance. Using a lipase catalyzed inter-esterification reaction as a model, we studied the controlling factors when water content in the organic solvent is such that a micro-aqueous phase is formed. In such an environment it was found that support particle aggregation is the major cause for decline in enzyme activity. We have shown that particle porosity, as well as the use of a particular non-woven fabric as an enzyme support, could alleviate this problem. These findings are being translated into a bioreactor design. We have also studied two {open_quotes}dry{close_quotes} non-aqueous systems, where a water phase is not formed since the water content is below its solubility in the organic solvent. In one of the systems, Subtilisin catalyzed trans-esterification of vinyl acrylate with a chiral alcohol, we have demonstrated that the use of a proprietary fabric support provides a significant boost in enzyme activity. We suggest that this particular fabric with its hydrophilic fibers acts as a lyoprotectant in the process of drying the enzyme. The benefits of this material as an enzyme support and its use in a lab scale bioreactor are being studied. Preliminary experiments have also been performed with a second {open_quotes}dry{close_quotes} reaction. This is the lipase catalyzed synthesis of AlliedSignal`s new product, VEctomer 4010.

  17. Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini).

    PubMed

    Erthal, Milton; Peres Silva, Carlos; Ian Samuels, Richard

    2007-11-01

    The digestive physiology and biochemistry of larvae of the leaf-cutting ant Acromyrmex subterraneus were investigated here. The activity of digestive enzymes was evaluated in the labial glands, midgut epithelium (soluble and particulate fractions), and in the lumen contents, separated into endo and ectoperitrophic regions. Enzymes with high levels of activity were partially characterised using chromatography and electrophoresis techniques. Microscope observations were carried out and the anatomy of the larval digestive tract was described here for the first time. Larvae fed with pH indicator solutions showed the anterior portion of the midgut to be acidic and the posterior portion neutral to alkaline, indicating that the pH of the different regions of the midgut could optimise certain enzyme activities, whilst inhibiting others. The flow rate of the intestinal contents was also evaluated in larvae fed with a dye solution. The slow flow rate is probably due to closure of the rear end of the larval midgut. No compartmentalisation of digestive enzymes acting on oligosaccharides and disaccharides in the ectoperitrophic space and on polysaccharides in the endoperitrophic space was observed here, which could also be related to the closure of the midgut. The digestive physiology of these larvae is therefore similar to ancestral Holometabola, a paradox when considering the highly evolved nature of these insects. The larval midgut demonstrated a large diversity of enzyme activities with high levels of alpha-amylase, alpha-mannosidase, chitinase, alpha-glucosidase, beta-glucosidase and proteinase. High levels of chitinase and amylase activities were detected in the labial glands of larvae. The enzyme profile reflected the necessity of the larvae to degrade the fungal substrate, their sole diet, and a probable source of some of the digestive enzymes detected here. When compared to adults, the larvae had a greater diversity and higher levels of enzyme activity, highlighting their importance as the "digestive caste" of the colony. PMID:17681527

  18. Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions

    USGS Publications Warehouse

    Ives, J.A.; Moffett, J.R.; Arun, P.; Lam, D.; Todorov, T.I.; Brothers, A.B.; Anick, D.J.; Centeno, J.; Namboodiri, M.A.A.; Jonas, W.B.

    2010-01-01

    Objectives: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. Methods: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. Results: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. Conclusions: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects. ?? 2009 The Faculty of Homeopathy.

  19. [Production of ligninolytic enzymes in bioreactor].

    PubMed

    Gao, Da-wen; Wen, Xiang-hua; Qian, Yi

    2006-02-01

    Production of ligninolytic enzymes under nitrogen limited conditions(C/N = 56/2.2) was studied in a 5-L stirred tank bioreactor with a working volume of 2 L for obtaining higher production of ligninolytic enzymes by white rot fungus Phanerochaete chrysosporium BKM-F-1767 and its control strategy. Results show that the manganese peroxidase (MnP) and laccase (Lac) reached peak at the sixth day and the seventh day, respectively, and the variation of them with time in a batch cultivation are similar to the results by agitated Erlenmeyer flasks; however higher enzyme activity was not achieved by applying a fed-batch strategy, in which nitrogen limited medium was fed to the reactor. In addition, variation of pH during cultivation was related to the growth of P. chrysosporium and enzymes production during both batch and fed-batch cultivation. The pH value of liquid medium began to decline when the enzyme activity occurred in the system, and the decline became more and more slow along with the decrease of enzyme activity at the end of fermentation. So, pH would be as a control parameter to find out the growth of P. chrysosporium and enzymes production during incubating P. chrysosporium. However, fed-batch strategy still need further study. PMID:16686200

  20. A survey of orphan enzyme activities

    PubMed Central

    Pouliot, Yannick; Karp, Peter D

    2007-01-01

    Background Using computational database searches, we have demonstrated previously that no gene sequences could be found for at least 36% of enzyme activities that have been assigned an Enzyme Commission number. Here we present a follow-up literature-based survey involving a statistically significant sample of such "orphan" activities. The survey was intended to determine whether sequences for these enzyme activities are truly unknown, or whether these sequences are absent from the public sequence databases but can be found in the literature. Results We demonstrate that for ~80% of sampled orphans, the absence of sequence data is bona fide. Our analyses further substantiate the notion that many of these enzyme activities play biologically important roles. Conclusion This survey points toward significant scientific cost of having such a large fraction of characterized enzyme activities disconnected from sequence data. It also suggests that a larger effort, beginning with a comprehensive survey of all putative orphan activities, would resolve nearly 300 artifactual orphans and reconnect a wealth of enzyme research with modern genomics. For these reasons, we propose that a systematic effort to identify the cognate genes of orphan enzymes be undertaken. PMID:17623104

  1. Enzyme reaction annotation using cloud techniques.

    PubMed

    Huang, Chuan-Ching; Lin, Chun-Yuan; Chang, Cheng-Wen; Tang, Chuan Yi

    2013-01-01

    An understanding of the activities of enzymes could help to elucidate the metabolic pathways of thousands of chemical reactions that are catalyzed by enzymes in living systems. Sophisticated applications such as drug design and metabolic reconstruction could be developed using accurate enzyme reaction annotation. Because accurate enzyme reaction annotation methods create potential for enhanced production capacity in these applications, they have received greater attention in the global market. We propose the enzyme reaction prediction (ERP) method as a novel tool to deduce enzyme reactions from domain architecture. We used several frequency relationships between architectures and reactions to enhance the annotation rates for single and multiple catalyzed reactions. The deluge of information which arose from high-throughput techniques in the postgenomic era has improved our understanding of biological data, although it presents obstacles in the data-processing stage. The high computational capacity provided by cloud computing has resulted in an exponential growth in the volume of incoming data. Cloud services also relieve the requirement for large-scale memory space required by this approach to analyze enzyme kinetic data. Our tool is designed as a single execution file; thus, it could be applied to any cloud platform in which multiple queries are supported. PMID:24222895

  2. ACTS of Education

    NASA Technical Reports Server (NTRS)

    Bauer, Robert; Krawczyk, Richard; Gargione, Frank; Kruse, Hans; Vrotsos, Pete (Technical Monitor)

    2002-01-01

    Now in its ninth year of operations, the Advanced Communications Technology Satellite (ACTS) program has continued, although since May 2000 in a new operations arrangement involving a university based consortium, the Ohio Consortium for Advanced Communications Technology (OCACT), While NASA has concluded its experimental intentions of ACTS, the spacecraft's ongoing viability has permitted its further operations to provide educational opportunities to engineering and communications students interested in satellite operations, as well as a Ka-band test bed for commercial interests in utilizing Kaband space communications. The consortium has reached its first year of operations. This generous opportunity by NASA has already resulted in unique educational opportunities for students in obtaining "hands-on" experience, such as, in satellite attitude control. An update is presented on the spacecraft and consortium operations.

  3. Catalysis by desolvation: the catalytic prowess of SAM-dependent halide-alkylating enzymes.

    PubMed

    Lohman, Danielle C; Edwards, David R; Wolfenden, Richard

    2013-10-01

    In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I(-), Br(-), Cl(-), F(-), HO(-), and H2O in water at elevated temperatures. Comparison of the resulting second-order rate constants, extrapolated to 25 °C, with the values of k(cat)/K(m) reported for fluorinase and chlorinase indicates that these enzymes enhance the rates of alkyl halide formation by factors of 2 × 10(15)- and 1 × 10(17)-fold, respectively. These rate enhancements, achieved without the assistance of cofactors, metal ions, or general acid-base catalysis, are the largest that have been reported for an enzyme that acts on two substrates. PMID:24041082

  4. Mathematical Modeling of Biosensors Based on an Array of Enzyme Microreactors

    PubMed Central

    Baronas, Romas; Ivanauskas, Feliksas; Kulys, Juozas

    2006-01-01

    This paper presents a two-dimensional-in-space mathematical model of biosensors based on an array of enzyme microreactors immobilised on a single electrode. The modeling system acts under amperometric conditions. The microreactors were modeled by particles and by strips. The model is based on the diffusion equations containing a non-linear term related to the Michaelis-Menten kinetics of the enzymatic reaction. The model involves three regions: an array of enzyme microreactors where enzyme reaction as well as mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region, where the analyte concentration is maintained constant. Using computer simulation, the influence of the geometry of the microreactors and of the diffusion region on the biosensor response was investigated. The digital simulation was carried out using the finite difference technique.

  5. ACTE Wing Loads Analysis

    NASA Technical Reports Server (NTRS)

    Horn, Nicholas R.

    2015-01-01

    The Adaptive Compliant Trailing Edge (ACTE) project modified a Gulfstream III (GIII) aircraft with a new flexible flap that creates a seamless transition between the flap and the wing. As with any new modification, it is crucial to ensure that the aircraft will not become overstressed in flight. To test this, Star CCM a computational fluid dynamics (CFD) software program was used to calculate aerodynamic data for the aircraft at given flight conditions.

  6. Toxic Substances Control Act

    SciTech Connect

    Not Available

    1992-05-15

    This Reference Book contains a current copy of the Toxic Substances Control Act and those regulations that implement the statute and appear to be most relevant to DOE activities. The document is provided to DOE and contractor staff for informational purposes only and should not be interpreted as legal guidance. Questions concerning this Reference Book may be directed to Mark Petts, EH-231 (202/586-2609).

  7. Freedom of Information Act

    USGS Publications Warehouse

    Newman, D.J.

    2012-01-01

    The Freedom of Information Act( FOIA), 5 U.S.C.§ 552, as amended, generally provides that any person has a right to request access to Federal agency records. The USGS proactively promotes information disclosure as inherent to its mission of providing objective science to inform decisionmakers and the general public. USGS scientists disseminate up-to-date and historical scientific data that are critical to addressing national and global priorities.

  8. The ACTS multibeam antenna

    NASA Technical Reports Server (NTRS)

    Regier, Frank A.

    1992-01-01

    The Advanced Communications Technology Satellite (ACTS) to be launched in 1993 introduces several new technologies including a multibeam antenna (MBA) operating at Ka-band. The satellite is introduced briefly, and then the MBA, consisting of electrically similar 30 GHz received and 20 GHz transmit offset Cassegrain systems utilizing orthogonal linear polarizations, is described. Dual polarization is achieved by using one feed assembly for each polarization in conjunction with nested front and back subreflectors, the gridded front subreflector acting as a window for one polarization and a reflector for the other. The antennas produce spot beams with approximately 0.3 deg beamwidth and gains of approximately 50 dbi. High surface accuracy and high edge taper produce low sidelobe levels and high cross-polarization isolation. A brief description is given of several Ka-band components fabricated for ACTS. These include multiflare antenna feedhorns, beam-forming networks utilizing latching ferrite waveguide switches, a 30 GHz high mobility electron transmitter (HEMT) low-noise amplifier and a 20 GHz TWT power amplifier.

  9. The Mismetallation of Enzymes during Oxidative Stress*

    PubMed Central

    Imlay, James A.

    2014-01-01

    Mononuclear iron enzymes can tightly bind non-activating metals. How do cells avoid mismetallation? The model bacterium Escherichia coli may control its metal pools so that thermodynamics favor the correct metallation of each enzyme. This system is disrupted, however, by superoxide and hydrogen peroxide. These species oxidize ferrous iron and thereby displace it from many iron-dependent mononuclear enzymes. Ultimately, zinc binds in its place, confers little activity, and imposes metabolic bottlenecks. Data suggest that E. coli compensates by using thiols to extract the zinc and by importing manganese to replace the catalytic iron atom. Manganese resists oxidants and provides substantial activity. PMID:25160623

  10. Milk-Clotting Enzymes From Microorganisms

    PubMed Central

    Srinivasan, R. A.; Iyengar, M. K. K.; Babbar, I. J.; Chakravorty, S. C.; Dudani, A. T.; Iya, K. K.

    1964-01-01

    A total of 230 cultures of fungi and 43 cultures of bacteria, isolated from such sources as soil, butter, and milk, were screened for their milk-clotting activity. The fungi were cultivated on semisolid media, and the bacteria were grown in milk media in shake culture. Phytic acid, added as calcium phytate, was found to stimulate production of the enzyme in most of the bacterial isolates. Proteolytic activity was invariably found to be associated with the milk-clotting enzyme in bacterial isolates. There was considerable variation in the ratio of the two enzymes from strain to strain. PMID:14239577

  11. Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

    PubMed

    Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

    2015-03-01

    Cellulase and ?-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 ?g (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. ?-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for ?-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and ?-glucosidases present in cellulase mixtures. When loading ?-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of ?-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme. PMID:25564204

  12. Sulfate Activation Enzymes: Phylogeny and Association with Pyrophosphatase

    E-print Network

    proteins led us to hypothesize that pyrophosphatase enzymes and sulfate activation enzymes physically by all sulfotransferase enzymes to attach sulfate to free hydroxyl groups on pre-existing carbohy- drate

  13. Flow Injection Amperometric Enzyme Biosensor for Direct Determination

    E-print Network

    Chen, Wilfred

    Flow Injection Amperometric Enzyme Biosensor for Direct Determination of Organophosphate Nerve A flow injection amperometric biosensor for the deter- mination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme

  14. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Milk-clotting enzymes, microbial. 173.150 Section 173...PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting...

  15. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 2011-04-01 false Enzyme analyzer for clinical use. 862.2500...Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a...

  16. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Milk-clotting enzymes, microbial. 173.150 Section 173...PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting...

  17. 21 CFR 864.9400 - Stabilized enzyme solution.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 2014-04-01 false Stabilized enzyme solution. 864.9400 Section 864...Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for...

  18. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100...Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  19. 21 CFR 864.9400 - Stabilized enzyme solution.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 2013-04-01 false Stabilized enzyme solution. 864.9400 Section 864...Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for...

  20. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device...

  1. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2009-04-01 true Insoluble glucose isomerase enzyme preparations. 184.1372 Section 184...184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the...

  2. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100...Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  3. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500...Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a...

  4. 21 CFR 864.9400 - Stabilized enzyme solution.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 2012-04-01 false Stabilized enzyme solution. 864.9400 Section 864...Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for...

  5. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100...Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  6. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Insoluble glucose isomerase enzyme preparations. 184.1372 Section 184...184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the...

  7. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2009-04-01 true Milk-clotting enzymes, microbial. 173.150 Section 173...PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting...

  8. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 false Insoluble glucose isomerase enzyme preparations. 184.1372 Section 184...184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the...

  9. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  10. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  11. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 2013-04-01 false Enzyme analyzer for clinical use. 862.2500...Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a...

  12. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Milk-clotting enzymes, microbial. 173.150 Section 173...PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting...

  13. 21 CFR 864.9400 - Stabilized enzyme solution.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 2011-04-01 false Stabilized enzyme solution. 864.9400 Section 864...Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for...

  14. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100...Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  15. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...2014-04-01 2014-04-01 false Enzyme analyzer for clinical use. 862.2500...Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a...

  16. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  17. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Milk-clotting enzymes, microbial. 173.150 Section 173...PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting...

  18. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...2011-04-01 false Drug metabolizing enzyme genotyping system. 862.3360 Section...Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification . A drug metabolizing enzyme genotyping system is a device...

  19. 21 CFR 864.9400 - Stabilized enzyme solution.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864...Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for...

  20. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 2012-04-01 false Enzyme analyzer for clinical use. 862.2500...Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a...

  1. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100...Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure...

  2. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...2013-04-01 false Insoluble glucose isomerase enzyme preparations. 184.1372 Section 184...184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the...

  3. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...2012-04-01 false Insoluble glucose isomerase enzyme preparations. 184.1372 Section 184...184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the...

  4. High Throughput Screening and Selection Methods for Directed Enzyme Evolution

    E-print Network

    Zhao, Huimin

    High Throughput Screening and Selection Methods for Directed Enzyme Evolution Han Xiao,, Zehua Bao at Urbana-Champaign, Urbana, Illinois 61801, United States ABSTRACT: Successful evolutionary enzyme enzyme evolution. Lastly, certain limitations of current methods, as well as future developments

  5. Enzyme Genetic Programming Modelling Biological Evolvability in Genetic Programming

    E-print Network

    Fernandez, Thomas

    Enzyme Genetic Programming Modelling Biological Evolvability in Genetic Programming Michael Adam.3 Evolvability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.4 Enzyme Genetic.1.1 Proteins and Enzymes . . . . . . . . . . . . . . . . . . . . . . . . 29 3.1.2 Nucleic Acids

  6. Cascaded Acts: Conscious Sequential Acting for Embodied Agents

    E-print Network

    Krovi, Venkat

    Cascaded Acts: Conscious Sequential Acting for Embodied Agents CSE Technical Report 99­10 Haythem O present a model for interleaving action, reasoning, and perception in order to allow for the conscious/atelic distinction. 2 #12; 1 Introduction Cognitive agents should act consciously. When carrying out a sequence

  7. Microbial Enzymes: Tools for Biotechnological Processes

    PubMed Central

    Adrio, Jose L.; Demain, Arnold L.

    2014-01-01

    Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi. PMID:24970208

  8. Enzyme Reactions in Nanoporous, Picoliter Volume Containers

    SciTech Connect

    Siuti, Piro; Retterer, Scott T; Choi, Chang Kyoung; Doktycz, Mitchel John

    2012-01-01

    Advancements in nanoscale fabrication allow creation of small volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ~19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate Amplex Red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics and high-throughput screening.

  9. Biotechnological relevance of starch-degrading enzymes

    SciTech Connect

    Stewart, G.G.

    1987-01-01

    Traditional enzymes, such as the amylases and the proteases, have been improved, novel applications have been found and new and valuable products have been marketed. The enzymatic hydrolysis of starch is described in some detail. (Refs. 8).

  10. Staphylococcal L-asparaginase: enzyme kinetics.

    PubMed

    Sobi?, M; Mikucki, J

    1991-01-01

    The ph optimum of purified staphylococcal L-asparaginase (EC 3.5.1.1) was found to be between 8.6 and 8.8. The temperature optimum was 30 degrees-32 degrees C and the highest reaction rate occurred at 30 degrees C. The KM of the enzyme calculated from Lineweaver-Burk plot was 3.71 x 10(-2) M. Besides L-asparaginase, the substrate specificity of enzyme was restricted to N-alpha-acetyl-L-asparagine. D-asparagine, L-aspartic acid and D-glutamic acid were competitive inhibitors. Hg2+ and Cu2+ cations strongly inhibited the enzyme while Na+ and K+ cations strongly stimulated activity. Two SH-groups could be detected after enzyme denaturation with guanidine. PMID:1726615

  11. Archaeal Enzymes and Applications in Industrial Biocatalysts

    PubMed Central

    Littlechild, Jennifer A.

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches. PMID:26494981

  12. Introduction Pancreatic cholesterol esterase (CEase), an enzyme,

    E-print Network

    Lee, Keun Woo

    535 Introduction Pancreatic cholesterol esterase (CEase), an enzyme, which is also known as bile pancreatic cholesterol esterase inhibitors using pharmacophore modelling, virtual screening, and optimization in potent CEase inhibitor designing. Keywords: Pancreatic cholesterol esterase, common feature pharmacophore

  13. On the hydrodynamics of swimming enzymes

    NASA Astrophysics Data System (ADS)

    Bai, Xiaoyu; Wolynes, Peter G.

    2015-10-01

    Several recent experiments suggest that rather generally the diffusion of enzymes may be augmented through their activity. We demonstrate that such swimming motility can emerge from the interplay between the enzyme energy landscape and the hydrodynamic coupling of the enzyme to its environment. Swimming thus occurs during the transit time of a transient allosteric change. We estimate the velocity during the transition. The analysis of such a swimming motion suggests the final stroke size is limited by the hydrodynamic size of the enzyme. This limit is quite a bit smaller than the values that can be inferred from the recent experiments. We also show that one proposed explanation of the experiments based on reaction heat effects can be ruled out using an extended hydrodynamic analysis. These results lead us to propose an alternate explanation of the fluorescence correlation measurements.

  14. Escherichia coli photoreactivating enzyme: purification and properties

    SciTech Connect

    Snapka, R.M.; Sutherland, B.M.

    1980-01-01

    Researchers have purified large quantities of Escherichia coli photoreactivating enzyme to apparent homogeneity and have studied its physical and chemical properties. The enzyme has a molecular weight of 36,800 and a S/sub 20,w//sup 0/ of 3.72 S. Amino acid analysis revealed an apparent absence of tryptophan, a low content of aromatic residues, and the presence of no unusual amino acids. The N terminus is arginine. The purified enzyme contained up to 13% carbohydrate by weight. The carbohydrate was composed of mannose, galactose, glucose, and N-acetylglucosamine. The enzyme is also associated with RNA containing uracil, adenine, guanine, and cytosine with no unusual bases detected.

  15. Enzyme clustering can induce metabolic channeling

    NASA Astrophysics Data System (ADS)

    Castellana, Michele

    2015-03-01

    Direct channeling of intermediates via a physical tunnel between enzyme active sites is an established mechanism to improve metabolic efficiency. In this talk, I will present a theoretical model that demonstrates that coclustering multiple enzymes into proximity can yield the full efficiency benefits of direct channeling. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with the spacing between coclusters in yeast and mammalian cells. The model also predicts that enzyme agglomerates can regulate steady-state flux division at metabolic branch points: we experimentally test this prediction for a fundamental branch point in Escherichia coli, and the results confirm that enzyme colocalization within an agglomerate can accelerate the processing of a shared intermediate by one branch. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.

  16. On the hydrodynamics of swimming enzymes

    E-print Network

    Xiaoyu Bai; Peter G. Wolynes

    2015-10-07

    Several recent experiments suggest that rather generally the diffusion of enzymes may be augmented through their activity. We demonstrate that such swimming motility can emerge from the interplay between the enzyme energy landscape and the hydrodynamic coupling of the enzyme to its environment. Swimming thus occurs during the transit time of a transient allosteric change. We estimate the velocity during the transition. The analysis of such a swimming motion suggests the final stroke size is limited by the hydrodynamic size of the enzyme. This limit is quite a bit smaller than the values that can be inferred from the recent experiments. We also show that one proposed explanation of the experiments based on reaction heat effects can be ruled out using an extended hydrodynamic analysis. These results lead us to propose an alternate explanation of the fluorescence correlation measurements.

  17. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...REGULATIONS ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act...the foreign trade of the United States in apples to protect the reputation of...

  18. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...REGULATIONS ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act...the foreign trade of the United States in apples to protect the reputation of...

  19. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...REGULATIONS ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act...the foreign trade of the United States in apples to protect the reputation of...

  20. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...REGULATIONS ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act...the foreign trade of the United States in apples to protect the reputation of...

  1. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...REGULATIONS ISSUED UNDER AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act...the foreign trade of the United States in apples to protect the reputation of...

  2. Extracellular enzyme kinetics scale with resource availability

    USGS Publications Warehouse

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  3. Tissue enzyme studies in Macaca nemestrina monkeys.

    NASA Technical Reports Server (NTRS)

    Hubbard, R. W.; Hoffman, R. A.; Jenkins, D.

    1971-01-01

    Total enzyme activities in fresh tissue specimens from major organs of Macaca nemestrina were analyzed for lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and aldolase. The concentration of these enzymes varied among the different tissue with skeletal muscle, heart, and brain having the highest activities. LDH isozymes determinations for the various tissues were also made. The spectrum of LDH isozyme distribution appears to be quite specific and characteristic for at least some of the tissues analyzed.

  4. Dry-Enzyme Test For Gaseous Chemicals

    NASA Technical Reports Server (NTRS)

    Barzana, Eduardo; Karel, Marcus; Klibanov, Alexander

    1990-01-01

    Simple, dry-chemical test detects ethanol in human breath. Method of test also adapted to detection of such toxic chemicals as formaldehyde in airstreams. Used qualitatively to detect chemical compounds above present level; for example, ethanol above legal level for driving. Also used to indicate quantitatively concentrations of compounds. Involves dry enzyme and color indicator. Adapted to detect any gaseous compound transformed by enzymes to produce change evident to human eye or to instrument.

  5. Rhamnogalacturonan I modifying enzymes: an update.

    PubMed

    Silva, Inês R; Jers, Carsten; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

    2016-01-25

    Rhamnogalacturonan I (RGI) modifying enzymes catalyse the degradation of the RGI backbone and encompass enzymes specific for either the ?1,2-bond linking galacturonic acid to rhamnose or the ?1,4-bond linking rhamnose to galacturonic acid in the RGI backbone. The first microbial enzyme found to be able to catalyse the degradation of the RGI backbone, an endo-hydrolase (EC 3.2.1.171) derived from Aspergillus aculeatus, was discovered 25 years ago. Today the group of RGI modifying enzymes encompasses endo- and exo-hydrolases as well as lyases. The RGI hydrolases, EC 3.2.1.171-EC 3.2.1.174, have been described to be produced by Aspergillus spp. and Bacillus subtilis and are categorized in glycosyl hydrolase families 28 and 105. The RGI lyases, EC 4.2.2.23-EC 4.2.2.24, have been isolated from different fungi and bacterial species and are categorized in polysaccharide lyase families 4 and 11. This review brings together the available knowledge of the RGI modifying enzymes and provides a detailed overview of biocatalytic reaction characteristics, classification, structure-function traits, and analyses the protein properties of these enzymes by multiple sequence alignments in neighbour-joining phylogenetic trees. Some recently detected unique structural features and dependence of calcium for activity of some of these enzymes (notably the lyases) are discussed and newly published results regarding improvement of their thermostability by protein engineering are highlighted. Knowledge of these enzymes is important for understanding microbial plant cell wall degradation and for advancing enzymatic processing and biorefining of pectinaceous plant biomass. PMID:26255130

  6. Glycolytic and Related Enzymes in Clostridial Classification

    PubMed Central

    Kot?e, J. P.

    1969-01-01

    The activities of 15 glycolytic and related enzymes were determined in clostridia. All contained 1-phosphofructokinase; three of them lacked 6-phosphofructokinase and mannitol 1-phosphate dehydrogenase. Glucose 6-phosphate dehydrogenase was found in six clostridia, thus demonstrating the presence of hexose monophosphate shunt. Only parts of the citric acid cycle were found to be present in most clostridia with an indication of the full cycle in Clostridium septicum. The intermediary enzyme activities were used to differentiate between the different clostridia. PMID:4244302

  7. Clean Water Act: A primer

    SciTech Connect

    Not Available

    1992-04-01

    The primer is based on documents prepared by the EPA and the Bureau of National Affairs, and covers a number of the significant provisions of the Clean Water Act as amended by the Water Quality Act of 1987.

  8. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (?-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of ?-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  9. Advanced Communications Technology Satellite (ACTS)

    NASA Technical Reports Server (NTRS)

    Schertler, Ronald J.; Gedney, Richard T.

    1992-01-01

    An overview of the NASA ACTS program is presented. The key technologies of ACTS include spot beams, on-board baseband processing and routing, wide bandwidth (900 MHz), and Ka-band transponders. The discussion covers system description, current status of the spacecraft development, ACTS earth stations, NGS traffic terminal, USAT, land and aeronautical mobiles, high data rate and propagation receive only terminals, and ACTS experiments program.

  10. Speech Acts and Conversational Interaction.

    ERIC Educational Resources Information Center

    Geis, Michael L.

    This book unites speech act theory and conversation analysis to advance a theory of conversational competence, called the Dynamic Speech Act Theory (DSAT). In contrast to traditional speech act theory that focuses almost exclusively on intuitive assessments of isolated, constructed examples, this theory is predicated on the assumption that speech…

  11. Family Educational Rights & Privacy Act

    E-print Network

    Spirtes, Peter

    or handicap in violation of Title VI of the Civil Rights Act of 1964, Title IX of the Educational AmendmentsFamily Educational Rights & Privacy Act (FERPA) FOR PARENTS Statement of Assurance Carnegie Mellon Educational Rights and Privacy Act (also called FERPA or the Buckley Amendment) that sets privacy standards

  12. Family Educational Rights & Privacy Act

    E-print Network

    Spirtes, Peter

    or handicap in violation of Title VI of the Civil Rights Act of 1964, Title IX of the Educational AmendmentsFamily Educational Rights & Privacy Act (FERPA) Statement of Assurance Carnegie Mellon University complying fully with the Family Educational Rights and Privacy Act (FERPA). FERPA covers the release

  13. Mr. Manny Pangelinan Acting Secretary

    E-print Network

    by the Magnuson-Stevens Fishery Conservation and Management Act, as amended by the Shark Conservation Act of2010 no directed federal shark fisheries exist as permitted under the Magnuson- Stevens Fishery Conservation#12;#12;Mr. Manny Pangelinan Acting Secretary Department of Land and Natural Resources Commonwealth

  14. Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities.

    PubMed Central

    Tomme, P; Kwan, E; Gilkes, N R; Kilburn, D G; Warren, R A

    1996-01-01

    The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45 degrees C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulose belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities. PMID:8763951

  15. Production of keratinolytic enzyme by an indigenous feather-degrading strain Bacillus cereus Wu2.

    PubMed

    Lo, Wei-Hsun; Too, Jui-Rze; Wu, Jane-Yii

    2012-12-01

    A novel feather-degrading microorganism was isolated from a poultry farm in Taiwan, and was identified Bacillus cereus Wu2 according to 16S rRNA sequencing. The isolated strain produces keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source. The experimental results indicated that the extra carbon sources (glucose, fructose, starch, sucrose, or lactose) could act as a catabolite repressor to the enzyme secretion or keratinolytic activity when keratinous substrates were employed as protein sources. However, addition of 2 g/L of NH(4)Cl to the feather medium increased the enzyme production. The optimum temperature and initial pH for enzyme production were 30°C and 7.0, respectively. The maximum yield of the enzyme was 1.75 kU/mL in the optimal chicken feather medium; this value was about 17-fold higher than the yield in the basal hair medium. The B. cereus Wu2 possessed disulfide reductase activity along with keratinolytic activity. The amino acid contents of feathers degradated by B. cereus Wu2 were higher, especially for lysine, methionine and threonine which were nutritionally essential amino acids and usually deficient in the feather meal. Thus, B. cereus Wu2 could be not only used to enhance the nutritional value of feather meal but is also a potential bioinoculant in agricultural environments. PMID:22999356

  16. Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

    PubMed

    Markmann, Sandra; Thelen, Melanie; Cornils, Kerstin; Schweizer, Michaela; Brocke-Ahmadinejad, Nahal; Willnow, Thomas; Heeren, Joerg; Gieselmann, Volkmar; Braulke, Thomas; Kollmann, Katrin

    2015-07-01

    Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes. PMID:25786328

  17. Stability and reaction characteristics of reverse-phase evaporation vesicles (REVs) as enzyme containers.

    PubMed

    Sada, E; Katoh, S; Terashima, M; Shiraga, H; Miura, Y

    1990-10-01

    The ion-dependent enzymes, activities of which are affected by ions, were entrapped in the inside aqueous phase of reverse-phase evaporation vesicles (REVs) to protect the enzyme activities from inhibitory cations. Their activities were still preserved in the presence of the inhibitory cations because the permeabilities of the inhibitory cations through the lipid membranes of REVs were much lower than those of substrates and products. The REVs were relatively stable in hypertonic condition, while they were unstable in hypotonic condition. Changes in osmotic pressure difference largely affected solute permeabilities of REVs. The shrinking and swelling of REVs resulting from osmotic pressure differences led to these changes in stabilities and solute permeabilities. Although the entrapment of the enzyme into REVs showed good protective effects against divalent cations, it was not effective against univalent cations. The measurements of cation permeabilities revealed that the enzyme trapped in the bilayer regions of REVs acts as a selective channel for the univalent cations. The REVs containing the enzyme could be used without any activity losses in a hollow fiber module. PMID:18597257

  18. ACTS broadband aeronautical experiment

    NASA Technical Reports Server (NTRS)

    Abbe, Brian S.; Jedrey, Thomas C.; Estabrook, Polly; Agan, Martin J.

    1993-01-01

    In the last decade, the demand for reliable data, voice, and video satellite communication links between aircraft and ground to improve air traffic control, airline management, and to meet the growing demand for passenger communications has increased significantly. It is expected that in the near future, the spectrum required for aeronautical communication services will grow significantly beyond that currently available at L-band. In anticipation of this, JPL is developing an experimental broadband aeronautical satellite communications system that will utilize NASA's Advanced Communications Technology Satellite (ACTS) as a satellite of opportunity and the technology developed under JPL's ACTS Mobile Terminal (AMT) Task to evaluate the feasibility of using K/Ka-band for these applications. The application of K/Ka-band for aeronautical satellite communications at cruise altitudes is particularly promising for several reasons: (1) the minimal amount of signal attenuation due to rain; (2) the reduced drag due to the smaller K/Ka-band antennas (as compared to the current L-band systems); and (3) the large amount of available bandwidth. The increased bandwidth available at these frequencies is expected to lead to significantly improved passenger communications - including full-duplex compressed video and multiple channel voice. A description of the proposed broadband experimental system will be presented including: (1) applications of K/Ka-band aeronautical satellite technology to U.S. industry; (2) the experiment objectives; (3) the experiment set-up; (4) experimental equipment description; and (5) industrial participation in the experiment and the benefits.

  19. Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

    PubMed Central

    Payne, Christina M.; Resch, Michael G.; Chen, Liqun; Crowley, Michael F.; Himmel, Michael E.; Taylor, Larry E.; Sandgren, Mats; Ståhlberg, Jerry; Stals, Ingeborg; Tan, Zhongping; Beckham, Gregg T.

    2013-01-01

    Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. PMID:23959893

  20. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1)

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good knowledge of the mechanisms of herbal drug interactions is necessary for assessing and minimizing clinical risks. These processes help prediction of interactions between herbal supplements and prescription drugs. Healthcare professionals should remain vigilant for potential interactions between herbal supplements/medicines and prescription drugs, especially for drugs with a narrow therapeutic index are used. PMID:26417265

  1. Structural Variation in Bacterial Glyoxalase I Enzymes

    PubMed Central

    Suttisansanee, Uthaiwan; Lau, Kelvin; Lagishetty, Satyanarayana; Rao, Krishnamurthy N.; Swaminathan, Subramanyam; Sauder, J. Michael; Burley, Stephen K.; Honek, John F.

    2011-01-01

    The glyoxalase system catalyzes the conversion of toxic, metabolically produced ?-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn2+ and non-Zn2+ activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni2+/Co2+-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn2+-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements. PMID:21914803

  2. Geometric and electronic structure contributions to function in non-heme iron enzymes.

    PubMed

    Solomon, Edward I; Light, Kenneth M; Liu, Lei V; Srnec, Martin; Wong, Shaun D

    2013-11-19

    Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)?O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for EAS without O-O cleavage. This activation is important in one class of NHFe enzymes that utilizes a HS Fe(III)-OOH intermediate in dioxygenation. For Fe(IV)?O intermediates, the LS form has a ?-type FMO activated for attack perpendicular to the Fe-O bond. However, the HS form (present in the NHFe enzymes) has a ? FMO activated perpendicular to the Fe-O bond and a ? FMO positioned along the Fe-O bond. For the NHFe enzymes, the presence of ? and ? FMOs enables enzymatic control in determining the type of reactivity: EAS or H-atom extraction for one substrate with different enzymes and halogenation or hydroxylation for one enzyme with different substrates. PMID:24070107

  3. Thermophilic Fungi: Their Physiology and Enzymes

    PubMed Central

    Maheshwari, Ramesh; Bharadwaj, Girish; Bhat, Mahalingeshwara K.

    2000-01-01

    Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes. PMID:10974122

  4. Triple acting radial seal

    DOEpatents

    Ebert, Todd A (West Palm Beach, FL); Carella, John A (Jupiter, FL)

    2012-03-13

    A triple acting radial seal used as an interstage seal assembly in a gas turbine engine, where the seal assembly includes an interstage seal support extending from a stationary inner shroud of a vane ring, the interstage seal support includes a larger annular radial inward facing groove in which an outer annular floating seal assembly is secured for radial displacement, and the outer annular floating seal assembly includes a smaller annular radial inward facing groove in which an inner annular floating seal assembly is secured also for radial displacement. A compliant seal is secured to the inner annular floating seal assembly. The outer annular floating seal assembly encapsulates the inner annular floating seal assembly which is made from a very low alpha material in order to reduce thermal stress.

  5. Federal Employees' Compensation Act.

    PubMed

    Ladou, Joseph

    2009-01-01

    The Federal Employees' Compensation Act (FECA) program provides wage loss compensation and payments for medical treatment to federal civilian employees. Administered by the Department of Labor (DOL), FECA covers over 2.7 million federal employees in more than 70 different agencies. FECA costs rose from $1.4 billion in 1990 to $2.6 in 2006, while the federal workforce remained essentially unchanged. While federal civilian employees represent only 2.1% of all workers eligible for workers' compensation benefits, federal programs account for 6% of the benefits paid. Disability benefits under FECA are far greater than those in the state workers' compensation programs. The benefit payments often exceed the former salary of the injured employee. The last congressional hearings on the FECA program were held over thirty years ago. It is unlikely that Congressional review will occur any time soon, as the entrenched bureaucracy that benefits from the FECA program defines and protects its future. PMID:19496485

  6. ACTS mobile propagation campaign

    NASA Technical Reports Server (NTRS)

    Goldhirsh, Julius; Vogel, Wolfhard J.; Torrence, Geoffrey W.

    1994-01-01

    Preliminary results are presented for three propagation measurement campaigns involving a mobile receiving laboratory and 20 GHz transmissions from the Advanced Communications Technology Satellite (ACTS). Four 1994 campaigns were executed during weekly periods in and around Austin, Texas in February and May, in Central Maryland during March, and in Fairbanks, Alaska and environs in June. Measurements tested the following effects at 20 GHz: (1) attenuation due to roadside trees with and without foliage, (2) multipath effects for scenarios in which line-of-sight paths were unshadowed, (3) fades due to terrain and roadside obstacles, (4) fades due to structures in urban environs, (5) single tree attenuation, and (6) effects of fading at low elevation angles (8 deg in Fairbanks, Alaska) and high elevation angles (55 deg in Austin, Texas). Results presented here cover sampled measurements in Austin, Texas for foliage and non-foliage cases and in Central Maryland for non-foliage runs.

  7. Type I restriction enzymes and their relatives

    PubMed Central

    Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.

    2014-01-01

    Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes. PMID:24068554

  8. Enzyme-Operated DNA-Based Nanodevices.

    PubMed

    Del Grosso, Erica; Dallaire, Anne-Marie; Vallée-Bélisle, Alexis; Ricci, Francesco

    2015-12-01

    Functional molecular nanodevices and nanomachines have attracted a growing interest for their potential use in life science and nanomedicine. In particular, due to their versatility and modularity DNA-based nanodevices appear extremely promising. However, a limitation of such devices is represented by the limited number of molecular stimuli and cues that can be used to control and regulate their function. Here we demonstrate the possibility to rationally control and regulate DNA-based nanodevices using biocatalytic reactions catalyzed by different enzymes. To demonstrate the versatility of our approach, we have employed three model DNA-based systems and three different enzymes (belonging to several classes, i.e., transferases and hydrolases). The possibility to use enzymes and enzymatic substrates as possible cues to operate DNA-based molecular nanodevices will expand the available toolbox of molecular stimuli to be used in the field of DNA nanotechnology and could open the door to many applications including enzyme-induced drug delivery and enzyme-triggered nanostructures assembly. PMID:26600418

  9. Sensitive radioenzymatic assay for epinephrine forming enzymes

    SciTech Connect

    Ziegler, M.G.; Kennedy, B.; Elayan, H.

    1988-01-01

    Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces /sup 3/H-E from norepinephrine (NE) and /sup 3/H-S-adenosylmethionine. The /sup 3/H-E is isolated on alumina, /sup 3/H-S-adenosylmethionine is precipitated and the /sup 3/H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.

  10. Enzyme-Operated DNA-Based Nanodevices

    PubMed Central

    2015-01-01

    Functional molecular nanodevices and nanomachines have attracted a growing interest for their potential use in life science and nanomedicine. In particular, due to their versatility and modularity DNA-based nanodevices appear extremely promising. However, a limitation of such devices is represented by the limited number of molecular stimuli and cues that can be used to control and regulate their function. Here we demonstrate the possibility to rationally control and regulate DNA-based nanodevices using biocatalytic reactions catalyzed by different enzymes. To demonstrate the versatility of our approach, we have employed three model DNA-based systems and three different enzymes (belonging to several classes, i.e., transferases and hydrolases). The possibility to use enzymes and enzymatic substrates as possible cues to operate DNA-based molecular nanodevices will expand the available toolbox of molecular stimuli to be used in the field of DNA nanotechnology and could open the door to many applications including enzyme-induced drug delivery and enzyme-triggered nanostructures assembly. PMID:26600418

  11. Development of enzymes and enzyme systems by genetic engineering to convert biomass to sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TITLE Development of Enzymes and Enzyme Systems by Genetic Engineering to Convert Biomass to Sugars ABSTRACT Plant cellulosic material is one of the most viable renewable resources for the world’s fuel and chemical feedstock needs. Currently ethanol derived from corn starch is the most common li...

  12. Structural studies of the pigeon cytosolic -dependent malic enzyme

    E-print Network

    Tong, Liang

    Structural studies of the pigeon cytosolic NADP+ -dependent malic enzyme ZHIRU YANG,1 HAILONG ZHANG decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP+ -dependent malic enzyme, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One

  13. Understanding bistability in complex enzyme-driven reaction networks

    E-print Network

    Craciun, Gheorghe

    to variations in enzyme transcription activity (or even to enzyme malformation) when, in fact, those changesUnderstanding bistability in complex enzyme-driven reaction networks Gheorghe Craciun* , Yangzhong for enzyme catalysis of a single overall reaction. We present a theorem that distinguishes between those mass

  14. Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes

    E-print Network

    Goddard III, William A.

    Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes Patrick L. Wintrode1 basis for the remarkable stability of enzymes isolated from thermophilic organisms has been the subject enzymes with those of homologous enzymes from mesophilic organisms.1,2 These studies have found many types

  15. Seeing & Feeling How Enzymes Work Using Tangible Models

    ERIC Educational Resources Information Center

    Lau, Kwok-chi

    2013-01-01

    This article presents a tangible model used to help students tackle some misconceptions about enzyme actions, particularly the induced-fit model, enzyme-substrate complementarity, and enzyme inhibition. The model can simulate how substrates induce a change in the shape of the active site and the role of attraction force during enzyme-substrate…

  16. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement...

  17. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement...

  18. Stochastic Simulation of Enzyme-Catalyzed Reactions with Disparate Timescales

    E-print Network

    Paul, Mark

    Stochastic Simulation of Enzyme-Catalyzed Reactions with Disparate Timescales Debashis Barik-steady-state approximation'' for enzyme-catalyzed reactions provides a useful framework for efficient and accurate stochastic simulations. The method is applied to three examples: a simple enzyme-catalyzed reaction where enzyme

  19. Directed Enzyme Evolution and High-Throughput Screening

    E-print Network

    Zhao, Huimin

    3 Directed Enzyme Evolution and High-Throughput Screening Michael J. McLachlan,1 Ryan P. Sullivan2, and Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3.1 Introduction Early enzyme of a complex mixture of secreted enzymes produced at low yields. Now, over 90% of industrial enzymes

  20. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement...

  1. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for clinical use is a device intended to measure enzymes in plasma or serum by nonkinetic or kinetic measurement...

  2. PURIFICATION AND ISOLATION OF THE PHOSPHOGLYCERATE KINASE ENZYME

    E-print Network

    Collins, Gary S.

    PURIFICATION AND ISOLATION OF THE PHOSPHOGLYCERATE KINASE ENZYME IN RICE PLANTS Cynthia Bach (NADH). The enzyme, phosphoglycerate kinase (PGKase), catalyzes the reaction that results to purify the enzyme from developing rice seeds. Isolation of the enzyme was obtained by obtaining a crude

  3. Controlling reaction specificity in pyridoxal phosphate enzymes

    PubMed Central

    Toney, Michael D.

    2012-01-01

    Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly ?-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at C? of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. PMID:21664990

  4. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  5. Contributions of Human Enzymes in Carcinogen Metabolism

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2012-01-01

    Considerable support exists for roles of metabolism in modulating the carcinogenic properties of chemicals. In particular, many of these compounds are procarcinogens that require activation to electrophilic forms to exert genotoxic effects. We systematically analyzed the existing literature on metabolism of carcinogens by human enzymes, which has been developed largely in the past 25 years. The metabolism and especially bioactivation of carcinogens are dominated by cytochrome P450 enzymes (66% of bioactivations). Within this group, six P450s—1A1, 1A2, 1B1, 2A6, 2E1, and 3A4—accounted for 77% of the P450 activation reactions. The roles of these P450s can be compared with those estimated for drug metabolism and should be considered in issues involving enzyme induction, chemoprevention, molecular epidemiology, inter-individual variations, and risk assessment. PMID:22531028

  6. Oral enzyme therapy for celiac sprue

    PubMed Central

    Bethune, Michael T; Khosla, Chaitan

    2012-01-01

    Celiac sprue is an inflammatory disease of the small intestine caused by dietary gluten and treated by adherence to a lifelong gluten-free diet. The recent identification of immunodominant gluten peptides, the discovery of their cogent properties, and the elucidation of the mechanisms by which they engender immunopathology in genetically-susceptible individuals have advanced our understanding of the molecular pathogenesis of this complex disease, enabling the rational design of new therapeutic strategies. The most clinically advanced of these is oral enzyme therapy, in which enzymes capable of proteolyzing gluten (i.e. glutenases) are delivered to the alimentary tract of a celiac sprue patient to detoxify ingested gluten in situ. In this chapter, we discuss the key challenges for discovery and preclinical development of oral enzyme therapies for celiac sprue. Methods for lead identification, assay development, gram-scale production and formulation, and lead optimization for next-generation proteases are described and critically assessed. PMID:22208988

  7. Measuring enzyme activity in single cells

    PubMed Central

    Kovarik, Michelle L.; Allbritton, Nancy L.

    2011-01-01

    Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range of techniques to obtain these data, including image-, flow- and separation-based assays. Research to date has focused on easy-to-measure glycosylases and clinically-relevant kinases. Expansion of these techniques to a wider range and larger number of enzymes will answer contemporary questions in proteomics and glycomics, specifically with respect to biological noise and cellular heterogeneity. PMID:21316781

  8. Measuring enzyme activity in single cells.

    PubMed

    Kovarik, Michelle L; Allbritton, Nancy L

    2011-05-01

    Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range of techniques to obtain these data, including image-, flow- and separation-based assays. Research to date has focused on easy-to-measure glycosylases and clinically-relevant kinases. Expansion of these techniques to a wider range and larger number of enzymes will answer contemporary questions in proteomics and glycomics, specifically with respect to biological noise and cellular heterogeneity. PMID:21316781

  9. Enzyme Nanoparticles-Based Electronic Biosensor

    SciTech Connect

    Liu, Guodong; Lin, Yuehe; Ostatna, V.; Wang, Joseph

    2005-06-28

    A novel method for fabricating electronic biosensors based on coupling enzyme nanoparticles and self assembly technology is illustrated. Redox horseradish peroxidase nanoparticles were prepared by desolvation with ethanol and subsequent crosslinking with glutaraldehyde. The cross-linked enzyme nanoparticles were functionalized by cysteine to introduce thiol groups on the nanoparticle surface. Immobilized enzyme nanoparticle on the gold electrode by self-assembly kept redox and electrocatalytic activities, and was used to develop reagentless biosensors for H2O2 detection without promoters and mediators. The new approach is simple, low cost and circumvents complications associated with solution systems. It is a universal immobilization method for biosensor, biomedical devices, biofuel cells and enzymatic bioreactors fabrication and expected to open new opportunities for biosensor, clinical diagnostics, and for bioanalysis, in general.

  10. 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

  11. Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.

    PubMed Central

    Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

    1995-01-01

    The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511

  12. Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties*

    PubMed Central

    Schalk, Amanda M.; Nguyen, Hien-Anh; Rigouin, Coraline; Lavie, Arnon

    2014-01-01

    The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with l-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial l-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of l-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes. PMID:25320094

  13. Inhibition of secretory phospholipase A(2) enzyme by bilirubin: a new role as endogenous anti-inflammatory molecule.

    PubMed

    Jameel, Noor Mohamed; Frey, Brigitte M; Frey, Felix J; Gowda, T Veerabasappa; Vishwanath, Bannikuppe S

    2005-08-01

    Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA(2) enzyme is not known. Inhibition of several secretory phospholipase A(2) (sPLA(2)) enzyme activities by bilirubin was studied using (14)C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA(2) enzyme from Vipera russellii and Naja naja venom and partially purified sPLA(2) enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC(50) values calculated for these enzymes ranges from 1.75 to 10.5 microM. Inflammatory human sPLA(2) enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA(2)s. Inhibition of sPLA(2) activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA(2) by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA(2) in a dose dependent manner in the same concentration range at which in vitro sPLA(2) inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA(2) was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA(2) induced mouse paw edema by bilirubin confirms its sPLA(2) inhibitory activity in vivo also. These findings indicate that inhibition of sPLA(2) by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA(2) enzyme activity. PMID:16132704

  14. Double acting bit holder

    SciTech Connect

    Morrell, Roger J.; Larson, David A.; Ruzzi, Peter L.

    1994-01-01

    A double acting bit holder that permits bits held in it to be resharpened during cutting action to increase energy efficiency by reducing the amount of small chips produced. The holder consist of: a stationary base portion capable of being fixed to a cutter head of an excavation machine and having an integral extension therefrom with a bore hole therethrough to accommodate a pin shaft; a movable portion coextensive with the base having a pin shaft integrally extending therefrom that is insertable in the bore hole of the base member to permit the moveable portion to rotate about the axis of the pin shaft; a recess in the movable portion of the holder to accommodate a shank of a bit; and a biased spring disposed in adjoining openings in the base and moveable portions of the holder to permit the moveable portion to pivot around the pin shaft during cutting action of a bit fixed in a turret to allow front, mid and back positions of the bit during cutting to lessen creation of small chip amounts and resharpen the bit during excavation use.

  15. Acting to gain information

    NASA Technical Reports Server (NTRS)

    Rosenchein, Stanley J.; Burns, J. Brian; Chapman, David; Kaelbling, Leslie P.; Kahn, Philip; Nishihara, H. Keith; Turk, Matthew

    1993-01-01

    This report is concerned with agents that act to gain information. In previous work, we developed agent models combining qualitative modeling with real-time control. That work, however, focused primarily on actions that affect physical states of the environment. The current study extends that work by explicitly considering problems of active information-gathering and by exploring specialized aspects of information-gathering in computational perception, learning, and language. In our theoretical investigations, we analyzed agents into their perceptual and action components and identified these with elements of a state-machine model of control. The mathematical properties of each was developed in isolation and interactions were then studied. We considered the complexity dimension and the uncertainty dimension and related these to intelligent-agent design issues. We also explored active information gathering in visual processing. Working within the active vision paradigm, we developed a concept of 'minimal meaningful measurements' suitable for demand-driven vision. We then developed and tested an architecture for ongoing recognition and interpretation of visual information. In the area of information gathering through learning, we explored techniques for coping with combinatorial complexity. We also explored information gathering through explicit linguistic action by considering the nature of conversational rules, coordination, and situated communication behavior.

  16. 77 FR 27532 - No FEAR Act Notice

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-10

    ...GENERAL FOR AFGHANISTAN RECONSTRUCTION No FEAR Act Notice AGENCY: Special Inspector General...Afghanistan Reconstruction's (SIGAR) ``No FEAR Act Notice'' Federal Register publication...Antidiscrimination and Retaliation Act of 2002 (No FEAR) Act and by the Office of Personnel...

  17. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER General Provisions Definitions § 63.1 Act. Act means section 375 of the...

  18. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER General Provisions Definitions § 63.1 Act. Act means section 375 of the...

  19. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER General Provisions Definitions § 63.1 Act. Act means section 375 of the...

  20. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER General Provisions Definitions § 63.1 Act. Act means section 375 of the...

  1. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...STANDARD CONTAINER REGULATIONS EXPORT GRAPES AND PLUMS Definitions § 35.1 Act. Act or Export Grape and Plum Act means “An Act to promote the foreign trade of the United States in grapes and plums, to protect the...

  2. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...STANDARD CONTAINER REGULATIONS EXPORT GRAPES AND PLUMS Definitions § 35.1 Act. Act or Export Grape and Plum Act means “An Act to promote the foreign trade of the United States in grapes and plums, to protect the...

  3. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...STANDARD CONTAINER REGULATIONS EXPORT GRAPES AND PLUMS Definitions § 35.1 Act. Act or Export Grape and Plum Act means “An Act to promote the foreign trade of the United States in grapes and plums, to protect the...

  4. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...STANDARD CONTAINER REGULATIONS EXPORT GRAPES AND PLUMS Definitions § 35.1 Act. Act or Export Grape and Plum Act means “An Act to promote the foreign trade of the United States in grapes and plums, to protect the...

  5. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...STANDARD CONTAINER REGULATIONS EXPORT GRAPES AND PLUMS Definitions § 35.1 Act. Act or Export Grape and Plum Act means “An Act to promote the foreign trade of the United States in grapes and plums, to protect the...

  6. Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1).

    PubMed

    O'Neill, Ellis C; Stevenson, Clare E M; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I; Rejzek, Martin; Nepogodiev, Sergey A; Limpaseni, Tipaporn; Field, Robert A; Lawson, David M

    2015-12-11

    The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-?-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms. PMID:26504082

  7. Non-homologous isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution

    PubMed Central

    2010-01-01

    Background Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. Results We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. Conclusions These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity. Reviewers This article was reviewed by Andrei Osterman, Keith F. Tipton (nominated by Martijn Huynen) and Igor B. Zhulin. For the full reviews, go to the Reviewers' comments section. PMID:20433725

  8. Investigation of Lasso Peptides Maturation Enzymes

    E-print Network

    Petta, Jason

    · Therapeutic effect · Receptor antagonism activity · Form of a lasso or a slipknot which the N in vitro to study kinetic parameters of the enzymes in detail. · Successful cloning. · Successful in this project were E. coli. · XL1 ­ Blue o Cloning · BL21 o Protein Expression #12;#12;Gene Expression Western

  9. An NMR Study of Enzyme Activity.

    ERIC Educational Resources Information Center

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  10. CO2 Capture with Enzyme Synthetic Analogue

    SciTech Connect

    Harry Cordatos

    2010-03-01

    Project overview provides background on carbonic anhydrase transport mechanism for CO2 in the human body and proposed approach for ARPA-E project to create a synthetic enzyme analogue and utilize it in a membrane for CO2 capture from flue gas.

  11. Targeting Mycobacterial Enzymes with Natural Products.

    PubMed

    Sieniawska, Elwira

    2015-10-22

    Tuberculosis (TB) is a recurring threat to contemporary civilization. It affects not only those within developing countries, but has also appeared again in places where it was once considered eradicated. TB co-infection in patients infected by HIV is, at the time of writing, the most common cause of death. In the field of searching for new antimycobacterial drug leads, compounds of natural origin still remain a promising source. The review is intended to gather information about natural products (metabolites of plants, fungi, bacteria, and marine sponges) that show activity against mycobacterial enzymes. Here, natural metabolites are presented as being inhibitors/activators of the mycobacterial enzymes involved in mycobacterial growth in vitro (ClpC1, ClpP, MurE ligase, mycothiol S-conjugate amidase, ?-ketoacyl-ACP synthase, InhA) and in vivo, as regards the host cell (PtpB). Each enzyme is briefly described so as to generate an understanding of its role in mycobacterial growth and engender a perception of the mechanism of action of the studied natural compounds. Furthermore, after the introduction of the enzyme, its inhibitors are listed and exactly characterized. PMID:26441042

  12. Computationally designed libraries for rapid enzyme stabilization

    PubMed Central

    Wijma, Hein J.; Floor, Robert J.; Jekel, Peter A.; Baker, David; Marrink, Siewert J.; Janssen, Dick B.

    2014-01-01

    The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10–12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution. PMID:24402331

  13. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme preparations. 864.4400 Section 864.4400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400...

  14. 21 CFR 864.4400 - Enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enzyme preparations. 864.4400 Section 864.4400 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400...

  15. Enzyme Kinetics: The Use of Amylose Azure.

    ERIC Educational Resources Information Center

    Cusimano, Vincent J.

    1978-01-01

    Amylose azure can be used as a chromogenic substrate for alpha-amylase in studying the effects of temperature and pH enzyme action. This is a model system which students can use to measure the energy of activation using the Arrhenius plot. (Author/BB)

  16. Computationally designed libraries for rapid enzyme stabilization.

    PubMed

    Wijma, Hein J; Floor, Robert J; Jekel, Peter A; Baker, David; Marrink, Siewert J; Janssen, Dick B

    2014-02-01

    The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution. PMID:24402331

  17. Enzyme Substrate Reactions in High Magnetic Fields

    PubMed Central

    Maling, J. E.; Weissbluth, M.; Jacobs, E. E.

    1965-01-01

    The reaction rates of two enzyme substrate systems, ribonuclease-RNA and succinate-cytochrome c reductase, were followed as a function of magnetic field from zero to 48,000 gauss. The reaction rates remained constant to within 10 per cent. PMID:5884011

  18. A Comprehensive Enzyme Kinetic Exercise for Biochemistry

    ERIC Educational Resources Information Center

    Barton, Janice S.

    2011-01-01

    This article describes a comprehensive treatment of experimental enzyme kinetics strongly coupled to electronic data acquisition and use of spreadsheets to organize data and perform linear and nonlinear least-squares analyses, all in a manner that promotes development of important reasoning skills. Kinetic parameters are obtained for the stable…

  19. 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)

  20. Tissue Dissociation Enzyme Neutral Protease Assessment

    PubMed Central

    Breite, A.G.; Dwulet, F.E.; McCarthy, R.C.

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate–conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. PMID:20692405

  1. The bioscouring performance of four polygalacturonase enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) analyses of greige cotton fabrics bioscoured with a combination of ultrasound and endo- and exo-polygalacturonase enzymes obtained from Rhizopus sp. fungi were used in a fractional factorial design experiment to examine their perform...

  2. Transition state theory for enzyme kinetics.

    PubMed

    Truhlar, Donald G

    2015-09-15

    This article is an essay that discusses the concepts underlying the application of modern transition state theory to reactions in enzymes. Issues covered include the potential of mean force, the quantization of vibrations, the free energy of activation, and transmission coefficients to account for nonequilibrium effect, recrossing, and tunneling. PMID:26008760

  3. Biofilm-degrading enzymes from Lysobacter gummosus

    PubMed Central

    Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

    2014-01-01

    Biofilm-degrading enzymes could be used for the gentle cleaning of industrial and medical devices and the manufacture of biofilm-resistant materials. We therefore investigated 20 species and strains of the bacterial genus Lysobacter for their ability to degrade experimental biofilms formed by Staphylococcus epidermidis, a common nosocomial pathogen typically associated with device-related infections. The highest biofilm-degradation activity was achieved by L. gummosus. The corresponding enzymes were identified by sequencing the L. gummosus genome. Partial purification of the biofilm-degrading activity from an extract of extracellular material followed by peptide mass fingerprinting resulted in the identification of two peptidases (?-lytic protease and ?-lytic metalloendopeptidase) that were predicted to degrade bacterial cell walls. In addition, we identified two isoforms of a lysyl endopeptidase and an enzyme similar to metalloproteases from Vibrio spp. Potential peptidoglycan-binding C-terminal fragments of two OmpA-like proteins also co-purified with the biofilm-degrading activity. The L. gummosus genome was found to encode five isoenzymes of ?-lytic protease and three isoenzymes of lysyl endopeptidase. These results indicated that the extracellular digestion of biofilms by L. gummosus depends on multiple bacteriolytic and proteolytic enzymes, which could now be exploited for biofilm control. PMID:24518560

  4. Biofilm-degrading enzymes from Lysobacter gummosus.

    PubMed

    Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

    2014-04-01

    Biofilm-degrading enzymes could be used for the gentle cleaning of industrial and medical devices and the manufacture of biofilm-resistant materials. We therefore investigated 20 species and strains of the bacterial genus Lysobacter for their ability to degrade experimental biofilms formed by Staphylococcus epidermidis, a common nosocomial pathogen typically associated with device-related infections. The highest biofilm-degradation activity was achieved by L. gummosus. The corresponding enzymes were identified by sequencing the L. gummosus genome. Partial purification of the biofilm-degrading activity from an extract of extracellular material followed by peptide mass fingerprinting resulted in the identification of two peptidases (?-lytic protease and ?-lytic metalloendopeptidase) that were predicted to degrade bacterial cell walls. In addition, we identified two isoforms of a lysyl endopeptidase and an enzyme similar to metalloproteases from Vibrio spp. Potential peptidoglycan-binding C-terminal fragments of two OmpA-like proteins also co-purified with the biofilm-degrading activity. The L. gummosus genome was found to encode five isoenzymes of ?-lytic protease and three isoenzymes of lysyl endopeptidase. These results indicated that the extracellular digestion of biofilms by L. gummosus depends on multiple bacteriolytic and proteolytic enzymes, which could now be exploited for biofilm control. PMID:24518560

  5. Divergence and Convergence in Enzyme Evolution: Parallel

    E-print Network

    Tawfik, Dan S.

    Divergence and Convergence in Enzyme Evolution: Parallel Evolution of Paraoxonases from Quorum 76100, Israel We discuss the basic features of divergent versus convergent evolution and of the common protein ancestors (9, 10). Divergent, Convergent, or Perhaps Parallel Evolution? Divergent evolution can

  6. Assay for Angiotensin-Converting Enzyme.

    ERIC Educational Resources Information Center

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  7. Kemp elimination catalysts by computational enzyme design

    E-print Network

    Tawfik, Dan S.

    extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon--a process that is normally restricted by high activation-energy barriers7,8 . Computational catalysis. Here we describe the computational design of eight enzymes that use two different catalytic

  8. 3-Keto-5-aminohexanoate Cleavage Enzyme

    PubMed Central

    Bellinzoni, Marco; Bastard, Karine; Perret, Alain; Zaparucha, Anne; Perchat, Nadia; Vergne, Carine; Wagner, Tristan; de Melo-Minardi, Raquel C.; Artiguenave, François; Cohen, Georges N.; Weissenbach, Jean; Salanoubat, Marcel; Alzari, Pedro M.

    2011-01-01

    The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to catalyze already known reactions might still wait to be identified. Here, we provide a structural and biochemical characterization of the 3-keto-5-aminohexanoate cleavage enzyme (Kce), an enzymatic activity long known as being involved in the anaerobic fermentation of lysine but whose catalytic mechanism has remained elusive so far. Although the enzyme shows the ubiquitous triose phosphate isomerase (TIM) barrel fold and a Zn2+ cation reminiscent of metal-dependent class II aldolases, our results based on a combination of x-ray snapshots and molecular modeling point to an unprecedented mechanism that proceeds through deprotonation of the 3-keto-5-aminohexanoate substrate, nucleophilic addition onto an incoming acetyl-CoA, intramolecular transfer of the CoA moiety, and final retro-Claisen reaction leading to acetoacetate and 3-aminobutyryl-CoA. This model also accounts for earlier observations showing the origin of carbon atoms in the products, as well as the absence of detection of any covalent acyl-enzyme intermediate. Kce is the first representative of a large family of prokaryotic hypothetical proteins, currently annotated as the “domain of unknown function” DUF849. PMID:21632536

  9. A Qualitative Approach to Enzyme Inhibition

    ERIC Educational Resources Information Center

    Waldrop, Grover L.

    2009-01-01

    Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…

  10. Rapid purification of fluorescent enzymes by ultrafiltration

    NASA Technical Reports Server (NTRS)

    Benjaminson, M. A.; Satyanarayana, T.

    1983-01-01

    In order to expedite the preparation of fluorescently tagged enzymes for histo/cytochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

  11. Rapid purification of fluorescent enzymes by ultrafiltration

    NASA Technical Reports Server (NTRS)

    Benjaminson, M. A.; Satyanarayana, T.

    1983-01-01

    In order to expedite the preparation of fluorescently tagged enzymes for histo-cyctochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

  12. Ultrasound stimulated production of a fibrinolytic enzyme.

    PubMed

    Avhad, Devchand N; Rathod, Virendra K

    2014-01-01

    The present study is aimed at enhanced production of a fibrinolytic enzyme from Bacillus sphaericus MTCC 3672 under ultrasonic stimulation. Various process parameters viz; irradiation at different growth phases, ultrasonication power, irradiation duration, duty cycle and multiple irradiation were studied for enhancement of fibrinolytic enzyme productivity. The optimum conditions were found as follows, irradiation of ultrasonic waves to fermentation broth at 12 h of growth phase with 25 kHz frequency, 160 W ultrasound power, 20% duty cycle for 5 min. The productivity of fibrinolytic enzyme was increased 1.82-fold from 110 to 201 U/mL compared with the non sonicated control fermentation. Drop in glucose concentration from 0.41% to 0.12% w/v in ultrasonicated batch implies that, ultrasonication increases the cell permeability, improves substrate intake and progresses metabolism of microbial cell. Microscopic images before and after ultrasonic stimulation clearly signifies the impact of duty cycle on decreasing biomass concentration. However, environmental scanning electron micrograph does not show any cell lysis at optimum ultrasonic irradiation. Offshoots of our results will contribute to fulfill the demand of enhancement of microbial therapeutic enzyme productivity, through ultrasonication stimulation. PMID:23810338

  13. Ferredoxin-linked chloroplast enzymes. Progress report

    SciTech Connect

    1993-12-31

    This report summarizes research on ferredoxin:NADP{sup +} oxidoreductase and ferredoxin:thioredoxin reductase. One of the primary goals of the original proposal was to map the ferredoxin-binding sites on three soluble enzymes that are located in spinach chloroplasts and utilize ferredoxin as an electron donor:Ferredoxin:NADP{sup +} oxidoreductase (FNR); ferredoxin:thioredoxin reductase (FTR) and glutamate synthase. As the availability of amino acid sequences for the enzymes are important in such studies, it was proposed that the amino acid sequence of glutamate synthase be determined. The amino acid sequences of FNR, FTR and ferredoxin are already known. An aim related to elucidating the binding sites on these enzymes for ferredoxin was to determine whether there is a common site on ferredoxin involved in binding to all of these ferredoxin-dependent chloroplast enzymes and, if so, to map it. One additional aim was to characterize thioredoxin binding by FTR and determine whether the same site on FTR is involved in binding both ferredoxin and thioredoxin. Considerable progress has been made on most of these original projects, although work conducted on FTR is still in its preliminary stages.

  14. Chimeric Restriction Enzymes: What Is Next?

    PubMed Central

    Smith, Jeff

    2014-01-01

    Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-FN was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy. PMID:10494832

  15. Enzymes in stereoselective pharmacokinetics of endogenous substances.

    PubMed

    Marzo, A; Cardace, G; Arrigoni Martelli, E

    1992-01-01

    The use of enzymes to assay individual components of the L-carnitine family in pharmaceuticals, foodstuffs, and biological fluids with various forms of detection is reviewed. The most useful enzyme in the assay of compounds of the L-carnitine family is carnitine acetyl transferase (CAT), which catalyses the reversible interconversion of L-carnitine and its short-chain acyl esters. CAT can be used in one or more coupled reactions combined with U.V., or radiolabelled detection, or combined with HPLC, allowing, enantioselective, structurally specific, and, in the case of radiolabelled tracing, highly sensitive assays to be carried out. When compared with chromatographic separation of enantiomers or diastereoisomers, enantioselective enzyme mediated assays may be cheaper, more sensitive, and simpler, but they do not allow the nonpreferred isomer to be assayed. Consequently, they are appropriate for the specific assay of endogenous enantiomeric substrates of the enzyme concerned, in biological samples. The analysis of the other enantiomer in raw materials or in pharmaceuticals must be more properly approached by enantioselective chromatographic methods. PMID:1389962

  16. Enzyme specific activity in functionalized nanoporous supports

    NASA Astrophysics Data System (ADS)

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-01

    Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (PLD) in functionalized nanoporous supports so that the enzyme immobilization efficiency (Ie, defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH2- and HOOC-functionalized mesoporous silica (300 Å, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing PLD. With decreasing PLD, Ie of GOX in FMS increased from<35% to>150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing PLD. With increasing PLD, the corresponding Ie of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high PLD, consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high PLD and may promote a more favorable confinement environment that enhances the OPH activity.

  17. Dynamics of Radical-Mediated Enzyme Catalyses

    NASA Astrophysics Data System (ADS)

    Warncke, Kurt

    1997-11-01

    An emergent class of enzymes harnesses the extreme reactivity of electron-deficient free radical species to perform some of the most difficult reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. The common primary step in these catalyses is metal- or metallocenter-assisted generation of an electron-deficient organic "initiator radical". The initiator radical abstracts a hydrogen atom from the substrate, opening a new reaction channel for rearrangement to the product. Our aim is to elucidate the detailed molecular mechanisms of the radical pair separation and radical rearrangement steps. Radical pair separation and substrate radical rearrangement are tracked by using time-resolved (10-7 to 10-3 s) techniques of pulsed-electron paramagnetic resonance spectroscopy (FT-EPR, ESEEM). Synchronous time-evolution of the reactions is attained by triggering with a visible laser pulse. Transient non-Boltzmann population of the states of the spin-coupled systems, and resultant electron spin polarization, facilitates study at or near room temperature under conditions where the enzymes are operative. The systems examined include ethanolamine deaminase, a vitamin B12 coenzyme-dependent enzyme, ribonucleotide reductase and photosynthetic reaction centers. The electronic and nuclear structural and kinetic information obtained from the pulsed-EPR studies is used to address how the initiator radicals are stabilized against deleterious recombination with the metal, and to distinguish the participation of concerted versus sequential rearrangement pathways.

  18. FOR THE RECORD Lipopolysaccharide phosphorylating enzymes encoded

    E-print Network

    Srinivasan, N.

    of cross-linking to adjacent LPS molecules via divalent cations (Yethon and Whitfield 2001). Lack and hydrophobic antibiotics, and also being less virulent (Ye- thon and Whitfield 2001). Two key enzymes involved-D-manno-octulosonic acid) kinase, have been well characterized (White et al. 1999; Yethon and Whitfield 2001). These two

  19. Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier

    PubMed Central

    Urayama, Akihiko; Dohgu, Shinya; Robinson, Sandra M.; Sly, William S.; Grubb, Jeffery H.; Banks, William A

    2015-01-01

    The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse) of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy. PMID:26545208

  20. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    SciTech Connect

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-03-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

  1. Probing enzyme location in water-in-oil microemulsion using enzyme-carbon dot conjugates.

    PubMed

    Das, Krishnendu; Maiti, Subhabrata; Das, Prasanta Kumar

    2014-03-11

    This article delineates the formation and characterization of different enzyme-carbon dot conjugates in aqueous medium (pH = 7.0). We used soybean peroxidase (SBP), Chromobacterium viscosum (CV) lipase, trypsin, and cytochrome c (cyt c) for the formation of conjugate either with cationic carbon dot (CCD) or anionic carbon dot (ACD) depending on the overall charge of the protein at pH 7.0. These nanobioconjugates were used to probe the location of enzymes in water-in-oil (w/o) microemulsion. The size of the synthesized water-soluble carbon dots were of 2-3 nm with distinctive emission property. The formation of enzyme/protein-carbon dot conjugates in aqueous buffer was confirmed via fluorescence spectroscopy and zeta potential measurement, and the structural alteration of enzyme/protein was monitored by circular dichroism spectroscopy. Biocatalytic activities of protein/enzymes in conjugation with carbon dots were found to be decreased in aqueous phosphate buffer (pH 7.0, 25 mM). Interestingly, the catalytic activity of the nanobioconjugates of SBP, CV lipase, and cyt c did not reduce in cetyltrimethylammonium bromide (CTAB)-based reverse micelle. It indicates different localization of carbon dots and the enzymes inside the reverse micelle. The hydrophilic carbon dots always preferred to be located in the water pool of reverse micelle, and thus, enzyme must be located away from the water pool, which is the interface. However, in case of trypsin-carbon dot conjugate, the enzyme activity notably decreased in reverse micelle in the presence of carbon dot in a similar way that was observed in water. This implies that trypsin and carbon dots both must be located at the same place, which is the water pool of reverse micelle. Carbon dot induced deactivation was not observed for those enzymes which stay away from the water pool and localized at the interfacial domain while deactivation is observed for those enzymes which reside at the water pool. Thus, the location of enzymes in the microdomain of w/o microemulsion can be predicted by comparing the activity profile of enzyme-carbon dot conjugate in water and w/o microemulsion. PMID:24528191

  2. 76 FR 14439 - No FEAR Act Notice

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... TRANSPARENCY BOARD No FEAR Act Notice AGENCY: Recovery Accountability and Transparency Board. ACTION: Notice... and Retaliation Act (No FEAR Act or Act), as implemented by Office of Personnel Management (OPM... No FEAR Act. See Public Law 107-174, codified at 5 U.S.C. 2301 note. One purpose of the Act is...

  3. Nurse Reinvestment Act. Public Law.

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC.

    This document contains the text of the Nurse Reinvestment Act, which amends the Public Health Service Act to address the increasing shortage of registered nurses by instituting a series of policies to improve nurse recruitment and nurse retention. Title I details two initiatives to boost recruitment of nurses. The first initiative includes the…

  4. Biomass Program Recovery Act Factsheet

    SciTech Connect

    2010-03-01

    The Biomass Program has awarded about $718 million in American Recovery and Reinvestment Act (Recovery Act) funds. The projects the Program is supporting are intended to: Accelerate advanced biofuels research, development, and demonstration; Speed the deployment and commercialization of advanced biofuels and bioproducts; Further the U.S. bioindustry through market transformation and creating or saving a range of jobs.

  5. Workforce Investment Act of 1998.

    ERIC Educational Resources Information Center

    Employment and Training Administration (DOL), Washington, DC.

    This document provides an overview of the Workforce Investment Act, which provides the framework for a national work force preparation and employment system designed to meet simultaneously the needs of the nation's businesses and of job seekers wishing to further their careers. The document begins with a brief summary of the act's five titles,…

  6. 76 FR 59073 - Privacy Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-23

    ... From the Federal Register Online via the Government Printing Office CENTRAL INTELLIGENCE AGENCY 32 CFR Part 1901 Privacy Act AGENCY: Central Intelligence Agency. ACTION: Proposed rule. SUMMARY: Consistent with the Privacy Act (PA), the Central Intelligence Agency (CIA) has undertaken and completed...

  7. Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C

    PubMed Central

    Watanabe, Shinya; Tan, Dongyan; Lakshminarasimhan, Mahadevan; Washburn, Michael P.; Hong, Eun-Jin Erica; Walz, Thomas; Peterson, Craig L.

    2015-01-01

    INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodeling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional (2D) class averaging to demonstrate that these remodeling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic ‘tail’ domain and a compact ‘head’ that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome binding affinity but is largely dispensable for remodeling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodeling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodeling. PMID:25964121

  8. A Genetic Analysis of the Pteridine Biosynthetic Enzyme, Guanosine Triphosphate Cyclohydrolase, in DROSOPHILA MELANOGASTER

    PubMed Central

    Mackay, William J.; O'Donnell, Janis M.

    1983-01-01

    Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues. PMID:6413298

  9. Investigation at the atomic level of homologous enzymes reveals distinct reaction paths

    NASA Astrophysics Data System (ADS)

    Zoi, Ioanna; Schwartz, Steven D.

    2015-03-01

    Bacterial enzymes Escherichia coli and Vibrio cholerae 5' -Methylthioadenosine nucleosidases (MTANs) have different binding affinities for the same transition state analogue. This was surprising as these enzymes share 60% sequence identity, have almost identical active sites and act under the same mechanism. We performed Transition Path Sampling simulations of both enzymes to reveal the atomic details of the catalytic chemical step, to explain the inhibitor affinity differences. Unlike EcMTAN, VcMTAN has multiple distinct transition states, which is an indication that multiple sets of coordinated protein motions can reach a transition state. We also identified the important residues that participate in each enzyme's reaction coordinate and explained their contribution. Subtle dynamic differences manifest in difference of reaction coordinate and transition state structure and also suggest that MTANs differ from most ribosyl transferases. As experimental approaches report averages regarding reaction coordinate information, this study offers, previously unavailable, detailed knowledge to the explanation of bacterial MTANs catalytic mechanism, and could have a significant impact on pharmaceutical design. We acknowledge the support of the National Institutes of Health through Grant GM068036.

  10. Functional Diversity of Carbohydrate-Active Enzymes Enabling a Bacterium to Ferment Plant Biomass

    PubMed Central

    Boutard, Magali; Cerisy, Tristan; Nogue, Pierre-Yves; Alberti, Adriana; Weissenbach, Jean; Salanoubat, Marcel; Tolonen, Andrew C.

    2014-01-01

    Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass. PMID:25393313

  11. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the ?-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  12. Acetylglutamate synthase in Neurospora crassa: characterization, localization, and genetic behavior of a regulatory enzyme of arginine biosynthesis

    SciTech Connect

    Jacobson, J.A.

    1988-01-01

    This study describes the characterization and localization of the first enzyme of arginine biosynthesis in Neurospora crassa. A radioactive assay was developed to detect this enzyme whereby radioactive substrate and product molecules could be separated by ion-exchange chromatography. The enzyme was found to have a pH optimum of 9.0 and K/sub m/ values for glutamate and acetyl-CoA of approximately 4.7 and 0.45 mM, respectively. The enzyme was shown to be feedback inhibited by arginine. Half-maximal inhibition was observed at 0.13 mM arginine, a concentration which is similar to be in vivo cytosolic concentration of 0.2 mM. Arginine was found to act as a competitive inhibitor with respect to acetyl-CoA. Acetylglutamate synthase was localized to the mitochondrion. However, in contrast to the mitochondrial matrix location of the other ornithine biosynthetic enzymes, this enzyme was found to reside on the mitochondrial inner membrane.

  13. Acting to let someone die.

    PubMed

    McGee, Andrew

    2015-02-01

    This paper examines the recent prominent view in medical ethics that withdrawing life-sustaining treatment (LST) is an act of killing. I trace this view to the rejection of the traditional claim that withdrawing LST is an omission rather than an act. Although that traditional claim is not as problematic as this recent prominent view suggests, my main claim is that even if we accepted that withdrawing LST should be classified as an act rather than as an omission, it could still be classified as letting die rather than killing. Even though omissions are contrasted with acts, letting die need not be, for one can let die by means of acts. The remainder of the paper is devoted to establishing this claim and addresses certain objections to it. PMID:24320715

  14. Working with Enzymes - Where Is Lactose Digested? An Enzyme Assay for Nutritional Biochemistry Laboratories

    NASA Astrophysics Data System (ADS)

    Pope, Sandi R.; Tolleson, Tonya D.; Williams, R. Jill; Underhill, Russell D.; Deal, S. Todd

    1998-06-01

    At Georgia Southern University, we offer a sophomore-level introductory biochemistry course that is aimed at nutrition and chemistry education majors. The laboratory portion of this course has long lacked an experimental introduction to enzymes. We have developed a simple enzyme assay utilizing lactase enzyme from crushed LactAid tablets and a 5% lactose solution ("synthetic milk"). In the experiment, the students assay the activity of the enzyme on the "synthetic milk" at pHs of approximately 1, 6, and 8 with the stated goal of determining where lactose functions in the digestive tract. The activity of the lactase may be followed chromatographically or spectrophotometrically. The experiment, which is actually a simple pH assay, is easily implemented in allied health chemistry laboratory courses and readily lends itself to adaptation for more complex kinetic assays in upper-level biochemistry laboratory courses. The experimental details, including a list of required supplies and hints for implementation, are provided.

  15. Redesigning the monovalent cation specificity of an enzyme

    NASA Astrophysics Data System (ADS)

    Prasad, Swati; Wright, Kelly J.; Banerjee Roy, Dolly; Bush, Leslie A.; Cantwell, Angelene M.; di Cera, Enrico

    2003-11-01

    Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K+, whereas a few of them show preferential activation by Na+. The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na+-activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K+-specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K+-activated enzyme.

  16. Direct Fabrication of Enzyme-Carrying Polymer Nanofibers by Electrospinning

    SciTech Connect

    Herricks, Thurston E.; Kim, S. H.; Kim, Jungbae; Li, Dien; Kwak, Ja Hun; Grate, Jay W.; Kim, Samuel H.; Xia, Yuanxian

    2005-08-01

    Nanofibers of an enzyme-polymer composite were successfully electrospun with retained biocatalytic activity. The enzyme, ?-chymotrypsin, was solubilized in toluene via the aid of a surfactant and mixed with a polymer solution of polystyrene and poly(styrene-co-maleic anhydride). The enzyme-polymer solution could be directly electrospun to produce nanofibers with diameters of ~873 nm. The enzyme activity remained stable over the course of one week when nanofibers were treated with 0.1% glutaraldehyde solution. Untreated nanofibers rapidly lost the enzyme activity due to leaching of the enzyme from nanofibers. The nanofiber-based mats were durable and easily recovered from a solution.

  17. Production of an extracellular polyethylene-degrading enzyme(s) by Streptomyces species.

    PubMed

    Pometto, A L; Lee, B T; Johnson, K E

    1992-02-01

    Extracellular culture concentrates were prepared from Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2 shake flask cultures. Ten-day-heat-treated (70 degrees C) starch-polyethylene degradable plastic films were incubated with shaking with active or inactive enzyme for 3 weeks (37 degrees C). Active enzyme illustrated changes in the films' Fourier transform infrared spectra, mechanical properties, and polyethylene molecular weight distributions. PMID:1610196

  18. Extension and application of the "enzyme test bench" for oxygen consuming enzyme reactions.

    PubMed

    Rachinskiy, Kirill; Kunze, Martin; Graf, Careen; Schultze, Hergen; Boy, Matthias; Büchs, Jochen

    2014-02-01

    Within industrial process development, powerful screening techniques are required to select the optimal biocatalyst regarding such process characteristics as cost effectiveness, turnover number or space time yield. Conventional measurement of the initial enzyme activity, which is the established high throughput screening technique, disregards the long-term stability of an enzyme. A new model based technique called "enzyme test bench" was recently presented before by our group which addresses this issue. It combines the high throughput screening approach with an extensive enzyme characterization, focusing especially on the long-term stability. The technique is based on modeling enzyme activation and deactivation as temperature dependent reactions in accordance with the Arrhenius law. Controlling these reactions by tailor made temperature profiles, the slow long-term deactivation effects are accelerated and characterizing models are parameterized. Thus, the process properties of an enzyme can be predicted and included into the screening procedure. Moreover, the optimum process temperature as function of the envisaged operation time can be found by these means. In this work, the technique is extended to the important class of oxygen consuming reactions. For this aim, a suitable assay and a defined oxygen supply were established. This extended technique was applied to characterize and to optimize a complex, multi-stage laccase-mediator system (LMS). For the variation and optimization of the enzyme to mediator to substrate ratio, experiments in microtiter plates were performed. Predictions from this high throughput characterization were compared to long-term experiments in a RAMOS device (Respiration Activity Monitoring System), a technique for on-line monitoring of the oxygen transfer rate in shake flasks. Within the limits of the model validity, the enzyme test bench predictions are in good agreement with the long-term experiments. PMID:23928872

  19. Enzyme recovery during gas/liquid two-phase flow microfiltration of enzyme/yeast mixtures.

    PubMed

    Mercier-Bonin, Muriel; Fonade, Christian

    2002-12-20

    The effect of a gas/liquid two-phase flow on the recovery of an enzyme was evaluated and compared with standard crossflow operation when confronted with the microfiltration of a high-fouling yeast suspension. Ceramic tubular and flat sheet membranes were used. At constant feed concentration (permeate recycling) and transmembrane pressure, the results obtained with the tubular membrane were dependent on the two-phase flow pattern. In comparison with single-phase flow performances at the same liquid velocity, the enzyme transmission was maintained at a high level with a bubble flow pattern but it decreased by 70% with a slug flow, whatever the flow rate ratio. Identical results were obtained with flat sheet membranes: for the highest flow rate ratio, the enzyme transmission was reduced by 70% even though the permeate flux was improved by 240%. During diafiltration experiments with the tubular membrane, it was found that a bubble flow pattern led to a 13% higher enzyme recovery compared to single-phase flow conditions, whereas with a slug flow the enzyme recovery was strongly reduced. With bubble flow conditions, energy consumption was minimal, confirming that this flow pattern was the most suitable for enzyme recovery. PMID:12378602

  20. Non-Alignment Features Based Enzyme/Non-Enzyme Classification Using an Ensemble Method

    PubMed Central

    Davidson, Nicholas J; Wang, Xueyi

    2011-01-01

    As a growing number of protein structures are resolved without known functions, using computational methods to help predict protein functions from the structures becomes more and more important. Some computational methods predict protein functions by aligning to homologous proteins with known functions, but they fail to work if such homology cannot be identified. In this paper we classify enzymes/non-enzymes using non-alignment features. We propose a new ensemble method that includes three support vector machines (SVM) and two k-nearest neighbor algorithms (k-NN) and uses a simple majority voting rule. The test on a data set of 697 enzymes and 480 non-enzymes adapted from Dobson and Doig shows 85.59% accuracy in a 10-fold cross validation and 86.49% accuracy in a leave-one-out validation. The prediction accuracy is much better than other non-alignment features based methods and even slightly better than alignment features based methods. To our knowledge, our method is the first time to use ensemble methods to classify enzymes/non-enzymes and is superior over a single classifier. PMID:21572553

  1. Dual function of MIPS1 as a metabolic enzyme and transcriptional regulator.

    PubMed

    Latrasse, David; Jégu, Teddy; Meng, Pin-Hong; Mazubert, Christelle; Hudik, Elodie; Delarue, Marianne; Charon, Céline; Crespi, Martin; Hirt, Heribert; Raynaud, Cécile; Bergounioux, Catherine; Benhamed, Moussa

    2013-03-01

    Because regulation of its activity is instrumental either to support cell proliferation and growth or to promote cell death, the universal myo-inositol phosphate synthase (MIPS), responsible for myo-inositol biosynthesis, is a critical enzyme of primary metabolism. Surprisingly, we found this enzyme to be imported in the nucleus and to interact with the histone methyltransferases ATXR5 and ATXR6, raising the question of whether MIPS1 has a function in transcriptional regulation. Here, we demonstrate that MIPS1 binds directly to its promoter to stimulate its own expression by locally inhibiting the spreading of ATXR5/6-dependent heterochromatin marks coming from a transposable element. Furthermore, on activation of pathogen response, MIPS1 expression is reduced epigenetically, providing evidence for a complex regulatory mechanism acting at the transcriptional level. Thus, in plants, MIPS1 appears to have evolved as a protein that connects cellular metabolism, pathogen response and chromatin remodeling. PMID:23341037

  2. PEGylated silica-enzyme nanoconjugates: a new frontier in large scale separation of ?-amylase.

    PubMed

    Mohsen Dehnavi, Seyed; Pazuki, Gholamreza; Vossoughi, Manouchehr

    2015-01-01

    High resolution is nearly lost at the expense of throughput in most conventional bioseparation methods. Nanoparticles, due to their high surface to volume ratio, are attractiveenzyme carriers, which can boost the performance of extraction manifold. Here, wereport design and application ofa method highly capable of improving the partitioning of ?-amylase in aqueous two-phase system of polymer and salt. Silica nanoparticle introduced to the system acts as a bridge that connects the enzyme and polymer. Theconjugated nanoparticles form the major part of the upper phase and thus significantly enhance the protein recovery. A thorough investigation was performed on the structure of the nanoconjugatesas well as analyzing the conformational structure of the enzyme after conjugationto explore anypossible denaturation. PMID:26656308

  3. PEGylated silica-enzyme nanoconjugates: a new frontier in large scale separation of ?-amylase

    PubMed Central

    Mohsen Dehnavi, Seyed; Pazuki, Gholamreza; Vossoughi, Manouchehr

    2015-01-01

    High resolution is nearly lost at the expense of throughput in most conventional bioseparation methods. Nanoparticles, due to their high surface to volume ratio, are attractiveenzyme carriers, which can boost the performance of extraction manifold. Here, wereport design and application ofa method highly capable of improving the partitioning of ?-amylase in aqueous two-phase system of polymer and salt. Silica nanoparticle introduced to the system acts as a bridge that connects the enzyme and polymer. Theconjugated nanoparticles form the major part of the upper phase and thus significantly enhance the protein recovery. A thorough investigation was performed on the structure of the nanoconjugatesas well as analyzing the conformational structure of the enzyme after conjugationto explore anypossible denaturation. PMID:26656308

  4. Inside the trap: gland morphologies, digestive enzymes, and the evolution of plant carnivory in the Caryophyllales?

    PubMed Central

    Renner, Tanya; Specht, Chelsea D

    2013-01-01

    The digestion of prey by carnivorous plants is determined in part by suites of enzymes that are associated with morphologically and anatomically diverse trapping mechanisms. Chitinases represent a group of enzymes known to be integral to effective plant carnivory. In non-carnivorous plants, chitinases commonly act as pathogenesis-related proteins, which are either induced in response to insect herbivory and fungal elicitors, or constitutively expressed in tissues vulnerable to attack. In the Caryophyllales carnivorous plant lineage, multiple classes of chitinases are likely involved in both pathogenic response and digestion of prey items. We review what is currently known about trap morphologies, provide an examination of the diversity, roles, and evolution of chitinases, and examine how herbivore and pathogen defense mechanisms may have been coopted for plant carnivory in the Caryophyllales. PMID:23830995

  5. Enzyme-Coupled Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for Searching for Low-Mass Inhibitors of Enzymes in Complex Mixtures

    NASA Astrophysics Data System (ADS)

    Salwi?ski, Aleksander; Da Silva, David; Delépée, Raphaël; Maunit, Benoît

    2014-04-01

    In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: `ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and `ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

  6. Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.

    PubMed

    Liu, Hongbo; Yang, Xiaolan; Liu, Lin; Dang, Jizheng; Xie, Yanling; Zhang, Yi; Pu, Jun; Long, Gaobo; Li, Yuanli; Yuan, Yonghua; Liao, Juan; Liao, Fei

    2013-02-19

    Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (?(A)) much larger than that of Product B (?(B)) formed by Enzyme B action on Substrate B; ?(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (?(0)); (b) absorbance at ?(A) and ?(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with ?-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. ?-Glutamyltransferase releases p-nitroaniline from ?-glutamyl-p-nitroaniline with ?(0) at 344 nm and ?(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing ?(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of ?-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and ?-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with ?(0) at 405 nm and ?(A) at 458 nm, ?-D-galactosidase releases 4-nitrophenol from ?-D-(4-nitrophenyl)-galactoside with ?(B) at 405 nm. No interference from substrates/products made SDESA of ?-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising. PMID:23305208

  7. The role of deubiquitinating enzymes in spermatogenesis.

    PubMed

    Suresh, Bharathi; Lee, Junwon; Hong, Seok-Ho; Kim, Kye-Seong; Ramakrishna, Suresh

    2015-12-01

    Spermatogenesis is a complex process through which spermatogonial stem cells undergo mitosis, meiosis, and cell differentiation to generate mature spermatozoa. During this process, male germ cells experience several translational modifications. One of the major post-translational modifications in eukaryotes is the ubiquitination of proteins, which targets proteins for degradation; this enables control of the expression of enzymes and structural proteins during spermatogenesis. It has become apparent that ubiquitination plays a key role in regulating every stage of spermatogenesis starting from gonocytes to differentiated spermatids. It is understood that, where there is ubiquitination, deubiquitination by deubiquitinating enzymes (DUBs) also exists to counterbalance the ubiquitination process in a reversible manner. Normal spermatogenesis is dependent on the balanced actions of ubiquitination and deubiquitination. This review highlights the current knowledge of the role of DUBs and their essential regulatory contribution to spermatogenesis, especially during progression into meiotic phase, acrosome biogenesis, quality sperm production, and apoptosis of germ cells. PMID:26350476

  8. Enzyme immunoassay system for panel testing.

    PubMed

    Donohue, J; Bailey, M; Gray, R; Holen, J; Huang, T M; Keevan, J; Mattimiro, C; Putterman, C; Stalder, A; Defreese, J

    1989-09-01

    An immunoassay system based on enzyme immunoassay technology has been developed for quantitative panel testing. The system includes test card disposables, reagents, and an instrument. Patients' samples are processed semiautomatically in the instrument with minimum user intervention. The test card has multiple test areas at individual locations on a membrane solid phase so that simultaneous determinations from a single specimen are possible. Each panel also includes positive and negative reagent procedural controls. Factory-determined calibration curves for each analyte are provided in barcode form with each test kit. The reagents include a specimen dilution buffer, enzyme conjugate, and precipitogenic substrate. Up to 10 test cards at a time can be processed in random-access and continuous-access modes, with automated agitation of sample and reagents over the solid phase, temperature-controlled incubation, and membrane washing and reading, data reduction, and printout of results. The optical reader measures diffuse reflectance and features source intensity and wavelength compensation. PMID:2673584

  9. Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting

    PubMed Central

    Reid, Emma L.; Worthy, Charlotte A.; Probert, Ian; Ali, Sohail T.; Love, John; Napier, Johnathan; Littlechild, Jenny A.; Somerfield, Paul J.; Allen, Michael J.

    2011-01-01

    Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house’ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase. PMID:21731551

  10. Directed evolution of an RNA enzyme

    NASA Technical Reports Server (NTRS)

    Beaudry, Amber A.; Joyce, Gerald F.

    1992-01-01

    An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

  11. Enzyme-based fiber optic sensors

    SciTech Connect

    Kulp, T.J.; Camins, I.; Angel, S.M.

    1987-12-01

    Fiber optic chemical sensors capable of detecting glucose and penicillin were developed. Each consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme and a pH-sensitive fluorescent dye (fluorescein). A signal is produced when the enzyme catalyzes the conversion of the analyte (glucose or penicillin) into a product (gluconic or penicilloic acid, respectively) that lowers the microenvironmental pH of the membrane and consequently, lowers the fluorescence intensity of the dye. Each sensor is capable of responding to analyte concentrations in the range of approx.0.1 to 100 mM. The penicillin optrode response time is 40 to 60 s while that for glucose is approx.5 to 12 min. 7 figs.

  12. Enzyme-Based Fiber Optic Sensors

    NASA Astrophysics Data System (ADS)

    Kulp, Thomas J.; Camins, Irene; Angel, Stanley M.

    1988-06-01

    Fiber optic chemical sensors capable of detecting glucose and penicillin were developed. Each consists of a polymer membrane that is covalently attached to the tip of a glass optical fiber. The membrane contains the enzyme and a pH-sensitive fluorescent dye (fluorescein). A signal is produced when the enzyme catalyzes the conversion of the analyte (glucose or penicillin) into a product (gluconic or penicilloic acid, respectively) that lowers the microenvironmental pH of the membrane and, consequently, lowers the fluorescence intensity of the dye. Each sensor is capable of responding to analyte concentrations in the range of ~0.1 to 100 mM. The penicillin optrode response time is 40 to 60 s while that for glucose is ~5 to 12 min.

  13. (Enzyme use in the Jute Industry)

    SciTech Connect

    Niyogi, S.K.

    1991-03-15

    This report covers my official visit to the Indian Jute Industries' Research Association (IJIRA), Calcutta, India. The visit lasted a little over two weeks, including two trips to three jute mills outside Calcutta and a one-day visit to the library of the Indian Institute of Chemical Biology, Calcutta. The report describes the applications of enzymes (derived from a moldy wheat bran extract) in upgrading the jute fiber and in enhancing the quality of tamarind kernel powder used for sizing of jute. The various methodological developments in these processes are discussed in detail along with suggestions for possible improvements. The report also describes the visits to the jute mills where enzyme applications are being made. Interactions with the IJIRA research staff are described in detail. My contributions to the Project are described along with specific recommendations for future research.

  14. Substrates and method for determining enzymes

    DOEpatents

    Smith, R.E.; Bissell, E.R.

    1981-10-13

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

  15. Substrates and method for determining enzymes

    DOEpatents

    Smith, Robert E. (574 Escondido Cir., Livermore, CA 94550); Bissell, Eugene R. (101 Via Lucia, Alamo, CA 94507)

    1981-01-01

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.

  16. Assessment & Commitment Tracking System (ACTS)

    SciTech Connect

    2004-12-20

    The ACTS computer code provides a centralized tool for planning and scheduling assessments, tracking and managing actions associated with assessments or that result from an event or condition, and "mining" data for reporting and analyzing information for improving performance. The ACTS application is designed to work with the MS SQL database management system. All database interfaces are written in SQL. The following software is used to develop and support the ACTS application: Cold Fusion HTML JavaScript Quest TOAD Microsoft Visual Source Safe (VSS) HTML Mailer for sending email Microsoft SQL Microsoft Internet Information Server

  17. Assessment & Commitment Tracking System (ACTS)

    Energy Science and Technology Software Center (ESTSC)

    2004-12-20

    The ACTS computer code provides a centralized tool for planning and scheduling assessments, tracking and managing actions associated with assessments or that result from an event or condition, and "mining" data for reporting and analyzing information for improving performance. The ACTS application is designed to work with the MS SQL database management system. All database interfaces are written in SQL. The following software is used to develop and support the ACTS application: Cold Fusion HTMLmore »JavaScript Quest TOAD Microsoft Visual Source Safe (VSS) HTML Mailer for sending email Microsoft SQL Microsoft Internet Information Server« less

  18. Evolution of Enzyme Specificity in the OSBS Family 

    E-print Network

    Jones, Christine Michelle

    2013-02-04

    In addition to their primary biological function, many proteins are at least moderately capable of catalyzing secondary, promiscuous activities that may have a major role in enzyme evolution. Mounting evidence supports the idea that new enzymes can...

  19. ENZYME-BASED DETECTION OF CHLORINATED HYDROCARBONS IN WATER

    EPA Science Inventory

    An enzyme-based approach for detecting hazardous levels of high molecular weight chlorinated hydrocarbons in natural waters has been explored. An extensive review of the literature indicated that the enzymes, lactate dehydrogenase, carbonic anhydrase, hexokinase, phosphorylase an...

  20. Layer-by-Layer Assembly of Enzymes on Carbon Nanotubes

    SciTech Connect

    Wang, Jun; Liu, Guodong; Lin, Yuehe

    2008-06-01

    The use of Layer-by-layer techniques for immobilizing several types of enzymes, e.g. glucose oxidase (GOx), horse radish oxidases(HRP), and choline oxidase(CHO) on carbon nanotubes and their applications for biosenseing are presented. The enzyme is immobilized on the negatively charged CNT surface by alternatively assembling a cationic polydiallyldimethyl-ammonium chloride (PDDA) layer and a enzyme layer. The sandwich-like layer structure (PDDA/enzyme/PDDA/CNT) formed by electrostatic assembling provides a favorable microenvironment to keep the bioactivity of enzyme and to prevent enzyme molecule leakage. The morphologies and electrocatalytic acitivity of the resulted enzyme film were characterized using TEM and electrochemical techniques, respectively. It was found that these enzyme-based biosensors are very sensitive, selective for detection of biomolecules, e.g. glucose, choline.

  1. Purification and characterization of two fully deuterated enzymes

    NASA Technical Reports Server (NTRS)

    Crespi, H. L.; Katz, J. J.; Parmerter, S.; Rokop, S.

    1969-01-01

    Comparative data reveal little difference between kinetic and thermal stabilities of pure preparations of two ordinary enzymes and their fully deuterated counterparts. The effects of temperature on the enzymes proved to be consistent with earlier results.

  2. Mechanistic aspects of molybdenum-containing enzymes Russ Hille

    E-print Network

    Rétey, János

    Mechanistic aspects of molybdenum-containing enzymes Russ Hille , Jaènos Reètey , Ulrike . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489 2. The molybdenum hydroxylases molybdenum centers in their active sites has increased signi¢cantly, and well over 50 such enzymes have been

  3. 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

  4. Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.

    PubMed

    Sakuraba, Hitoshi; Sawada, Makoto; Matsuzawa, Fumiko; Aikawa, Sei-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Itoh, Kohji

    2006-08-01

    Lysosomal diseases comprise a group of inherited disorders resulting from defects of lysosomal enzymes and their cofactors, and in many of them the nervous system is affected. Recently, enzyme replacement therapy with recombinant lysosomal enzymes has been clinically available for several lysosomal diseases. Such enzyme replacement therapies can improve non-neurological disorders but is not effective for neurological ones. In this review, we discuss the molecular pathologies of lysosomal diseases from the protein structural aspect, current enzyme replacement therapies, and attempts to develop enzyme replacement therapies effective for lysosomal diseases associated with neurological disorders, i.e., production of enzymes, brain-specific delivery and incorporation of lysosomal enzymes into cells. PMID:16918392

  5. Structural analysis of hydroxypropylphosphonic acid epoxidase : a fosfomycin biosynthetic enzyme

    E-print Network

    Higgins, Luke J. (Luke James)

    2006-01-01

    An X-ray crystallographic study of the fosfomycin biosynthetic enzyme hydroxypropylphosphonic acid epoxidase (HppE) from Streptomyces wedmorensis is presented. Structural analysis of this cupin mononuclear iron enzyme in ...

  6. Quantitative Interpretation of the Randomness in Single Enzyme Turnover Times

    E-print Network

    Yang, Seongeun

    Fluctuating turnover times of a single enzyme become observable with the advent of modern cutting-edge, single enzyme experimental techniques. Although the conventional chemical kinetics and its modern generalizations could ...

  7. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...safe (GRAS). Enzyme-modified milk powder may be prepared with GRAS enzymes from reconstituted milk powder, whole milk, condensed or concentrated...whole milk, evaporated milk, or milk powder. The lipolysis is...

  8. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...safe (GRAS). Enzyme-modified milk powder may be prepared with GRAS enzymes from reconstituted milk powder, whole milk, condensed or concentrated...whole milk, evaporated milk, or milk powder. The lipolysis is...

  9. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...safe (GRAS). Enzyme-modified milk powder may be prepared with GRAS enzymes from reconstituted milk powder, whole milk, condensed or concentrated...whole milk, evaporated milk, or milk powder. The lipolysis is...

  10. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...safe (GRAS). Enzyme-modified milk powder may be prepared with GRAS enzymes from reconstituted milk powder, whole milk, condensed or concentrated...whole milk, evaporated milk, or milk powder. The lipolysis is...

  11. 21 CFR 184.1287 - Enzyme-modified fats.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...safe (GRAS). Enzyme-modified milk powder may be prepared with GRAS enzymes from reconstituted milk powder, whole milk, condensed or concentrated...whole milk, evaporated milk, or milk powder. The lipolysis is...

  12. Cholecystokinin-converting enzymes in brain.

    PubMed Central

    Malesci, A; Straus, E; Yalow, R S

    1980-01-01

    Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10(-6) M; the Km for trypsin cleavage of the same bond is 4.7 x 10(-6) M. The lower Vmax for the brain enzyme (1.5 x 10(-11) mol/min per g of extract) compared with trypsin (66 x 10(-11) mol/min per g of trypsin) simply reflects the lesser degree of purify of the brain extract than of the highly purified trypsin. Images PMID:6987659

  13. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  14. Dynamical contribution into enzyme catalytic efficiency

    E-print Network

    A. E. Sitnitsky

    2006-03-31

    A realistic physical model for the so called rate promoting vibration (RPV) at enzyme action is constructed. The origin of the RPV is assumed to be an oscillating electric field produced by long-lived localized vibrational modes in protein dynamics, namely, by the so called discrete breather (DB) in secondary structure. The strength of interaction of the RPV with the reaction coordinate is evaluated and its effect on the reaction acceleration is assessed within the framework of modern theory for thermally activated escape rate at periodic driving. We reveal the phenomenon of resonant activation in our model elucidating why the frequency of the RPV in the range $100\\div200 cm^{-1}$ was chosen by the evolution of enzymes as an optimal one. The effect of the RPV on the reaction acceleration is shown to vary from moderate one (up to $10^3\\div10^4$) in the case of three-site DB to enormous (up to $10^6\\div10^8$) in the case of five-site DB and thus can significantly contribute into enzyme catalytic efficiency. Also the model is shown to be compatible with the known functional dependence of enzymatic reaction rates on solvent viscosity.

  15. Enzyme-Gelatin Electrochemical Biosensors: Scaling Down

    PubMed Central

    Wael, Karolien De; Belder, Stijn De; Pilehvar, Sanaz; Steenberge, Geert Van; Herrebout, Wouter; Heering, Hendrik A.

    2012-01-01

    In this article we investigate the possibility of scaling down enzyme-gelatin modified electrodes by spin coating the enzyme-gelatin layer. Special attention is given to the electrochemical behavior of the selected enzymes inside the gelatin matrix. A glassy carbon electrode was used as a substrate to immobilize, in the first instance, horse heart cytochrome c (HHC) in a gelatin matrix. Both a drop dried and a spin coated layer was prepared. On scaling down, a transition from diffusion controlled reactions towards adsorption controlled reactions is observed. Compared to a drop dried electrode, a spin coated electrode showed a more stable electrochemical behavior. Next to HHC, we also incorporated catalase in a spin coated gelatin matrix immobilized on a glassy carbon electrode. By spincoating, highly uniform sub micrometer layers of biocompatible matrices can be constructed. A full electrochemical study and characterization of the modified surfaces has been carried out. It was clear that in the case of catalase, gluteraldehyde addition was needed to prevent leaking of the catalase from the gelatin matrix. PMID:25585635

  16. Rational design of a split-Cas9 enzyme complex

    SciTech Connect

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and ?-helical lobe are expressed as separate polypeptides. The lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

  17. Rational design of a split-Cas9 enzyme complex

    PubMed Central

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-01-01

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and ?-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications. PMID:25713377

  18. Sterilization of enzyme glucose sensors: problems and concepts.

    PubMed

    von Woedtke, Th; Jülich, W-D; Hartmann, V; Stieber, M; Abel, P U

    2002-05-01

    A useful method of enzyme glucose sensor sterilization has not only to ensure the needs of sterility assurance but has also to guarantee the functional stability of the sensors. The action of 2 or 3% alkalinized glutaraldehyde solution, as well as gamma irradiation with a dose of 25 kGy caused changes of the in vitro functionality and polymer material irritations, respectively. After a combined treatment by 0.6% hydrogen peroxide solution acting over 4 days with 7 kGy gamma irradiation only a slight loss of sensitivity must be registered. The combination of a specially designed universal homogeneous ultraviolet irradiation over 300 s with a 3 days lasting treatment by an inclusion compound of hydrogen peroxide with tensides in urea (0.15% effective hydrogen peroxide concentration) did not cause any influence on the glucose sensor function in vitro. With all methods tested here, a Bacillus subtilis spore reduction over 8 log(10) cycles from 10(6) to 10(-2) spores per test object on an average could be proved experimentally. In general, if non-thermal methods must be used it seems to be impossible to guarantee a sterility assurance level of 10(-6) as it is demanded by the pharmacopoeias. Consequently, effective concepts to produce sterile glucose biosensors for medical and biological applications should be based not only on final product treatments but should include germ reducing measures in every manufacturing step. PMID:11888727

  19. Rational design of a split-Cas9 enzyme complex

    DOE PAGESBeta

    Wright, Addison V.; Sternberg, Samuel H.; Taylor, David W.; Staahl, Brett T.; Bardales, Jorge A.; Kornfeld, Jack E.; Doudna, Jennifer A.

    2015-02-23

    Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and ?-helical lobe are expressed as separate polypeptides. The lobes do not interactmore »on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.« less

  20. Enteropancreatic reflexes mediating the pancreatic enzyme response to nutrients.

    PubMed

    Niebergall-Roth, Elke; Singer, Manfred V

    2006-04-30

    The observation that in dogs electrical stimulation of the vagus nerve elicited a strong secretory activity of the pancreas, prompted I. P. Pavlov in 1888 to conclude that the pancreatic secretory response to nutrients is mediated by enteropancreatic reflexes involving the vagus nerves. It took, however, more than 90 years until by studying the latency of pancreatic amylase response to exogenous and endogenous stimuli for the first time experimental evidence was provided for the actual existence of cholinergic vago-vagal enteropancreatic reflexes. Follow-up studies, based on stepwise extrinsic denervation of the pancreas, ruled out possible splanchnic pathways for enteropancreatic reflexes. In more recent years, experiments utilizing specific antagonists demonstrated a physiological role for both cholinergic M1 and cholecystokinin (CCK) receptors within the enteropancreatic reflex. At least a significant portion of the cholinergic fibres of the enteropancreatic reflex end on muscarinic receptors of the subtype M1. CCK, the most important hormone stimulating pancreatic enzyme secretion, appears to act at least in part on CCK receptors located on vagal afferent nerves, which in turn elicit a vago-vagal reflex, implying that CCK exerts its effect on the pancreas at least in part through vago-vagal reflexes. Furthermore, pharmacological blockade of CCK receptors totally abolished the early pancreatic amylase response to intestinal nutrients, suggesting that the activation of (probably vagal) CCK receptors is essential to run the enteropancreatic reflex. PMID:16490403

  1. Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

    PubMed Central

    Lee, S H; Johnson, J D; Walsh, M P; Van Lierop, J E; Sutherland, C; Xu, A; Snedden, W A; Kosk-Kosicka, D; Fromm, H; Narayanan, N; Cho, M J

    2000-01-01

    Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients. PMID:10926857

  2. Enzymes/non-enzymes classification model complexity based on composition, sequence, 3D and topological indices.

    PubMed

    Munteanu, Cristian Robert; González-Díaz, Humberto; Magalhães, Alexandre L

    2008-09-21

    The huge amount of new proteins that need a fast enzymatic activity characterization creates demands of protein QSAR theoretical models. The protein parameters that can be used for an enzyme/non-enzyme classification includes the simpler indices such as composition, sequence and connectivity, also called topological indices (TIs) and the computationally expensive 3D descriptors. A comparison of the 3D versus lower dimension indices has not been reported with respect to the power of discrimination of proteins according to enzyme action. A set of 966 proteins (enzymes and non-enzymes) whose structural characteristics are provided by PDB/DSSP files was analyzed with Python/Biopython scripts, STATISTICA and Weka. The list of indices includes, but it is not restricted to pure composition indices (residue fractions), DSSP secondary structure protein composition and 3D indices (surface and access). We also used mixed indices such as composition-sequence indices (Chou's pseudo-amino acid compositions or coupling numbers), 3D-composition (surface fractions) and DSSP secondary structure amino acid composition/propensities (obtained with our Prot-2S Web tool). In addition, we extend and test for the first time several classic TIs for the Randic's protein sequence Star graphs using our Sequence to Star Graph (S2SG) Python application. All the indices were processed with general discriminant analysis models (GDA), neural networks (NN) and machine learning (ML) methods and the results are presented versus complexity, average of Shannon's information entropy (Sh) and data/method type. This study compares for the first time all these classes of indices to assess the ratios between model accuracy and indices/model complexity in enzyme/non-enzyme discrimination. The use of different methods and complexity of data shows that one cannot establish a direct relation between the complexity and the accuracy of the model. PMID:18606172

  3. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different detection modes. The detection limits are 22, 15, and 3.6 {micro}M ADP for using fluorometer, ICCD and PMT respectively. The Michealis constant K{sub m}(ATP) for protein kinases ranges from 5 to 100 {micro}M. For inhibitor screening, in order to get the most accurate result, ATP concentration should be closed to K{sub m}. In this case, further lower the detection limit of ADP is needed before the direct detection of ADP can be actually used in kinase inhibitor screening.

  4. Monitoring EERE's Recovery Act Portfolio

    SciTech Connect

    2011-01-01

    Performance monitoring of Recovery Act projects within EERE has been an ongoing effort. Project recipients have been reporting technical and financial progress to project officers on a quarterly basis.

  5. The enzymic conversion of linoleic acid hydroperoxide by flax-seed hydroperoxide isomerase

    PubMed Central

    Veldink, G. A.; Vliegenthart, J. F. G.; Boldingh, J.

    1970-01-01

    1. The mode of action of flax-seed hydroperoxide isomerase was studied in vitro by using as substrates linoleic acid hydroperoxides formed by soya-bean lipoxygenase. 2. The enzyme converts only 13-hydroperoxyoctadeca-cis-9-trans-11-dienoic acid, whereas the 9-hydroperoxy isomer does not react. 3. The isomerization product was identified by chemical and spectroscopic methods as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. 4. 12,13-Epoxyoleic acid isomers do not act as intermediates in the isomerization reaction. 5. Suggestions for a functional relationship between hydroperoxide isomerase and lipoxygenase are discussed. PMID:5494229

  6. BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Cloning, characterization, and mutational analysis

    E-print Network

    Zhao, Huimin

    the enzyme the ability to accept D-sorbitol as a substrate, suggesting that the amino acids flankingBIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Cloning, characterization, and mutational analysis.1.1.12), a common enzyme found in yeasts and filamentous fungi, catalyzes the second step of the recently elucidated

  7. LEAPT: Lectin-directed enzyme-activated prodrug therapy

    E-print Network

    Davis, Ben G.

    LEAPT: Lectin-directed enzyme-activated prodrug therapy Mark A. Robinson*, Stuart T. Charlton) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply

  8. Identity between the Ca2 -independent Phospholipase A2 Enzymes

    E-print Network

    Dennis, Edward A.

    Identity between the Ca2 -independent Phospholipase A2 Enzymes from P388D1 Macrophages and Chinese., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227­9233). This enzyme appears to play a key role in regulating is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of c

  9. Origins of catalysis by computationally designed retroaldolase enzymes

    E-print Network

    Herschlag, Dan

    Origins of catalysis by computationally designed retroaldolase enzymes Jonathan K. Lassilaa , David enzymes with the goal of understanding the extent and the origins of their catalytic power. Direct comparison of the designed enzymes to primary amine catalysts in solution revealed a rate acceleration of 105

  10. This article is part of the 2010 `Enzymes & Proteins'

    E-print Network

    Zhao, Huimin

    This article is part of the 2010 `Enzymes & Proteins' web themed issue This issue showcases high quality research in the field of enzymes and proteins. Please visit the website to access the other papers is a novel P450pyr enzyme from Sphingomonas sp. HXN-2003 that belongs to the class I P450 proteins

  11. Better Enzymes for Biofuels and Green Chemistry: Solving the

    E-print Network

    RESEARCH HIGHLIGHTS Better Enzymes for Biofuels and Green Chemistry: Solving the Cofactor Imbalance Better Enzymes for Biofuels and Green Chemistry: Solving the Cofactor Imbalance Problem Global are constructed, a need has arisen for tools to rapidly and reliably optimize enzymes for new metabolic contexts

  12. Electrochemical biosensor based on immobilized enzymes and redox polymers

    DOEpatents

    Skotheim, Terje A. (Shoreham, NY); Okamoto, Yoshiyuki (Fort Lee, NJ); Hale, Paul D. (Northport, NY)

    1992-01-01

    The present invention relates to an electrochemical enzyme biosensor for use in liquid mixtures of components for detecting the presence of, or measuring the amount of, one or more select components. The enzyme electrode of the present invention is comprised of an enzyme, an artificial redox compound covalently bound to a flexible polymer backbone and an electron collector.

  13. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  14. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  15. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  16. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  17. Evolution of extracellular enzyme activities during manure composting

    E-print Network

    Tiquia-Arashiro, Sonia M.

    : API ZYMTM assay was used to monitor the activities of 19 extracellular enzymes during poultry and pig present. The relative abundance and activities of enzymes were higher in poultry manure than in pig manure enzymes in poultry manure, whereas it was N-acetyl-b-glucosaminidase for the pig manure. A number

  18. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  19. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  20. Method for synthesizing peptides with saccharide linked enzyme polymer conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-06-17

    A method is disclosed for synthesizing peptides using water soluble enzyme polymer conjugates. The method comprises catalyzing the peptide synthesis with enzyme which has been covalently bonded to a polymer through at least three linkers which linkers have three or more hydroxyl groups. The enzyme is conjugated at lysines or arginines. 19 figs.

  1. Directed Evolution Study of Temperature Adaptation in a Psychrophilic Enzyme

    E-print Network

    Arnold, Frances H.

    to identify enzymes that acquired greater thermostability without sacri®cing low- temperature activity enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efDirected Evolution Study of Temperature Adaptation in a Psychrophilic Enzyme Kentaro Miyazaki1

  2. Multifunctional nanoadditives for the thermodynamic and kinetic stabilization of enzymes.

    PubMed

    Clemons, Tristan D; Evans, Cameron W; Zdyrko, Bogdan; Luzinov, Igor; Fitzgerald, Melinda; Dunlop, Sarah A; Harvey, Alan R; Iyer, K Swaminathan; Stubbs, Keith A

    2011-10-01

    Stabilization of enzymes has become a major focus in the quest to improve the activity, sustainability and recyclability of enzymes for their successful integration into both industry and medicine. Here, we describe the kinetic and thermodynamic stabilization of a variety of enzymes in the presence of cationic multifunctional polymeric nanoparticles. PMID:21897968

  3. 21 CFR 862.2500 - Enzyme analyzer for clinical use.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enzyme analyzer for clinical use. 862.2500 Section... (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Laboratory Instruments § 862.2500 Enzyme analyzer for clinical use. (a) Identification. An enzyme analyzer for...

  4. A structural comparison of molybdenum cofactor-containing enzymes

    E-print Network

    Rétey, János

    A structural comparison of molybdenum cofactor-containing enzymes Caroline Kisker , Hermann have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes

  5. L. A NOTE ON THE KINETICS OF ENZYME ACTION.

    E-print Network

    Li, Tiejun

    L. A NOTE ON THE KINETICS OF ENZYME ACTION. BY GEORGE EDWARD BRIGGS AND JOHN BURDON SANDERSON cases of enzyme action. It is therefore desirable to examine its theoretical basis. Consider the irreversible reaction A -+ B, unimolecular as regards A, and catalysed by an enzyme. Suppose one molecule

  6. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  7. Effects of Long-Term Nitrogen Addition on Microbial Enzyme

    E-print Network

    Thomas, David D.

    Effects of Long-Term Nitrogen Addition on Microbial Enzyme Activity in Eight Forested and Grassland of microbially produced extracellu- lar enzymes involved in decomposition. Specifi- cally, it is hypothesized that adding N to N-limited ecosystems increases activity of cellulose degrading enzymes and decreases

  8. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  9. Method for synthesizing peptides with saccharide linked enzyme polymer conjugates

    DOEpatents

    Callstrom, Matthew R. (Columbus, OH); Bednarski, Mark D. (Berkeley, CA); Gruber, Patrick R. (St. Paul, MN)

    1997-01-01

    A method is disclosed for synthesizing peptides using water soluble enzyme polymer conjugates. The method comprises catalyzing the peptide synthesis with enzyme which has been covalently bonded to a polymer through at least three linkers which linkers have three or more hydroxyl groups. The enzyme is conjugated at lysines or arginines.

  10. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  11. Conformational Cycle of a Single Working Enzyme Noam Agmon*

    E-print Network

    Agmon, Noam

    Conformational Cycle of a Single Working Enzyme Noam Agmon* The Fritz Haber Research Center function of a single working cholesterol oxidase enzyme, a diffusional model reveals the coupling of conformation change with enzyme action. Active- site oxidation induces a conformational change that opens

  12. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  13. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid...

  14. Network of coupled promoting motions in enzyme catalysis

    E-print Network

    Hammes-Schiffer, Sharon

    Network of coupled promoting motions in enzyme catalysis Pratul K. Agarwal, Salomon R. Billeter, P. Benkovic, January 4, 2002 A network of coupled promoting motions in the enzyme dihydro- folate reductase and are found on the exterior of the enzyme as well as in the active site. This type of network has broad

  15. Critical Review Internal Friction in Enzyme Reactions Anna Rauscher1

    E-print Network

    Derényi, Imre

    Critical Review Internal Friction in Enzyme Reactions Anna Rauscher1 Imre Derenyi2,3 Laszlo Graf1 on the viscosity dependence of enzyme reactions. We also present speculations about the structural background and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions

  16. Development of the Enzyme-Substrate Interactions Concept Inventory

    ERIC Educational Resources Information Center

    Bretz, Stacey Lowery; Linenberger, Kimberly J.

    2012-01-01

    Enzyme function is central to student understanding of multiple topics within the biochemistry curriculum. In particular, students must understand how enzymes and substrates interact with one another. This manuscript describes the development of a 15-item Enzyme-Substrate Interactions Concept Inventory (ESICI) that measures student understanding…

  17. 21 CFR 173.150 - Milk-clotting enzymes, microbial.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Milk-clotting enzymes, microbial. 173.150 Section 173.150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

  18. UDP-(5F)-GlcNAc acts as a slow-binding inhibitor of MshA, a retaining glycosyltransferase.

    PubMed

    Frantom, Patrick A; Coward, James K; Blanchard, John S

    2010-05-19

    Glycosyltransferase enzymes play important roles in numerous cellular pathways. Despite their participation in many therapeutically relevant pathways, there is a paucity of information on how to effectively inhibit this class of enzymes. Here we report that UDP-(5F)-GlcNAc acts as a slow-binding, competitive inhibitor of the retaining glycosyltransferase MshA from Corynebacterium glutamicum (K(i) approximately 1.6 muM). The kinetic data are consistent with a single-step inhibition mechanism whose equilibration is slow relative to catalysis. We believe that this is the first slow-onset inhibitor to be reported for the glycosyltransferase family of enzymes. The potent inhibition of the enzyme by the fluoro-substituted substrate is consistent with the involvement of an oxocarbenium transition-state structure, which has been previously proposed for this family of enzymes. Additionally, although several members of the GT-B enzyme family, including MshA, have been shown to undergo a conformational change upon UDP-GlcNAc binding, the kinetic data are inconsistent with a two-step inhibition mechanism. This suggests that there may be other conformations of the enzyme that are useful for the design of inhibitors against the large family of GT-B glycosyltransferase enzymes. PMID:20411981

  19. 75 FR 7312 - No FEAR Act Notice

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-18

    ... FEAR Act Notice Summary: 5 CFR part 724.202 requires that each Federal agency provide notice to its... Register. No FEAR Act Notice On May 15, 2002, Congress enacted the Notification and Federal Employee Antidiscrimination and Retaliation Act of 2002, which is now known as the No FEAR Act. One purpose of the Act is...

  20. 76 FR 14439 - No FEAR Act Notice

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ...TRANSPARENCY BOARD [Doc. No. 11-002] No FEAR Act Notice AGENCY: Recovery Accountability...Antidiscrimination and Retaliation Act (No FEAR Act or Act), as implemented by Office...2002,'' which is now known as the No FEAR Act. See Public Law 107-174,...

  1. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act to promote the foreign trade of the United States in apples to protect the reputation of American-grown apples in foreign markets, to prevent deception or misrepresentation as to the quality...

  2. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act to promote the foreign trade of the United States in apples to protect the reputation of American-grown apples in foreign markets, to prevent deception or misrepresentation as to the quality...

  3. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act to promote the foreign trade of the United States in apples to protect the reputation of American-grown apples in foreign markets, to prevent deception or misrepresentation as to the quality...

  4. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act to promote the foreign trade of the United States in apples to protect the reputation of American-grown apples in foreign markets, to prevent deception or misrepresentation as to the quality...

  5. 7 CFR 33.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AUTHORITY OF THE EXPORT APPLE ACT Definitions § 33.1 Act. Act and Export Apple Act are synonymous and mean “An act to promote the foreign trade of the United States in apples to protect the reputation of American-grown apples in foreign markets, to prevent deception or misrepresentation as to the quality...

  6. Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

    NASA Technical Reports Server (NTRS)

    Gibeaut, David M.; Karuppiah, Nadarajah; Chang, S.-R.; Brock, Thomas G.; Vadlamudi, Babu; Kim, Donghern; Ghosheh, Najati S.; Rayle, David L.; Carpita, Nicholas C.; Kaufman, Peter B.

    1990-01-01

    The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response and asymmetric processes involving degradation of starch and cell wall synthesis. Cellular and biochemical events were studied by investigation of the activities of related enzymes and changes in cell walls and their constituents. It is suggested that an osmotic potential gradient acts as the driving factor for growth, while wall extensibility is a limiting factor in pulvinus growth.

  7. Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3.

    PubMed

    Cheng, Guangyan; He, Liying; Sun, Zhibin; Cui, Zhongli; Du, Yingxiang; Kong, Yi

    2015-09-28

    A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50°C and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40°C. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg(2+) and Ca(2+) and completely inhibited by Cu(2+), but slightly enhanced by Fe(2+). According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy. PMID:26017226

  8. Nematocidal activity of extracellular enzymes produced by the nematophagous fungus Duddingtonia flagrans on cyathostomin infective larvae.

    PubMed

    Braga, Fabio Ribeiro; Soares, Filippe Elias Freitas; Giuberti, Thais Zanotti; Carmen Garcias Lopes, Aline Del; Lacerda, Tracy; Ayupe, Tiago de Hollanda; Queiroz, Paula Viana; Gouveia, Angélica de Souza; Pinheiro, Larissa; Araújo, Andreia Luíza; Queiroz, José Humberto; Araújo, Jackson Victor

    2015-09-15

    Duddingtonia flagrans produces chitinases, however, optimization of the production of these enzymes still needs to be explored, and its nematocidal activity should still be the subject of studies. The objective of the present study was to optimize chitinase production, and evaluate the nematocidal activity of extracellular enzymes produced by the nematophagous fungus D. flagrans on cyathostomin infective larvae. An isolate from D. flagrans (AC001) was used in this study. For the production of enzymes (protease and chitinase), two different culture media were inoculated with AC001 conidia. Both enzymes were purified. The statistical Plackett-Burman factorial design was used to investigate some variables and their effect on the production of chitinases by D. flagrans. After that, the design central composite (CCD) was used in order to determine the optimum levels and investigate the interactions of these variables previously observed. Only two variables (moisture and incubation time), in the evaluated levels, had a significant effect (p<0.05) on chitinase production. The conditions of maximum chitinase activity were calculated, with the following values: incubation time 2 days, and moisture 511%. The protease and chitinase derived from D. flagrans, individually or together (after 24h), led to a significant reduction (p<0.01) in the number of intact cyathostomin L3, when compared to the control, with following reduction percentage values: 19.4% (protease), 15.5% (chitinase), and 20.5% (protease+chitinase). Significant differences were observed (p<0.05) between the group treated with proteases in relation to the group treated with proteases+chitinases. In this study, the assay with the cyathostomins showed that chitinase had a nematocidal effect, suggesting that this enzyme acts on the "fungus versus nematodes" infection process. It is known that nematode eggs are rich in chitin, and in this case, we could think of a greater employability for this chitinase. PMID:26319197

  9. 75 FR 29 - Privacy Act, Government in the Sunshine Act, Freedom of Information Act (“FOIA”), and Federal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-04

    ...and Federal Election Campaign Act (``FECA'') Rules; Corrections AGENCY: Federal...Government in the Sunshine Act, FOIA, and FECA rules. DATES: Effective January 4, 2010...the Federal Election Campaign Act (``FECA'') of 1971, as amended, 2...

  10. Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

    PubMed Central

    Ardiani, Andressa; Johnson, Adam J.; Ruan, Hongmei; Sanchez-Bonilla, Marilyn; Serve, Kinta; Black, Margaret E.

    2012-01-01

    Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects. PMID:22384805

  11. EFFECTS OF NUTRIENTS, TEMPERATURE AND AERATION STATUS ON ENZYME KINETIC PROPERTIES OF SUBTROPICAL WETLAND SOILS

    E-print Network

    Ma, Lena

    1 EFFECTS OF NUTRIENTS, TEMPERATURE AND AERATION STATUS ON ENZYME KINETIC PROPERTIES OF SUBTROPICAL Factors Regulating Soil Enzyme Activities.............................................................. 16............................................................................ 41 Extracellular Enzyme Activity Assays

  12. Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

    PubMed Central

    2011-01-01

    Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

  13. Enzyme-Responsive Nanomaterials for Controlled Drug Delivery

    PubMed Central

    Hu, Quanyin; Katti, Prateek S.; Gu, Zhen

    2015-01-01

    Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area. PMID:25251024

  14. Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays.

    PubMed

    Evangelista, R A; Pollak, A; Templeton, E F

    1991-08-15

    Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications. PMID:1952068

  15. 7 CFR 982.2 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.2 Act. Act means Public Act...

  16. 7 CFR 982.2 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.2 Act. Act means Public Act...

  17. 7 CFR 982.2 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.2 Act. Act means Public Act...

  18. 7 CFR 982.2 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.2 Act. Act means Public Act...

  19. 7 CFR 1210.302 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.302 Act. Act means the Watermelon Research and Promotion Act of 1985...

  20. 7 CFR 1210.302 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...COMMODITIES), DEPARTMENT OF AGRICULTURE WATERMELON RESEARCH AND PROMOTION PLAN Watermelon Research and Promotion Plan Definitions § 1210.302 Act. Act means the Watermelon Research and Promotion Act of 1985...