Science.gov

Sample records for alpha-glucan acting enzymes

  1. Aspergillus niger genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles.

    PubMed

    Yuan, Xiao-Lian; van der Kaaij, Rachel M; van den Hondel, Cees A M J J; Punt, Peter J; van der Maarel, Marc J E C; Dijkhuizen, Lubbert; Ram, Arthur F J

    2008-06-01

    The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of alpha-amylase, glucoamylase and alpha-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative alpha-glucosidase (AgdB) and an alpha-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative alpha-glucan modifying processes are discussed. PMID:18320228

  2. ApuA, a multifunctional alpha-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus.

    PubMed

    Ferrando, Maria Laura; Fuentes, Susana; de Greeff, Astrid; Smith, Hilde; Wells, Jerry M

    2010-09-01

    We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved alpha-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal alpha-amylase domain of ApuA was shown to have alpha-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase alpha-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host. PMID:20522493

  3. Effect of glucose on alpha-glucan degradation in Polyporus circinatus.

    PubMed Central

    Oliveira, M B; Zancan, G T

    1981-01-01

    The effect of glucose on the enzymes involved in the degradation of a reserve alpha-glucan in Polyporus circinatus was studied. The levels of phosphorylase activity, endoamylase, amylo-1,6-glucosidase were regulated by glucose concentration. PMID:6161914

  4. 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

  5. The structure of cell wall alpha-glucan from fission yeast.

    PubMed

    Grün, Christian H; Hochstenbach, Frans; Humbel, Bruno M; Verkleij, Arie J; Sietsma, J Hans; Klis, Frans M; Kamerling, Johannis P; Vliegenthart, Johannes F G

    2005-03-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis. PMID:15470229

  6. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

    PubMed Central

    Hochstenbach, Frans; Klis, Frans M.; van den Ende, Herman; van Donselaar, Elly; Peters, Peter J.; Klausner, Richard D.

    1998-01-01

    The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1+, which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis. PMID:9689051

  7. 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.

  8. The structural basis of alpha-glucan recognition by a family 41 carbohydrate-binding module from Thermotoga maritima.

    PubMed

    van Bueren, Alicia Lammerts; Boraston, Alisdair B

    2007-01-19

    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 6(3)-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. PMID:17095014

  9. 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 α-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 α-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 α-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

  10. Tailoring enzymes acting on carrier protein-tethered substrates in natural product biosynthesis.

    PubMed

    Lin, Shuangjun; Huang, Tingting; Shen, Ben

    2012-01-01

    Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-β-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products. PMID:23034236

  11. 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 ...

  12. Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal alpha-amylase enzymes.

    PubMed

    van der Kaaij, R M; Janecek, S; van der Maarel, M J E C; Dijkhuizen, L

    2007-12-01

    Currently known fungal alpha-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal alpha-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal alpha-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall alpha-glucan. To study the biochemical characteristics of this novel cluster of alpha-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg(-1)), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall alpha-glucan synthesis is discussed. PMID:18048915

  13. Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus

    PubMed Central

    Becker, Stephen C.; Roach, Dwayne R.; Chauhan, Vinita S.; Shen, Yang; Foster-Frey, Juli; Powell, Anne M.; Bauchan, Gary; Lease, Richard A.; Mohammadi, Homan; Harty, William J.; Simmons, Chad; Schmelcher, Mathias; Camp, Mary; Dong, Shengli; Baker, John R.; Sheen, Tamsin R.; Doran, Kelly S.; Pritchard, David G.; Almeida, Raul A.; Nelson, Daniel C.; Marriott, Ian; Lee, Jean C.; Donovan, David M.

    2016-01-01

    Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics. PMID:27121552

  14. Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus.

    PubMed

    Becker, Stephen C; Roach, Dwayne R; Chauhan, Vinita S; Shen, Yang; Foster-Frey, Juli; Powell, Anne M; Bauchan, Gary; Lease, Richard A; Mohammadi, Homan; Harty, William J; Simmons, Chad; Schmelcher, Mathias; Camp, Mary; Dong, Shengli; Baker, John R; Sheen, Tamsin R; Doran, Kelly S; Pritchard, David G; Almeida, Raul A; Nelson, Daniel C; Marriott, Ian; Lee, Jean C; Donovan, David M

    2016-01-01

    Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics. PMID:27121552

  15. Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

    PubMed

    van der Kaaij, R M; Yuan, X-L; Franken, A; Ram, A F J; Punt, P J; van der Maarel, M J E C; Dijkhuizen, L

    2007-07-01

    In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed. PMID:17496125

  16. 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

  17. A natural vanishing act: the enzyme-catalyzed degradation of carbon nanomaterials.

    PubMed

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

    2012-10-16

    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 and biodegradation. Finally, we outline potential future directions that could enhance our mechanistic understanding of the CNM oxidation and 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 and 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 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

  18. Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

    PubMed

    Kalousek, F; Hendrick, J P; Rosenberg, L E

    1988-10-01

    The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size. PMID:3050998

  19. Inspection of the activator binding site for 4-alpha-glucanotransferase in porcine liver glycogen debranching enzyme with fluorogenic dextrins.

    PubMed

    Yamamoto, Eriko; Watanabe, Yumiko; Makino, Yasushi; Omichi, Kaoru

    2009-05-01

    Recently, we found that alpha-, beta- and gamma-cyclodextrins accelerated the 4-alpha-glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), and proposed the presence of an activator binding site in the GDE molecule. In liver cells, the structures of alpha-glucans proximal to the site GDE acts are not cyclodextrins, but glycogen and its degradation products. To estimate the structural characteristics of intrinsic activators and to inspect the features of the activator binding site, we examined the effects of four fluorogenic dextrins, (Glcalpha1-6)(m)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (B5/51, m = 1, n = 3; B6/61, m = 1, n = 4; B7/71, m = 1, n = 5; G6PA, m = 0, n = 4), on the debranching of B5/84 by porcine liver GDE. The GDE 4-alpha-glucanotransferase removed the maltotriosyl residue from the maltotetraosyl branch of B5/84, producing Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G9PA). In the absence of G6PA, the removed maltotriosyl residue was transferred to water. B7/71, B6/61 and B5/51 did not undergo any changes by the GDE, but they accelerated the action of the 4-alpha-glucanotransferase in removing the maltotriosyl residue. Of the four fluorogenic dextrins examined, B6/61 most strongly accelerated the 4-alpha-glucanotransferase action. The activator binding site is likely to be a space that accommodates the structure of Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glc. PMID:19155269

  20. Enzyme-catalyzed formation of beta-peptides: beta-peptidyl aminopeptidases BapA and DmpA acting as beta-peptide-synthesizing enzymes.

    PubMed

    Heck, Tobias; Kohler, Hans-Peter E; Limbach, Michael; Flögel, Oliver; Seebach, Dieter; Geueke, Birgit

    2007-09-01

    In recent studies, we discovered that the three beta-peptidyl aminopeptidases, BapA from Sphingosinicella xenopeptidilytica 3-2W4, BapA from S. microcystinivorans Y2, and DmpA from Ochrobactrum anthropi LMG7991, possess the unique feature of cleaving N-terminal beta-amino acid residues from beta- and alpha/beta-peptides. Herein, we investigated the use of the same three enzymes for the reverse reaction catalyzing the oligomerization of beta-amino acids and the synthesis of mixed peptides with N-terminal beta-amino acid residues. As substrates, we employed the beta-homoamino acid derivatives H-beta hGly-pNA, H-beta3 hAla-pNA, H-(R)-beta3 hAla-pNA, H-beta3 hPhe-pNA, H-(R)-beta3 hPhe-pNA, and H-beta3 hLeu-pNA. All three enzymes were capable of coupling the six beta-amino acids to oligomers with chain lengths of up to eight amino acid residues. With the enzyme DmpA as the catalyst, we observed very high conversion rates, which correspond to dimer yields of up to 76%. The beta-dipeptide H-beta3 hAla-beta3 hLeu-OH and the beta/alpha-dipeptide H-beta hGly-His-OH (carnosine) were formed with almost 50% conversion, when a five-fold excess of beta3-homoleucine or histidine was incubated with H-beta3 hAla-pNA and H-beta hGly-pNA, respectively, in the presence of the enzyme BapA from S. microcystinivorans Y2. BapA from S. xenopeptidilytica 3-2W4 turned out to be a versatile catalyst capable of coupling various beta-amino acid residues to the free N-termini of beta- and alpha-amino acids and even to an alpha-tripeptide. Thus, these aminopeptidases might be useful to introduce a beta-amino acid residue as an N-terminal protecting group into a 'natural' alpha-peptide, thereby stabilizing the peptide against degradation by other proteolytic enzymes. PMID:17886858

  1. Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio.

    PubMed

    Kralj, Slavko; van Leeuwen, Sander S; Valk, Vincent; Eeuwema, Wieger; Kamerling, Johannis P; Dijkhuizen, Lubbert

    2008-12-01

    The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into alpha-d-glucans (labelled reuterans) with mainly alpha-(1-->4) glucosidic linkages (50% and 70%, respectively), plus alpha-(1-->6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1-3; O1-3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize alpha-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in alpha-(1-->4) and alpha-(1-->6) linkage ratios. PMID:19016850

  2. Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains.

    PubMed

    Kralj, S; van Geel-Schutten, G H; Dondorff, M M G; Kirsanovs, S; van der Maarel, M J E C; Dijkhuizen, L

    2004-11-01

    Members of the genera Streptococcus and Leuconostoc synthesize various alpha-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly alpha-(1-->4) linkages together with alpha-(1-->6) and alpha-(1-->4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. This paper reports, for the first time, isolation and characterization of dextransucrase and mutansucrase genes and enzymes from various Lactobacillus species and the characterization of the glucan products synthesized, which mainly have alpha-(1-->6)- and alpha-(1-->3)-glucosidic linkages. The four GTF enzymes characterized from three different Lb. reuteri strains are highly similar at the amino acid level, and consequently their protein structures are very alike. Interestingly, these four Lb. reuteri GTFs have relatively large N-terminal variable regions, containing RDV repeats, and relatively short putative glucan-binding domains with conserved and less-conserved YG-repeating units. The three other GTF enzymes, isolated from Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri, contain smaller variable regions and larger putative glucan-binding domains compared to the Lb. reuteri GTF enzymes. PMID:15528655

  3. Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells.

    PubMed

    Zhao, Lin; Liu, Zhongbo; Jia, Haiqun; Feng, Zhihui; Liu, Jiankang; Li, Xuesen

    2015-01-01

    In our previous study, we found that pretreatment with lipoamide (LM) more effectively than alpha-lipoic acid (LA) protected retinal pigment epithelial (RPE) cells from the acrolein-induced damage. However, the reasons and mechanisms for the greater effect of LM than LA are unclear. We hypothesize that LM, rather than the more direct antioxidant LA, may act more as an indirect antioxidant. In the present study, we treated ARPE-19 cells with LA and LM and compared their effects on activation of mitochondrial biogenesis and induction of phase II enzyme systems. It is found that LM is more effective than LA on increasing mitochondrial biogenesis and inducing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation to the nucleus, leading to an increase in expression or activity of phase II antioxidant enzymes (NQO-1, GST, GCL, catalase and Cu/Zn SOD). Further study demonstrated that mitochondrial biogenesis and phase II enzyme induction are closely coupled via energy requirements. These results suggest that LM, compared with the direct antioxidant LA, plays its protective effect on oxidative damage more as an indirect antioxidant to simultaneously stimulate mitochondrial biogenesis and induction of phase II antioxidant enzymes. PMID:26030919

  4. Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells

    PubMed Central

    Zhao, Lin; Liu, Zhongbo; Jia, Haiqun; Feng, Zhihui; Liu, Jiankang; Li, Xuesen

    2015-01-01

    In our previous study, we found that pretreatment with lipoamide (LM) more effectively than alpha-lipoic acid (LA) protected retinal pigment epithelial (RPE) cells from the acrolein-induced damage. However, the reasons and mechanisms for the greater effect of LM than LA are unclear. We hypothesize that LM, rather than the more direct antioxidant LA, may act more as an indirect antioxidant. In the present study, we treated ARPE-19 cells with LA and LM and compared their effects on activation of mitochondrial biogenesis and induction of phase II enzyme systems. It is found that LM is more effective than LA on increasing mitochondrial biogenesis and inducing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation to the nucleus, leading to an increase in expression or activity of phase II antioxidant enzymes (NQO-1, GST, GCL, catalase and Cu/Zn SOD). Further study demonstrated that mitochondrial biogenesis and phase II enzyme induction are closely coupled via energy requirements. These results suggest that LM, compared with the direct antioxidant LA, plays its protective effect on oxidative damage more as an indirect antioxidant to simultaneously stimulate mitochondrial biogenesis and induction of phase II antioxidant enzymes. PMID:26030919

  5. Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose.

    PubMed

    Rytioja, Johanna; Hildén, Kristiina; Hatakka, Annele; Mäkelä, Miia R

    2014-11-01

    The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-acting enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable transcript levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cellulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expression levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus. PMID:24394946

  6. The Catalytic Scaffold fo the Haloalkanoic Acid Dehalogenase Enzyme Superfamily Acts as a Mold for the Trigonal Bipyramidal Transition State

    SciTech Connect

    Lu,Z.; Dunaway-Mariano, D.; Allen, K.

    2008-01-01

    The evolution of new catalytic activities and specificities within an enzyme superfamily requires the exploration of sequence space for adaptation to a new substrate with retention of those elements required to stabilize key intermediates/transition states. Here, we propose that core residues in the large enzyme family, the haloalkanoic acid dehalogenase enzyme superfamily (HADSF) form a 'mold' in which the trigonal bipyramidal transition states formed during phosphoryl transfer are stabilized by electrostatic forces. The vanadate complex of the hexose phosphate phosphatase BT4131 from Bacteroides thetaiotaomicron VPI-5482 (HPP) determined at 1.00 Angstroms resolution via X-ray crystallography assumes a trigonal bipyramidal coordination geometry with the nucleophilic Asp-8 and one oxygen ligand at the apical position. Remarkably, the tungstate in the complex determined to 1.03 Angstroms resolution assumes the same coordination geometry. The contribution of the general acid/base residue Asp-10 in the stabilization of the trigonal bipyramidal species via hydrogen-bond formation with the apical oxygen atom is evidenced by the 1.52 Angstroms structure of the D10A mutant bound to vanadate. This structure shows a collapse of the trigonal bipyramidal geometry with displacement of the water molecule formerly occupying the apical position. Furthermore, the 1.07 Angstroms resolution structure of the D10A mutant complexed with tungstate shows the tungstate to be in a typical 'phosphate-like' tetrahedral configuration. The analysis of 12 liganded HADSF structures deposited in the protein data bank (PDB) identified stringently conserved elements that stabilize the trigonal bipyramidal transition states by engaging in favorable electrostatic interactions with the axial and equatorial atoms of the transferring phosphoryl group.

  7. X-ray structure of MalY from Escherichia coli: a pyridoxal 5'-phosphate-dependent enzyme acting as a modulator in mal gene expression.

    PubMed

    Clausen, T; Schlegel, A; Peist, R; Schneider, E; Steegborn, C; Chang, Y S; Haase, A; Bourenkov, G P; Bartunik, H D; Boos, W

    2000-03-01

    MalY represents a bifunctional pyridoxal 5'-phosphate-dependent enzyme acting as a beta-cystathionase and as a repressor of the maltose regulon. Here we present the crystal structures of wild-type and A221V mutant protein. Each subunit of the MalY dimer is composed of a large pyridoxal 5'-phosphate-binding domain and a small domain similar to aminotransferases. The structural alignment with related enzymes identifies residues that are generally responsible for beta-lyase activity and depicts a unique binding mode of the pyridoxal 5'-phosphate correlated with a larger, more flexible substrate-binding pocket. In a screen for MalY mutants with reduced mal repressor properties, mutations occurred in three clusters: I, 83-84; II, 181-189 and III, 215-221, which constitute a clearly distinguished region in the MalY crystal structure far away from the cofactor. The tertiary structure of one of these mutants (A221V) demonstrates that positional rearrangements are indeed restricted to regions I, II and III. Therefore, we propose that a direct protein-protein interaction with MalT, the central transcriptional activator of the maltose system, underlies MalY-dependent repression of the maltose system. PMID:10698925

  8. The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

    PubMed Central

    Lindenberg, Sandra; Klauck, Gisela; Pesavento, Christina; Klauck, Eberhard; Hengge, Regine

    2013-01-01

    C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling. PMID:23708798

  9. The catecholamine biosynthetic enzyme dopamine β-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

  10. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  11. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    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.

  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

    SciTech Connect

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

    1993-07-13

    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. In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate

    PubMed Central

    Savarino, Andrea

    2007-01-01

    Background HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain. Methods Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD). Results Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC50s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N-Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those

  15. Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells

    PubMed Central

    Ryan, Elizabeth J.; McNeela, Edel; Murphy, Geraldine A.; Stewart, Helen; O'hagan, Derek; Pizza, Mariagrazia; Rappuoli, Rino; Mills, Kingston H. G.

    1999-01-01

    Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1–Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive. PMID:10569737

  16. Mutants of Escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic AB complex and enzyme activity on Th1 and Th2 cells.

    PubMed

    Ryan, E J; McNeela, E; Murphy, G A; Stewart, H; O'hagan, D; Pizza, M; Rappuoli, R; Mills, K H

    1999-12-01

    Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1-Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive. PMID:10569737

  17. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs.

    PubMed

    Gasciolli, Virginie; Mallory, Allison C; Bartel, David P; Vaucheret, Hervé

    2005-08-23

    Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production. PMID:16040244

  18. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  19. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  20. 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...

  1. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  2. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  3. 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…

  4. 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)

  5. DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA)

    PubMed Central

    Erova, Tatiana E.; Kosykh, Valeri G.; Sha, Jian; Chopra, Ashok K.

    2013-01-01

    Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam+) and GM33 (Δdam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA+ strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ΔgidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation caused by

  6. 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.

  7. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  8. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  9. 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 ...

  10. 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)

  11. ACT Test

    MedlinePlus

    ... this page helpful? Also known as: ACT; Activated Coagulation Time Formal name: Activated Clotting Time Related tests: ... in the blood called platelets and proteins called coagulation factors are activated in a sequence of steps ...

  12. Engineering Cellulase Enzymes for Bioenergy

    NASA Astrophysics Data System (ADS)

    Atreya, Meera Elizabeth

    Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

  13. 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.

  14. The ENZYME data bank.

    PubMed Central

    Bairoch, A

    1994-01-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and it contains the following data for each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided: EC number Recommended name Alternative names (if any) Catalytic activity Cofactors (if any) Pointers to the SWISS-PROT protein sequence entrie(s) that correspond to the enzyme (if any) Pointers to human disease(s) associated with a deficiency of the enzyme (if any). PMID:7937072

  15. Enzyme Therapy: Current Perspectives.

    PubMed

    UmaMaheswari, Thiyagamoorthy; Hemalatha, Thiagarajan; Sankaranarayanan, Palavesam; Puvanakrishnan, Rengarajulu

    2016-01-01

    Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders. PMID:26891548

  16. Developments in Enzyme Technology.

    ERIC Educational Resources Information Center

    Chaplin, M. F.

    1984-01-01

    Enzyme technology has a well-established industrial base, with applications that have survived competition. The most prominent applications of enzymes in biotechnology are examined with an explanation of some theoretical background. Topics include extending an enzyme's useful life, partition and diffusion, industrial uses, and therapeutic uses.…

  17. 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 β-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.

  18. Novel enzymes for the degradation of cellulose.

    PubMed

    Horn, Svein Jarle; Vaaje-Kolstad, Gustav; Westereng, Bjørge; Eijsink, Vincent Gh

    2012-01-01

    The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix. PMID:22747961

  19. Novel enzymes for the degradation of cellulose

    PubMed Central

    2012-01-01

    The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix. PMID:22747961

  20. 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.

  1. Enzymes for improved biomass conversion

    DOEpatents

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  2. Divergence and Convergence in Enzyme Evolution*

    PubMed Central

    Galperin, Michael Y.; Koonin, Eugene V.

    2012-01-01

    Comparative analysis of the sequences of enzymes encoded in a variety of prokaryotic and eukaryotic genomes reveals convergence and divergence at several levels. Functional convergence can be inferred when structurally distinct and hence non-homologous enzymes show the ability to catalyze the same biochemical reaction. In contrast, as a result of functional diversification, many structurally similar enzyme molecules act on substantially distinct substrates and catalyze diverse biochemical reactions. Here, we present updates on the ATP-grasp, alkaline phosphatase, cupin, HD hydrolase, and N-terminal nucleophile (Ntn) hydrolase enzyme superfamilies and discuss the patterns of sequence and structural conservation and diversity within these superfamilies. Typically, enzymes within a superfamily possess common sequence motifs and key active site residues, as well as (predicted) reaction mechanisms. These observations suggest that the strained conformation (the entatic state) of the active site, which is responsible for the substrate binding and formation of the transition complex, tends to be conserved within enzyme superfamilies. The subsequent fate of the transition complex is not necessarily conserved and depends on the details of the structures of the enzyme and the substrate. This variability of reaction outcomes limits the ability of sequence analysis to predict the exact enzymatic activities of newly sequenced gene products. Nevertheless, sequence-based (super)family assignments and generic functional predictions, even if imprecise, provide valuable leads for experimental studies and remain the best approach to the functional annotation of uncharacterized proteins from new genomes. PMID:22069324

  3. 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.

  4. 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.

  5. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Pacold, Ivan

    1972-01-01

    Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf. PMID:16657968

  6. Human Photoreactivating Enzyme

    PubMed Central

    Sutherland, J. C.; Sutherland, B. M.

    1975-01-01

    The action spectrum for photoreactivation by enzymes from human leukocytes and fibroblasts extends from 300 to approximately 600 nm with a maximum near 400 nm. The ability of the human enzymes to utilize light of wavelengths greater than 500 nm suggested that yellow or gold lights conventionally used as safelights for photoreactivation might serve as sources of photoreactivating light for these enzymes. Experiments using lights with a range of spectral outputs confirm that the standard yellow “safe” lights do produce photoreactivation by the human but not the Escherichia coli enzyme. PMID:19211015

  7. Adenylate-forming enzymes.

    PubMed

    Schmelz, Stefan; Naismith, James H

    2009-12-01

    Thioesters, amides, and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture, and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  8. 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.

  9. 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

  10. Extracting enzyme processivity from kinetic assays

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    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.

  11. 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...

  12. Cellulose degradation by oxidative enzymes.

    PubMed

    Dimarogona, Maria; Topakas, Evangelos; Christakopoulos, Paul

    2012-01-01

    Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future. PMID:24688656

  13. Cellulose degradation by oxidative enzymes

    PubMed Central

    Dimarogona, Maria; Topakas, Evangelos; Christakopoulos, Paul

    2012-01-01

    Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future. PMID:24688656

  14. Enzyme nanoband electrodes

    SciTech Connect

    Wang, J.; Naser, N. ); Renschler, C.L. )

    1993-07-01

    Enzyme nanoelectrodes have been constructed by immobilizing glucose oxidase, alcohol oxidase or tyrosinase onto ultrathin carbon films (of 35-50 nm thickness). The enzyme immobilization is accomplished via entrapment within electropolymerized poly(o-phenylenediamine) coatings. Cyclic voltammetry and controlled-potential amperometry are used to characterize the performance of the new nanoscopic biosensors under different preparation and operation conditions. The resulting electrodes offer convenient and rapid measurements of millimolar substrate concentrations, and (to the best of the authors' knowledge) are the smallest enzyme probes reported to date. 10 refs., 7 figs.

  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. Enzyme Kinetics in Microgravity

    NASA Astrophysics Data System (ADS)

    Liu, C. C.; Licata, V. J.

    2010-04-01

    The kinetics of some enzymes have been found to be enhanced by the microgravity environment. This is a relatively small effect, but is sufficient to have physiological effects and to impact pharmaceutical therapy in microgravity.

  17. Enzymes in Analytical Chemistry.

    ERIC Educational Resources Information Center

    Fishman, Myer M.

    1980-01-01

    Presents tabular information concerning recent research in the field of enzymes in analytic chemistry, with methods, substrate or reaction catalyzed, assay, comments and references listed. The table refers to 128 references. Also listed are 13 general citations. (CS)

  18. 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)

  19. Overproduction of ligninolytic enzymes

    DOEpatents

    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.

  20. Enzyme and microbial sensors for environmental monitoring

    NASA Astrophysics Data System (ADS)

    Wollenberger, U.; Neumann, B.; Scheller, Frieder W.

    1993-03-01

    Biosensors employing the biocatalyst on a different level of integration have been developed for monitoring environmental pollution. These probes range from laboratory specimen to commercial detectors applied to analyzers. This paper presents a selection of recent developments on amperometric enzyme and microbial biosensors. A monoenzymatic bulk type carbon electrode is described for biosensing organic hydroperoxides in aqueous solutions. Here, peroxidase is immobilized within the electrode body and the direct electron transfer between electrode and enzyme is measured. Both, reversible and irreversible inhibitors of acetylcholinesterase have been quantified by using a kinetically controlled acetylcholine enzyme sequence electrode. The inhibitory effect of pesticides such as butoxycarboxime, dimethoate, and trichlorfon could be quantified within 6 min in micrometers olar concentrations. Different multi-enzyme electrodes have been developed for the determination of inorganic phosphate. These sensors represent examples of sequentially acting enzymes in combination with enzymatic analyte recycling. Using this type of amplification nanomolar concentrations could be measured. A very fast responding microbial sensor for biological oxygen demand has been developed by immobilizing Trichosporon cutaneum onto an oxygen electrode. With this whole cell sensor waste water can be assayed with a sample frequency of 20 per hour and a working stability of more than 30 days.

  1. 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

  2. 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.

  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. Lipid metabolizing enzyme activities modulated by phospholipid substrate lateral distribution.

    PubMed

    Salinas, Dino G; Reyes, Juan G; De la Fuente, Milton

    2011-09-01

    Biological membranes contain many domains enriched in phospholipid lipids and there is not yet clear explanation about how these domains can control the activity of phospholipid metabolizing enzymes. Here we used the surface dilution kinetic theory to derive general equations describing how complex substrate distributions affect the activity of enzymes following either the phospholipid binding kinetic model (which assumes that the enzyme molecules directly bind the phospholipid substrate molecules), or the surface-binding kinetic model (which assumes that the enzyme molecules bind to the membrane before binding the phospholipid substrate). Our results strongly suggest that, if the enzyme follows the phospholipid binding kinetic model, any substrate redistribution would increase the enzyme activity over than observed for a homogeneous distribution of substrate. Besides, enzymes following the surface-binding model would be independent of the substrate distribution. Given that the distribution of substrate in a population of micelles (each of them a lipid domain) should follow a Poisson law, we demonstrate that the general equations give an excellent fit to experimental data of lipases acting on micelles, providing reasonable values for kinetic parameters--without invoking special effects such as cooperative phenomena. Our theory will allow a better understanding of the cellular-metabolism control in membranes, as well as a more simple analysis of the mechanisms of membrane acting enzymes. PMID:21108012

  5. The ENZYME database in 2000.

    PubMed

    Bairoch, A

    2000-01-01

    The ENZYME database is a repository of information related to the nomenclature of enzymes. In recent years it has became an indispensable resource for the development of metabolic databases. The current version contains information on 3705 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/enzyme/ ). PMID:10592255

  6. The ENZYME database in 2000

    PubMed Central

    Bairoch, Amos

    2000-01-01

    The ENZYME database is a repository of information related to the nomenclature of enzymes. In recent years it has became an indispensable resource for the development of metabolic databases. The current version contains information on 3705 enzymes. It is available through the ExPASy WWW server (http://www. expasy.ch/enzyme/ ). PMID:10592255

  7. Toying with Enzyme Catalysis.

    ERIC Educational Resources Information Center

    Richards, Debbie

    1998-01-01

    Describes a set of manipulatives that are used to establish a secure understanding of the concepts related to the environmental factors that affect the activities of enzymes. Includes a description of the model components and procedures for construction of the model. (DDR)

  8. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  9. Computational enzyme design

    NASA Astrophysics Data System (ADS)

    Bolon, Daniel N.

    2002-08-01

    The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function. In order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate. Using a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies. Because polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been

  10. Monitoring enzyme kinetic behavior of enzyme-quantum dot bioconjugates

    NASA Astrophysics Data System (ADS)

    Claussen, Jonathan C.; Walper, Scott A.; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

    2014-05-01

    Luminescent semiconductor nanocrystals or quantum dots (QDs) hold tremendous promise for in vivo biosensing, cellular imaging, theranostics, and smart molecular sensing probes due to their small size and favorable photonic properties such as resistance to photobleaching, size-tunable PL, and large effective Stokes shifts. Herein, we demonstrate how QD-based bioconjugates can be used to enhance enzyme kinetics. Enzyme-substrate kinetics are analyzed for solutions containing both alkaline phosphatase enzymes and QDs with enzyme-to- QD molar ratios of 2, 12, and 24 as well as for a solution containing the same concentration of enzymes but without QDs. The enzyme kinetic paramters Vmax, KM, and Kcat/KM are extracted from the enzyme progress curves via the Lineweaver-Burk plot. Results demonstrate an approximate increase in enzyme efficiency of 5 - 8% for enzymes immobilized on the QD versus free in solution without QD immobilization.

  11. Synthesis of the Enzymes of the Mandelate Pathway by Pseudomonas putida I. Synthesis of Enzymes by the Wild Type

    PubMed Central

    Hegeman, G. D.

    1966-01-01

    Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140–1154. 1966.—The control of synthesis of the five enzymes responsible for the conversion of d(−)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(−)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation. PMID:5929747

  12. 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

  13. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    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 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.

  15. 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)

  16. Robust enzyme-silica composites made from enzyme nanocapsules.

    PubMed

    Li, Jie; Jin, Xin; Liu, Yang; Li, Fan; Zhang, Linlin; Zhu, Xianyuan; Lu, Yunfeng

    2015-06-14

    Novel enzyme composites are synthesized first by in situ polymerization around enzymes and a subsequent sol-gel process. Both the polymer shell and the silica shell with desired functional moieties provide not only great enzyme protection but also a favorable microenvironment, resulting in significantly enhanced activity and stability. PMID:25971337

  17. Sulfite oxidizing enzymes

    PubMed Central

    Feng, Changjian; Tollin, Gordon; Enemark, John H.

    2007-01-01

    Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme

  18. 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.

  19. Extracting enzyme processivity from kinetic assays

    SciTech Connect

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

    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.

  20. 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…

  1. 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.

  2. ACTS data center

    NASA Technical Reports Server (NTRS)

    Syed, Ali; Vogel, Wolfhard J.

    1993-01-01

    Viewgraphs on ACTS Data Center status report are included. Topics covered include: ACTS Data Center Functions; data flow overview; PPD flow; RAW data flow; data compression; PPD distribution; RAW Data Archival; PPD Audit; and data analysis.

  3. ACTS data center

    NASA Astrophysics Data System (ADS)

    Syed, Ali; Vogel, Wolfhard J.

    1993-08-01

    Viewgraphs on ACTS Data Center status report are included. Topics covered include: ACTS Data Center Functions; data flow overview; PPD flow; RAW data flow; data compression; PPD distribution; RAW Data Archival; PPD Audit; and data analysis.

  4. 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.

  5. 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.

  6. 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,…

  7. Anion-π Enzymes

    PubMed Central

    2016-01-01

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pKa of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pKa of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

  8. Anion-π Enzymes.

    PubMed

    Cotelle, Yoann; Lebrun, Vincent; Sakai, Naomi; Ward, Thomas R; Matile, Stefan

    2016-06-22

    In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pK a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pK a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization. PMID:27413782

  9. Characterising Complex Enzyme Reaction Data.

    PubMed

    Dönertaş, Handan Melike; Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-01-01

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

  10. Characterising Complex Enzyme Reaction Data

    PubMed Central

    Rahman, Syed Asad; Thornton, Janet M.

    2016-01-01

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

  11. Industrial use of immobilized enzymes.

    PubMed

    DiCosimo, Robert; McAuliffe, Joseph; Poulose, Ayrookaran J; Bohlmann, Gregory

    2013-08-01

    Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization. PMID:23436023

  12. A sensitive enzyme electrode for phenol monitoring

    SciTech Connect

    Kulys, J.; Schmid, R.D. )

    1990-01-01

    Tyrosinase (EC.1.14.18.1) was immobilized onto graphite electrodes, which had been modified with tetracyanoquinodimethane (TCNQ). The response time, 12 or 35 s, was dependent on the enzyme immobilization technique used. The electrodes showed a linear calibration function up to 25 or 65 {mu}M phenol, and a sensitivity of 0.36 or 2.2 A/M was achieved which was also dependent on the enzyme immobilization technique used. The detection limit for phenol was 0.23 {mu}M. The electrodes acted from potentials of {minus}200 to +180 mV (vs. a saturated Ag/AgCl electrode). The electrode signal was independent of pH within the pH range 4.5-6.0. The enzyme electrode responded to phenol (100%), p-cresol (93%) and catechol (330%), but not to o-cresol and L-tyrosine. The electrodes showed a stability for more than one week. The electrodes can be utilized for the sensitive assay of phenol in water.

  13. Paradoxical Roles of Antioxidant Enzymes: Basic Mechanisms and Health Implications.

    PubMed

    Lei, Xin Gen; Zhu, Jian-Hong; Cheng, Wen-Hsing; Bao, Yongping; Ho, Ye-Shih; Reddi, Amit R; Holmgren, Arne; Arnér, Elias S J

    2016-01-01

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated from aerobic metabolism, as a result of accidental electron leakage as well as regulated enzymatic processes. Because ROS/RNS can induce oxidative injury and act in redox signaling, enzymes metabolizing them will inherently promote either health or disease, depending on the physiological context. It is thus misleading to consider conventionally called antioxidant enzymes to be largely, if not exclusively, health protective. Because such a notion is nonetheless common, we herein attempt to rationalize why this simplistic view should be avoided. First we give an updated summary of physiological phenotypes triggered in mouse models of overexpression or knockout of major antioxidant enzymes. Subsequently, we focus on a series of striking cases that demonstrate "paradoxical" outcomes, i.e., increased fitness upon deletion of antioxidant enzymes or disease triggered by their overexpression. We elaborate mechanisms by which these phenotypes are mediated via chemical, biological, and metabolic interactions of the antioxidant enzymes with their substrates, downstream events, and cellular context. Furthermore, we propose that novel treatments of antioxidant enzyme-related human diseases may be enabled by deliberate targeting of dual roles of the pertaining enzymes. We also discuss the potential of "antioxidant" nutrients and phytochemicals, via regulating the expression or function of antioxidant enzymes, in preventing, treating, or aggravating chronic diseases. We conclude that "paradoxical" roles of antioxidant enzymes in physiology, health, and disease derive from sophisticated molecular mechanisms of redox biology and metabolic homeostasis. Simply viewing antioxidant enzymes as always being beneficial is not only conceptually misleading but also clinically hazardous if such notions underpin medical treatment protocols based on modulation of redox pathways. PMID:26681794

  14. 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

  15. Enzyme molecules in solitary confinement.

    PubMed

    Liebherr, Raphaela B; Gorris, Hans H

    2014-01-01

    Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities. PMID:25221867

  16. 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.

  17. Enzyme therapeutics for systemic detoxification.

    PubMed

    Liu, Yang; Li, Jie; Lu, Yunfeng

    2015-08-01

    Life relies on numerous biochemical processes working synergistically and correctly. Certain substances disrupt these processes, inducing living organism into an abnormal state termed intoxication. Managing intoxication usually requires interventions, which is referred as detoxification. Decades of development on detoxification reveals the potential of enzymes as ideal therapeutics and antidotes, because their high substrate specificity and catalytic efficiency are essential for clearing intoxicating substances without adverse effects. However, intrinsic shortcomings of enzymes including low stability and high immunogenicity are major hurdles, which could be overcome by delivering enzymes with specially designed nanocarriers. Extensive investigations on protein delivery indicate three types of enzyme-nanocarrier architectures that show more promise than others for systemic detoxification, including liposome-wrapped enzymes, polymer-enzyme conjugates, and polymer-encapsulated enzymes. This review highlights recent advances in these nano-architectures and discusses their applications in systemic detoxifications. Therapeutic potential of various enzymes as well as associated challenges in achieving effective delivery of therapeutic enzymes will also be discussed. PMID:25980935

  18. Negative cooperativity in regulatory enzymes.

    PubMed

    Levitzki, A; Koshland, D E

    1969-04-01

    Negative cooperativity has been observed in CTP synthetase, an allosteric enzyme which contains a regulatory site. Thus, the same enzyme exhibits negative cooperativity for GTP (an effector) and glutamine (a substrate) and positive cooperativity for ATP and UTP (both substrates). In the process of the delineation of these phenomena, diagnostic procedures for negative cooperativity were developed. Application of these procedures to other enzymes indicates that negative cooperativity is a characteristic of many of them. These findings add strong support for the sequential model of subunit interactions which postulates that ligand-induced conformational changes are responsible for regulatory and cooperative phenomena in enzymes. PMID:5256410

  19. Enzyme Mimics: Advances and Applications.

    PubMed

    Kuah, Evelyn; Toh, Seraphina; Yee, Jessica; Ma, Qian; Gao, Zhiqiang

    2016-06-13

    Enzyme mimics or artificial enzymes are a class of catalysts that have been actively pursued for decades and have heralded much interest as potentially viable alternatives to natural enzymes. Aside from having catalytic activities similar to their natural counterparts, enzyme mimics have the desired advantages of tunable structures and catalytic efficiencies, excellent tolerance to experimental conditions, lower cost, and purely synthetic routes to their preparation. Although still in the midst of development, impressive advances have already been made. Enzyme mimics have shown immense potential in the catalysis of a wide range of chemical and biological reactions, the development of chemical and biological sensing and anti-biofouling systems, and the production of pharmaceuticals and clean fuels. This Review concerns the development of various types of enzyme mimics, namely polymeric and dendrimeric, supramolecular, nanoparticulate and proteinic enzyme mimics, with an emphasis on their synthesis, catalytic properties and technical applications. It provides an introduction to enzyme mimics and a comprehensive summary of the advances and current standings of their applications, and seeks to inspire researchers to perfect the design and synthesis of enzyme mimics and to tailor their functionality for a much wider range of applications. PMID:27062126

  20. Act II of the Sunshine Act.

    PubMed

    Pham-Kanter, Genevieve

    2014-11-01

    To coincide with the introduction in the United States of the Sunshine Act, Genevieve Pham-Kanter discusses what we need to look for to fight hidden bias and deliberate or unconscious corruption. Please see later in the article for the Editors' Summary. PMID:25369363

  1. Enzyme actuated bioresponsive hydrogels

    NASA Astrophysics Data System (ADS)

    Wilson, Andrew Nolan

    Bioresponsive hydrogels are emerging with technological significance in targeted drug delivery, biosensors and regenerative medicine. Conferred with the ability to respond to specific biologically derived stimuli, the design challenge is in effectively linking the conferred biospecificity with an engineered response tailored to the needs of a particular application. Moreover, the fundamental phenomena governing the response must support an appropriate dynamic range and limit of detection. The design of these systems is inherently complicated due to the high interdependency of the governing phenomena that guide the sensing, transduction, and the actuation response of hydrogels. To investigate the dynamics of these materials, model systems may be used which seek to interrogate the system dynamics by uni-variable experimentation and limit confounding phenomena such as: polymer-solute interactions, polymer swelling dynamics and biomolecular reaction-diffusion concerns. To this end, a model system, alpha-chymotrypsin (Cht) (a protease) and a cleavable peptide-chromogen (pro-drug) covalently incorporated into a hydrogel, was investigated to understand the mechanisms of covalent loading and release by enzymatic cleavage in bio-responsive delivery systems. Using EDC and Sulfo-NHS, terminal carboxyl groups of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a cleavable chromogen, were conjugated to primary amines of a hydrated poly(HEMA)-based hydrogel. Hydrogel discs were incubated in buffered Cht causing enzyme-mediated cleavage of the peptide and concomitant release of the chromophore for monitoring. To investigate substrate loading and the effects of hydrogel morphology on the system, the concentration of the amino groups (5, 10, 20, and 30 mol%) and the cross-linked density (1, 5, 7, 9 and 12 mol%) were independently varied. Loading-Release Efficiency of the chromogen was shown to exhibit a positive relation to increasing amino groups (AEMA). The release rates demonstrated a

  2. Rapid electrochemical enzyme assay with enzyme-free calibration.

    PubMed

    Zhang, Maogen; Karra, Sushma; Gorski, Waldemar

    2013-06-18

    The internally calibrated electrochemical continuous enzyme assay (ICECEA, patent pending) was developed for the fast determination of enzyme activity unit (U). The assay depends on the integration of enzyme-free preassay calibration with the actual enzyme assay in one continuous experiment. Such integration resulted in a uniquely shaped amperometric trace that allowed for the selective picomolar determination of redox enzymes. The ICECEA worked because the preassay calibration did not interfere with the enzyme assay allowing both measurements to be performed in succession in the same solution and at the same electrode. The method displayed a good accuracy (relative error, <3%) and precision (relative standard deviation (RSD), <3%) when tested with different working electrodes (carbon nanotubes/chitosan, glassy carbon, platinum) and enzymes (alcohol dehydrogenase, ADH; lactate dehydrogenase, LDH; xanthine oxidase, XOx; glucose oxidase, GOx). The limit of detection for the ADH, LDH, XOx, and GOx was equal to 0.18, 0.14, 0.0031, and 0.11 U L(-1) (or 4.2, 0.72, 89, and 6.0 pM), respectively. The simplicity, reliability, and short analysis time make the ICECEA competitive with the optical enzyme assays currently in use. PMID:23697336

  3. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  4. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    PubMed

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  5. Enzyme catalysis: Evolution made easy

    NASA Astrophysics Data System (ADS)

    Wee, Eugene J. H.; Trau, Matt

    2014-09-01

    Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.

  6. 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.

  7. 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…

  8. 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.

  9. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    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.

  10. 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.

  11. Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

    PubMed

    Bailey, Megan E; Jiang, Nan; Dima, Ruxandra I; Ross, Jennifer L

    2016-08-01

    Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016. PMID:27037673

  12. Production of theabrownins using a crude fungal enzyme concentrate.

    PubMed

    Wang, Qiuping; Gong, Jiashun; Chisti, Yusuf; Sirisansaneeyakul, Sarote

    2016-08-10

    Theabrownins were produced from infusions of sun-dried green tea leaves using a crude enzyme concentrate of Aspergillus tubingensis TISTR 3647. This fungus had been isolated from a solid state fermentation of Pu-erh type tea. The crude enzyme concentrate contained activities of peroxidase, catechol oxidase and laccase. The enzyme concentrate effectively oxidized the phenolic compounds in green tea infusion to theabrownins. A theabrownins concentration of 56.0g/L was obtained in 44h. The reaction mixture contained the green tea infusion and crude enzyme concentrate in the volume ratio of 1: 0.205. The tea infusion had been produced using 200g of tea leaves per liter of distilled water. The reaction was carried out in a stirred bioreactor at 37°C with an aeration rate of 1 vvm, an agitation speed of 250rpm and a controlled pH of 7.0. Peroxidase, catechol oxidase, and laccase acted synergistically to convert the phenolic compounds in green tea infusion to theabrownins. Previously, theabrownins had been produced from green tea infusions only by using live fungal cultures. Production using the microorganism-free enzyme concentrate was comparable to production using the fungus A. tubingensis TISTR 3647. The proposed novel production process using the fungal crude enzymes and green tea infusion, offers a more controlled, reproducible and highly productive option for commercial production of theabrownins. PMID:27318175

  13. 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

  14. Registration of heavy metal ions and pesticides with ATR planar waveguide enzyme sensors

    NASA Astrophysics Data System (ADS)

    Nabok, Alexei; Haron, Saharudin; Ray, Asim

    2004-11-01

    The proposed novel type of enzyme optical sensors is based on a combination of SiO2/Si3N4/SiO2 planar waveguide ATR (attenuated total reflection) transducer, fabricated by standard silicon planar technology, with the composite polyelectrolyte self-assembled coating containing both organic chromophores and enzyme molecules. Such devices were deployed to monitor typical industrial and agricultural water pollutants, such as heavy metal ions and pesticides, acting as inhibitors of enzyme reactions. The sensitivity of registration of these pollutants in the range of 1 ppb was achieved. The use of different enzymes in the sensitive membrane provides a background for pattern recognition of the above pollutants.

  15. 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.

  16. Metabolic regulation via enzyme filamentation

    PubMed Central

    Aughey, Gabriel N.; Liu, Ji-Long

    2016-01-01

    Abstract Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease. PMID:27098510

  17. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  18. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  19. Assertive Community Treatment (ACT)

    MedlinePlus

    ... community treatment? Assertive community treatment (ACT) is a model of psychiatric care that can be very effective ... it the most. Similar to the “treatment team” model of an inpatient psychiatric unit, which includes nurses, ...

  20. The ACTS propagation program

    NASA Technical Reports Server (NTRS)

    Chakraborty, Dayamoy; Davarian, Faramaz

    1991-01-01

    The purpose of the Advanced Communications Technology Satellite (ACTS) is to demonstrate the feasibility of the Ka-band (20 and 30 GHz) spectrum for satellite communications, as well as to help maintain U.S. leadership in satellite communications. ACTS incorporates such innovative schemes as time division multiple access (TDMA), microwave and baseband switching, onboard regeneration, and adaptive application of coding during rain-fade conditions. The success or failure of the ACTS experiment will depend on how accurately the rain-fade statistics and fade dynamics can be predicted in order to derive an appropriate algorithm that will combat weather vagaries, specifically for links with small terminals, such as very small aperture terminals (VSAT's) where the power margin is a premium. This article describes the planning process and hardware development program that will comply with the recommendations of the ACTS propagation study groups.

  1. 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.

  2. The ACTS propagation program

    NASA Technical Reports Server (NTRS)

    Chakraborty, D.; Davarian, Faramaz

    1992-01-01

    The success or failure of the ACTS experiment will depend on how accurately the rain-fade statistics and fade dynamics can be predicted in order to derive an appropriate algorithm that will combat weather vagaries, specifically for links with small terminals, such as very small aperture terminals (VSAT's) where the power margin is a premium. The planning process and hardware development program that will comply with the recommendations of the ACTS propagation study groups are described.

  3. [Angiotensin-converting enzyme inhibitors as neutralizers of hydroxyl radical].

    PubMed

    Mira, M L; Silva, M M; Queirós, M J; Manso, C

    1992-05-01

    Angiotensin converting enzyme inhibitors are utilized in the treatment of essential hypertension and of chronic cardiac failure. They are also employed in the treatment of the myocardial lesion of ischemia-reperfusion, which involves oxygen free radicals. In the present study we investigated the possibility of three angiotensin converting enzyme inhibitors (captopril, enalapril, lisinopril) to act as hydroxyl radical scavengers. The rate constants for reactions of those compounds with .OH were determined using the deoxyribose method. All there compounds proved to be good scavengers of .OH with rate constants of about 10(10)M-1s-1 and are iron chelators specially enalapril. The fact that captopril possesses a thiol group does not confer an higher antioxidative capacity. These results suggest that scavenging of oxygen free radicals may be a possible mechanism contributing to the therapeutic effect of angiotensin converting enzyme inhibitors. PMID:1325814

  4. Flow-cell fibre-optic enzyme sensor for phenols

    SciTech Connect

    Papkovsky, D.B.; Ghindilis, A.L.; Kurochkin, I.N. )

    1993-07-01

    A solid-state fibre-optic luminescent oxygen sensor was used for flow-through measurements. It acts as a transducer in a new flow-cell enzyme sensor arrangement. This arrangement comprises a flow path, sample injector, microcolumn with the immobilized enzyme, oxygen membrane and fibre-optic connector joined together to form an integral unit. Laccase enzyme was used as a recognition system which provided specific oxidation of the substrates with the dissolved oxygen being monitored. The assay procedure was optimized and performance of the new system studied. The sensor was applied to the determination polyphenol content in tea, brandy, etc. (quality control test). The sensitivity to some important phenolic compounds was tested with the view of industrial wastewater control applications. 5 refs., 6 figs., 1 tab.

  5. L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?

    PubMed Central

    Verri, A; Montecucco, A; Gosselin, G; Boudou, V; Imbach, J L; Spadari, S; Focher, F

    1999-01-01

    We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy. PMID:9895305

  6. Nanoporous gold for enzyme immobilization.

    PubMed

    Stine, Keith J; Jefferson, Kenise; Shulga, Olga V

    2011-01-01

    Nanoporous gold (NPG) is a material of emerging interest for immobilization of biomolecules and -especially enzymes. NPG materials provide a high gold surface area onto which biomolecules can either be directly physisorbed or covalently linked after first modifying the NPG with a self-assembled monolayer. The material can be used as a high surface area electrode and with immobilized enzymes can be used for amperometric detection schemes. NPG can be prepared in a variety of formats from alloys containing less than 50 atomic% gold by dealloying procedures. Related high surface area gold structures have been prepared using templating approaches. Covalent enzyme immobilization can be achieved by first forming a self-assembled monolayer on NPG bearing a terminal reactive functional group followed by conjugation to the enzyme through amide linkages to lysine residues. PMID:20865389

  7. 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

  8. 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

  9. 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

  10. PIXE analysis of Zn enzymes

    NASA Astrophysics Data System (ADS)

    Solís, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J. L.; Romero, I.; Celis, H.

    1999-04-01

    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillumrubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy.

  11. Enzymes: principles and biotechnological applications

    PubMed Central

    Robinson, Peter K.

    2015-01-01

    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

  12. Homogeneous enzyme immunoassay for netilmicin.

    PubMed Central

    Wenk, M; Hemmann, R; Follath, F

    1982-01-01

    A newly developed homogeneous enzyme immunoassay for the determination of netilmicin in serum was evaluated and compared with a radioenzymatic assay. A total of 102 serum samples from patients treated with netilmicin were measured by both methods. This comparison showed an excellent correlation (r = 0.993). The enzyme immunoassay has proved to be precise, accurate, and specific. Because of its rapidity and the ease of performance, this method is a useful alternative to current assays for monitoring serum netilmicin concentrations. PMID:6760807

  13. The CEO's second act.

    PubMed

    Nadler, David A

    2007-01-01

    When a CEO leaves because of performance problems, the company typically recruits someone thought to be better equipped to fix what the departing executive couldn't--or wouldn't. The board places its confidence in the new person because of the present dilemma's similarity to some previous challenge that he or she dealt with successfully. But familiar problems are inevitably succeeded by less familiar ones, for which the specially selected CEO is not quite so qualified. More often than not, the experiences, skills, and temperament that yielded triumph in Act I turn out to be unequal to Act II's difficulties. In fact, the approaches that worked so brilliantly in Act I may be the very opposite of what is needed in Act II. The CEO has four choices: refuse to change, in which case he or she will be replaced; realize that the next act requires new skills and learn them; downsize or circumscribe his or her role to compensate for deficiencies; or line up a successor who is qualified to fill a role to which the incumbent's skills and interests are no longer suited. Hewlett-Packard's Carly Fiorina exemplifies the first alternative; Merrill Lynch's Stanley O'Neal the second; Google's Sergey Brin and Larry Page the third; and Quest Diagnostics' Ken Freeman the fourth. All but the first option are reasonable responses to the challenges presented in the second acts of most CEOs' tenures. And all but the first require a power of observation, a propensity for introspection, and a strain of humility that are rare in the ranks of the very people who need those qualities most. There are four essential steps executives can take to discern that they have entered new territory and to respond accordingly: recognition that their leadership style and approach are no longer working; acceptance of others' advice on why performance is faltering; analysis and understanding of the nature of the Act II shift; and, finally, decision and action. PMID:17286076

  14. [The rise of enzyme engineering in China].

    PubMed

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China. PMID:26672358

  15. The ACTS multibeam antenna

    NASA Astrophysics Data System (ADS)

    Regier, Frank A.

    1992-06-01

    The Advanced Communications Technology Satellite (ACTS) to be launched in 1993 is briefly introduced. Its multibeam antenna, consisting of electrically similar 30 GHz receive and 20 GHz transmit offset Cassegrain systems, both utilizing orthogonal 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 degree 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 HEMT low-noise amplifier and a 20 GHz TWT power amplifier.

  16. 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 is briefly introduced. Its multibeam antenna, consisting of electrically similar 30 GHz receive and 20 GHz transmit offset Cassegrain systems, both utilizing orthogonal 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 degree 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 HEMT low-noise amplifier and a 20 GHz TWT power amplifier.

  17. Enzyme exposure and enzyme sensitisation in the baking industry.

    PubMed Central

    Vanhanen, M; Tuomi, T; Hokkanen, H; Tupasela, O; Tuomainen, A; Holmberg, P C; Leisola, M; Nordman, H

    1996-01-01

    OBJECTIVES: To assess the exposure to enzymes and prevalence of enzyme sensitisation in the baking industry. METHODS: A cross sectional study was conducted in four bakeries, one flour mill, and one crispbread factory. Sensitisation to enzymes, flours, and storage mites was examined by skin prick and radioallergosorbent (RAST) tests. 365 workers were tested. The workers were interviewed for work related respiratory and skin symptoms. Total dust concentrations were measured by a gravimetric method, and the concentration of alpha-amylase in air was measured by a catalytic method. An immunochemical method was used for measuring cellulase and xylanase in air. RESULTS: Total measured dust concentrations were from 0.1 to 18 mg/m3, with highest values in dough making areas of bakeries. The alpha-amylase concentrations generally followed the total dust concentrations and reached the highest values < 6.6 micrograms/m3 in the same areas. Cellulase and xylanase varied with concentrations < 180 ng/m3 and < 40 ng/m3, respectively, in the flour mill and the crispbread factory. No cellulase, but concentrations of 1-200 ng/m3 xylanase, were found in the bakeries, probably indicating the natural xylanase activity of wheat. 12 workers (8%) in the bakeries, three (5%) in the flour mill, and four (3%) in the crispbread factory were skin prick positive to enzymes. The corresponding percentages of positive reactions to flours were 12%, 5%, and 8%. CONCLUSIONS: The study confirmed that industrial enzymes in baking used as additives in a powdered form pose a risk of sensitisation. The no effect air concentrations for industrial enzymes are not known. Based on present knowledge, however, lowering exposures and eliminating short and high peaks by technical measures would lower the risk of sensitisation. This would be most effectively accomplished by shifting to non-dusty products. PMID:8943831

  18. Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment.

    PubMed

    Masran, Ruqayyah; Zanirun, Zuraidah; Bahrin, Ezyana Kamal; Ibrahim, Mohamad Faizal; Lai Yee, Phang; Abd-Aziz, Suraini

    2016-06-01

    Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process. PMID:27115758

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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…

  4. 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…

  5. 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.

  6. 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:…

  7. 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.

  8. Immunomodulatory Effects of Chitotriosidase Enzyme

    PubMed Central

    Elmonem, Mohamed A.; van den Heuvel, Lambertus P.; Levtchenko, Elena N.

    2016-01-01

    Chitotriosidase enzyme (EC: 3.2.1.14) is the major active chitinase in the human body. It is produced mainly by activated macrophages, in which its expression is regulated by multiple intrinsic and extrinsic signals. Chitotriosidase was confirmed as essential element in the innate immunity against chitin containing organisms such as fungi and protozoa; however, its immunomodulatory effects extend far beyond innate immunity. In the current review, we will try to explore the expanding spectrum of immunological roles played by chitotriosidase enzyme in human health and disease and will discuss its up-to-date clinical value. PMID:26881065

  9. Acts of kindness and acts of novelty affect life satisfaction.

    PubMed

    Buchanan, Kathryn E; Bardi, Anat

    2010-01-01

    The present experiment was designed to establish the effects of acts of kindness and acts of novelty on life satisfaction. Participants aged 18-60 took part on a voluntary basis. They were randomly assigned to perform either acts of kindness, acts of novelty, or no acts on a daily basis for 10 days. Their life satisfaction was measured before and after the 10-day experiment. As expected, performing acts of kindness or acts of novelty resulted in an increase in life satisfaction. PMID:20575332

  10. Call for an enzyme genomics initiative

    PubMed Central

    Karp, Peter D

    2004-01-01

    I propose an Enzyme Genomics Initiative, the goal of which is to obtain at least one protein sequence for each enzyme that has previously been characterized biochemically. There are 1,437 enzyme activities for which Enzyme Commission (EC) numbers have been assigned but no sequence can be found in public protein-sequence databases. PMID:15287973

  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. 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.

  13. 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.

  14. 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.

  15. Phage lytic enzymes targeting streptococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Streptococcal pathogens contribute to a wide variety of human and livestock diseases. There is a need for new antimicrobials to replace over-used conventional antibiotics. Bacteriophage (viruses that infect bacteria) endolysins (enzymes that help degrade the bacterial cell wall) are ideal candidat...

  16. 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...

  17. 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…

  18. Biological abatement of enzyme inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulose pretreatments release phenolic compounds that cause enzyme inhibition and deactivation. Bio-abatement, the biological removal of furfurals, acetic acid and phenolics, may utilize fungal fermentation to metabolize these compounds to CO2, water, cell mass, and heat. Our work with Coni...

  19. Organoclay-enzyme film electrodes.

    PubMed

    Mbouguen, Justin Kemmegne; Ngameni, Emmanuel; Walcarius, Alain

    2006-09-25

    This paper aims at showing the interest of organoclays (clay minerals containing organic groups covalently attached to the inorganic particles) as suitable host matrices likely to immobilize enzymes onto electrode surfaces for biosensing applications. The organoclays used in this work were natural Cameroonian smectites grafted with either aminopropyl (AP) or trimethylpropylammonium (TMPA) groups. The first ones were exploited for their ability to anchor biomolecules by covalent bonding while the second category exhibited favorable electrostatic interactions with negatively charged enzymes due to ion exchange properties that were pointed out here by means of multisweep cyclic voltammetry. AP-clay materials were applied to the immobilization of glucose oxidase (GOD) and TMPA-clays for polyphenol oxidase (PPO) anchoring. When deposited onto the surface of platinum or glassy carbon electrodes as enzyme/organoclay films, these systems were evaluated as biosensing electrochemical devices for detection of glucose and catechol chosen as model analytes. The advantageous features of these organoclays were discussed by comparison to the performance of related film electrodes made of non-functionalized clays. It appeared that organoclays provide a favorable environment to enzymes activity, as highlighted from the biosensors characteristics and determination of Michaelis-Menten constants. PMID:17723706

  20. 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.

  1. ACTS TDMA network control

    NASA Astrophysics Data System (ADS)

    Inukai, T.; Campanella, S. J.

    This paper presents basic network control concepts for the Advanced Communications Technology Satellite (ACTS) System. Two experimental systems, called the low-burst-rate and high-burst-rate systems, along with ACTS ground system features, are described. The network control issues addressed include frame structures, acquisition and synchronization procedures, coordinated station burst-time plan and satellite-time plan changes, on-board clock control based on ground drift measurements, rain fade control by means of adaptive forward-error-correction (FEC) coding and transmit power augmentation, and reassignment of channel capacities on demand. The NASA ground system, which includes a primary station, diversity station, and master control station, is also described.

  2. Purification and characterization of konjac glucomannan degrading enzyme from anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group.

    PubMed

    Nakajima, N; Matsuura, Y

    1997-10-01

    Konjac glucomannan degrading enzyme was purified to homogeneity from the culture broth of an anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group. The enzyme was composed of a single polypeptide chain with a molecular weight of 50,000-53,000. The enzyme was an endo-beta-mannanase that acted specifically on the polysaccharides such as konjac glucomannan and coffee mannan, producing exclusively their smaller oligosaccharides and the monosaccharides. The optimal pH of the enzyme for the hydrolysis of konjac glucomannan was around 7-8 and the enzyme was stable in rather alkaline pH range of 8-10. The enzyme reaction was activated by the addition of CaCl2 and dithiothreitol. It was suggested that the enzyme might contribute to the decomposition of konjac glucomannan in human digestive tract. PMID:9362121

  3. [Patients' Rights Act].

    PubMed

    Haier, A J

    2016-09-01

    The new Patients' Rights Act does not reflect rights of patients as professional obligations of physicians for the first time. It adopted common longtime jurisdiction, but in some respects it is going beyond. This law clearly extended the documentation requirements of physicians, especially concerning the extent of documentation. In surgical fields the requirements for enlightening physicians were more strongly worded than in previous jurisdiction. In medical facilities it is now mandatory to establish an internal quality management system. PMID:27626814

  4. 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).

  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. 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.

  7. The ACTS multibeam antenna

    NASA Astrophysics Data System (ADS)

    Regier, Frank A.

    1992-04-01

    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.

  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. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine.

    PubMed

    Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G

    2014-09-01

    The oldest animals on Earth, sponges, form both the calcareous and the siliceous matrices of their spicules enzymatically. Until recently, it has been neglected that enzymes play crucial roles during formation of these biominerals. This paradigm shift occurred after the discovery that the enzyme silicatein, which catalyzes the polycondensation of silica, and the enzyme carbonic anhydrase (CA), which catalyzes the formation of bicarbonate (HCO3(-)/CaCO3), produce solid amorphous bioglass or biocalcite. This suggests that in mammals, biosilica and biocalcite can act anabolically during hydroxyapatite (HA) synthesis and bone formation. Biosilica and biocalcite are thus promising candidates for the fabrication of biomaterials for regenerative medicine. PMID:24908383

  15. Delicate conformational balance of the redox enzyme cytochrome P450cam.

    PubMed

    Skinner, Simon P; Liu, Wei-Min; Hiruma, Yoshitaka; Timmer, Monika; Blok, Anneloes; Hass, Mathias A S; Ubbink, Marcellus

    2015-07-21

    The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction. PMID:26130807

  16. Delicate conformational balance of the redox enzyme cytochrome P450cam

    PubMed Central

    Skinner, Simon P.; Liu, Wei-Min; Hiruma, Yoshitaka; Timmer, Monika; Blok, Anneloes; Hass, Mathias A. S.; Ubbink, Marcellus

    2015-01-01

    The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction. PMID:26130807

  17. Feasibility of using an isolated intestinal segment as an artificial organ for enzyme replacement therapy.

    PubMed

    Shelt, D; Walton, D; Sato, P

    1982-01-01

    Guinea pigs fed an ascorbic acid-deficient diet develop scurvy because of the absence of the enzyme L-gulonolactone oxidase. In theory if this enzyme is provided and its substrate L-gulonolactone is present at adequate concentrations ascorbic acid will be synthesized and the development of scurvy prevented. Using this model we tested whether a viable segment of intestine could be used to contain the administered enzyme and act as an artificial organ for the production of ascorbic acid. A surgical procedure was developed to prepare an externalized pouch of intestine with its circulation left intact. When enzyme is inserted in this intestinal bag it is not toxic and not antigenic in some animals, whereas, enzyme injected intraperitoneally is clearly antigenic. Synthesis of ascorbic acid by this artificial organ could not, however, be detected by elevation of plasma concentrations of the vitamin. PMID:7104431

  18. EzCatDB: the enzyme reaction database, 2015 update.

    PubMed

    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

  19. 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.

  20. Enzyme catalysis in ionic liquids.

    PubMed

    Kragl, Udo; Eckstein, Marrit; Kaftzik, Nicole

    2002-12-01

    Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed. PMID:12482515

  1. Metrological aspects of enzyme production

    NASA Astrophysics Data System (ADS)

    Kerber, T. M.; Dellamora-Ortiz, G. M.; Pereira-Meirelles, F. V.

    2010-05-01

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies.

  2. 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.

  3. Quick acting gimbal joint

    NASA Technical Reports Server (NTRS)

    Wood, William B. (Inventor); Krch, Gary D. (Inventor)

    1993-01-01

    The present invention relates to an adjustable linkage assembly for selectively retaining the position of one member pivotable with respect to another member. More specifically, the invention relates to a linkage assembly commonly referred to as a gimbal joint, and particularly to a quick release or quick acting gimbal joint. The assembly is relatively simple in construction, compact in size, and has superior locking strength in any selected position. The device can be quickly and easily actuated, without separate tooling, by inexperienced personnel or by computer controlled equipment. It also is designed to prevent inadvertent actuation.

  4. Substrate Mediated Enzyme Prodrug Therapy

    PubMed Central

    Fejerskov, Betina; Zelikin, Alexander N.

    2012-01-01

    In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT) as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s) into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol), β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose – dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering. PMID:23152927

  5. Enzyme immunoassays in diagnostic medicine

    PubMed Central

    Voller, A.; Bidwell, D. E.; Bartlett, Ann

    1976-01-01

    Serological methods are playing an increasingly important role in the diagnosis and epidemiological assessment of diseases. Simple, inexpensive methods for large-scale application are urgently needed. The enzyme immunoassay methods developed recently and reviewed here hold great promise for application in a wide variety of conditions. Under laboratory conditions they can be as sensitive as radio-immunoassay, but they can also be adapted as simple field screening procedures. These methods are based on the use of antibodies or antigens that are linked to an insoluble carrier surface. This is then used to “capture” the relevant antigen or antibody in the test solution and the complex is detected by means of an enzyme-labelled antibody or antigen. The degradation of the enzyme substrate, measured photometrically, is proportional to the concentration of the unknown “antibody” or “antigen” in the test solution. The application of these techniques to endocrinology, immunopathology, haematology, microbiology, and parasitology is reviewed. PMID:1085667

  6. 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

  7. Enzyme-enabled responsive surfaces for anti-contamination materials.

    PubMed

    Wu, Songtao; Buthe, Andreas; Jia, Hongfei; Zhang, Minjuan; Ishii, Masahiko; Wang, Ping

    2013-06-01

    Many real-life stains have origins from biological matters including proteins, lipids, and carbohydrates that act as gluing agents binding along with other particulates or microbes to exposed surfaces of automobiles, furniture, and fabrics. Mimicking naturally occurring self-defensive processes, we demonstrate in this work that a solid surface carrying partially exposed enzyme granules protected the surface in situ from contamination by biological stains and fingerprints. Attributed to the activities of enzymes which can be made compatible with a wide range of materials, such anti-contamination and self-cleaning functionalities are highly selective and efficient toward sticky chemicals. This observation promises a new mechanism in developing smart materials with desired anti-microbial, self-reporting, self-cleaning, or self-healing functions. PMID:23335427

  8. 75 FR 63703 - Privacy Act of 1974; Privacy Act Regulation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-18

    ...The Board of Governors of the Federal Reserve System (Board) is issuing a final rule to amend its regulation implementing the Privacy Act of 1974 (Privacy Act). The primary changes concern the waiver of copying fees charged to current and former Board employees, and applicants for Board employment, for access to their records under the Privacy Act; the amendment of special procedures for the......

  9. 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

  10. The Amborella vacuolar processing enzyme family.

    PubMed

    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. 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.

  12. Affordable Care Act and Women

    MedlinePlus

    ... Privacy Policy FOIA Plain Writing Act No Fear Act Disclaimers Viewers & Players Assistant Secretary for Planning and Evaluation, Room 415F U.S. Department of Health and Human Services 200 Independence Avenue, SW Washington, D.C. ...

  13. 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...

  14. The Nurse Reinvestment Act revisited.

    PubMed

    Luther, Ann P

    2007-01-01

    The United States is in the midst of a widely recognized critical nursing shortage. In 2002 the "Nurse Reinvestment Act" was passed with overwhelming bipartisan support in an effort to address this serious public health threat. The Act is due for reauthorization of funding in 2007. This paper provides a brief overview of the programs contained within the Act and describes practical ways in which members of the nursing community can take action to insure renewed support for the Act. PMID:17691598

  15. 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.

  16. 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.

  17. Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

    PubMed

    Brooks, S P; Storey, K B

    1993-05-12

    The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell. PMID:8350861

  18. 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.

  19. ACT/SAT College Survey.

    ERIC Educational Resources Information Center

    Stafford, John E.

    1998-01-01

    Reports on findings of a survey designed to discover whether higher education institutions' admission standards accept SAT I or ACT and if there is preference for either, and whether ACT could be submitted in lieu of SAT II subject tests. Eighty-six percent of the reporting schools indicated no preference; 28 schools indicated that the ACT was an…

  20. Defining Acts of Journalistic Deception.

    ERIC Educational Resources Information Center

    Elliott, Deni; Culver, Charles M.

    To determine when, if ever, deceptive acts can be morally justified in investigative reporting, it is important to distinguish a deceptive act that is morally justified from an act that is not deceptive in the first place. This paper seeks to provide an account of what counts as deception and identify the kinds of journalistic practice that are…

  1. Digestive Enzyme Supplementation in Gastrointestinal Diseases

    PubMed Central

    Ianiro, Gianluca; Pecere, Silvia; Giorgio, Valentina; Gasbarrini, Antonio; Cammarota, Giovanni

    2016-01-01

    Background: Digestive enzymes are able to break down proteins and carbohydrates and lipids, and their supplementation may play a role in the management of digestive disorders, from lactose intolerance to cystic fibrosis. To date, several formulations of digestive enzymes are available on the market, being different each other in terms of enzyme type, source and origin, and dosage. Methods: This review, performed through a non-systematic search of the available literature, will provide an overview of the current knowledge of digestive enzyme supplementation in gastrointestinal disorders, discussion of the use of pancreatic enzymes, lactase (β-galactosidase) and conjugated bile acids, and also exploring the future perspective of digestive enzyme supplementation. Results: Currently, the animal-derived enzymes represent an established standard of care, however the growing study of plant-based and microbe-derived enzymes offers great promise in the advancement of digestive enzyme therapy. Conclusion: New frontiers of enzyme replacement are being evaluated also in the treatment of diseases not specifically related to enzyme deficiency, whereas the combination of different enzymes might constitute an intriguing therapeutic option in the future. PMID:26806042

  2. The ENZYME data bank in 1995.

    PubMed Central

    Bairoch, A

    1996-01-01

    The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. The current version (October 1995) contains information relevant to 3594 enzymes. It is available from a variety of file and ftp servers as well as through the ExPASy World Wide Web server (http://expasy.hcuge.ch/). PMID:8594586

  3. 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)…

  4. 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.

  5. 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...

  6. 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...

  7. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  8. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4400 Enzyme preparations. (a) Identification. Enzyme preparations are products that are used in the...

  9. 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...

  10. 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)

  11. The ENZYME data bank in 1999.

    PubMed Central

    Bairoch, A

    1999-01-01

    The ENZYME data bank is a repository of information related to the nomenclature of enzymes. In recent years it has become an indispensable resource for the development of metabolic databases. The current version contains information on 3704 enzymes. It is available through the ExPASy WWW server (http://www.expasy.ch/). PMID:9847212

  12. 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 β- 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

  13. 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.

  14. FAST ACTING CURRENT SWITCH

    DOEpatents

    Batzer, T.H.; Cummings, D.B.; Ryan, J.F.

    1962-05-22

    A high-current, fast-acting switch is designed for utilization as a crowbar switch in a high-current circuit such as used to generate the magnetic confinement field of a plasma-confining and heat device, e.g., Pyrotron. The device particularly comprises a cylindrical housing containing two stationary, cylindrical contacts between which a movable contact is bridged to close the switch. The movable contact is actuated by a differential-pressure, airdriven piston assembly also within the housing. To absorb the acceleration (and the shock imparted to the device by the rapidly driven, movable contact), an adjustable air buffer assembly is provided, integrally connected to the movable contact and piston assembly. Various safety locks and circuit-synchronizing means are also provided to permit proper cooperation of the invention and the high-current circuit in which it is installed. (AEC)

  15. Triple acting radial seal

    DOEpatents

    Ebert, Todd A; Carella, John A

    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.

  16. Caught in the Act

    NASA Technical Reports Server (NTRS)

    2005-01-01

    5 September 2005 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a dust devil caught in the act of creating a dark streak on the floor of the large, south mid-latitude crater, Mendel. Dozens of other dark streaks mark the paths of earlier dust devils. Dust devil streaks at southern middle and high latitudes are seasonal features; they are erased each winter by thin deposits of dust and frost, and they are re-created each spring and summer by new dust devils.

    Location near: 58.9oS, 199.4oW Image width: width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Spring

  17. Enzymes involved in the biodegradation of hexachlorocyclohexane: a mini review.

    PubMed

    Camacho-Pérez, Beni; Ríos-Leal, Elvira; Rinderknecht-Seijas, Noemí; Poggi-Varaldo, Héctor M

    2012-03-01

    The scope of this paper encompasses the following subjects: (i) aerobic and anaerobic degradation pathways of γ-hexachlorocyclohexane (HCH); (ii) important genes and enzymes involved in the metabolic pathways of γ-HCH degradation; (iii) the instrumental methods for identifying and quantifying intermediate metabolites, such as gas chromatography coupled to mass spectrometry (GC-MS) and other techniques. It can be concluded that typical anaerobic and aerobic pathways of γ-HCH are well known for a few selected microbial strains, although less is known for anaerobic consortia where the possibility of synergism, antagonism, and mutualism can lead to more particular routes and more effective degradation of γ-HCH. Conversion and removals in the range 39%-100% and 47%-100% have been reported for aerobic and anaerobic cultures, respectively. Most common metabolites reported for aerobic degradation of lindane are γ-pentachlorocyclohexene (γ-PCCH), 2,5-dichlorobenzoquinone (DCBQ), Chlorohydroquinone (CHQ), chlorophenol, and phenol, whereas PCCH, isomers of trichlorobenzene (TCB), chlorobenzene, and benzene are the most typical metabolites found in anaerobic pathways. Enzyme and genetic characterization of the involved molecular mechanisms are in their early infancy; more work is needed to elucidate them in the future. Advances have been made on identification of enzymes of Sphingomonas paucimobilis where the gene LinB codifies for the enzyme haloalkane dehalogenase that acts on 1,3,4,6-tetrachloro 1,4-cyclohexadiene, thus debottlenecking the pathway. Other more common enzymes such as phenol hydroxylase, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase are also involved since they attack intermediate metabolites of lindane such as catechol and less substituted chlorophenols. Chromatography coupled to mass spectrometric detector, especially GC-MS, is the most used technique for resolving for γ-HCH metabolites, although there is an increased participation of HPLC

  18. 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.

  19. [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

  20. 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.

  1. Enzyme Analysis to Determine Glucose Content

    NASA Astrophysics Data System (ADS)

    Carpenter, Charles; Ward, Robert E.

    Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

  2. Stabilization of enzymes through encapsulation in liposomes.

    PubMed

    Yoshimoto, Makoto

    2011-01-01

    Phospholipid vesicle (liposome) offers an aqueous compartment surrounded by lipid bilayer membranes. Various enzyme molecules were reported to be encapsulated in liposomes. The liposomal enzyme shows peculiar catalytic activity and selectivity to the substrate in the bulk liquid, which are predominantly derived from the substrate permeation resistance through the membrane. We reported that the quaternary structure of bovine liver catalase and alcohol dehydrogenase was stabilized in liposomes through their interaction with lipid membranes. The method and condition for preparing the enzyme-containing liposomes with well-defined size, lipid composition, and enzyme content are of particular importance, because these properties dominate the catalytic performance and stability of the liposomal enzymes. PMID:20865384

  3. [The synthesis of specific enzyme inhibitors].

    PubMed

    Iakovleva, G M

    1987-04-01

    The review deals with directed synthesis of specific enzyme inhibitors. They are classified within the framework of the mechanistic approach, namely, stable analogues of substrates, which form enzyme complexes mimicking the Michaelis complex or those which influence the chemical stages of enzyme catalysis; conformational inhibitors; substrate analogues participating in enzyme reactions and producing modified products; suicide inhibitors; stage inhibitors (inhibitors influencing certain stages of enzyme reaction); transition state analogues; multisubstrate analogues and collected substrates. Types of chemical modification used in synthesis of the specific inhibitors are discussed. Some possibilities of the quantity structure-activity relationship methods, computer modelling and molecular graphics in designing the optimal structure of inhibitors are mentioned. PMID:3300658

  4. Smart mud and sensitive enzymes

    SciTech Connect

    Knott, D.

    1993-04-19

    Environmental legislation is increasingly preventing use of oil base mud. Most recently, Marathon Oil U.K. Ltd. won a U.K. production license that specified oil base mud cannot be used on the license blocks. The goal is to protect sea-birds. Unfortunately, water base mud, the green' alternative, does not have a performance to match oil base mud. But an Aberdeen chemist thinks he has found the answer with Smart Mud, an emulsion drilling mud that becomes water soluble as soon as it hits the sea. Smart Mud passed the laboratory test stage and is ready for field trials this year. Another researcher is using enzymes and organisms to detect gases that are hard to monitor and cause problems for the oil and gas industry: phenol vapors, methane, and sulfur and nitrous oxides. The methane sensor, for example, uses methanotrophic organisms. They metabolize methane, producing chemicals that can be detected by electrochemical sensors, which relay signals to instruments. Enzymes perform a similar task for phenol and oxide detection. The main problem is to keep the biosensors alive and detect their by-products, while maintaining contact with the toxic gases. To do this, the team invented a polymer matrix in which the biosensors can live.

  5. Enzyme extraction by ultrasound from sludge flocs.

    PubMed

    Yu, Guanghui; He, Pinjing; Shao, Liming; Zhu, Yishu

    2009-01-01

    Enzymes play essential roles in the biological processes of sludge treatment. In this article, the ultrasound method to extract enzymes from sludge flocs was presented. Results showed that using ultrasound method at 20 kHz could extract more types of enzymes than that at 40 kHz and ethylenediamine tetraacetic acid (EDTA) methods. The optimum parameters of ultrasound extraction at 20 kHz were duration of 10 min and intensity of 552 W/g TSS. Under the optimum condition, ultrasound could break the cells and extract both the extracellular and a small part of intercellular enzymes. Ultrasound intensity was apparently more susceptive to enzyme extraction than duration, suggesting that the control of intensity during ultrasound extraction was more important than that of duration. The Pearson correlation analysis between enzyme activities and cation contents revealed that the different types of enzymes had distinct cation binding characteristics. PMID:19402423

  6. 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

  7. Fast-Acting Valve

    NASA Technical Reports Server (NTRS)

    Wojciechowski, Bogdan V. (Inventor); Pegg, Robert J. (Inventor)

    2003-01-01

    A fast-acting valve includes an annular valve seat that defines an annular valve orifice between the edges of the annular valve seat, an annular valve plug sized to cover the valve orifice when the valve is closed, and a valve-plug holder for moving the annular valve plug on and off the annular valve seat. The use of an annular orifice reduces the characteristic distance between the edges of the valve seat. Rather than this distance being equal to the diameter of the orifice, as it is for a conventional circular orifice, the characteristic distance equals the distance between the inner and outer radii (for a circular annulus). The reduced characteristic distance greatly reduces the gap required between the annular valve plug and the annular valve seat for the valve to be fully open, thereby greatly reducing the required stroke and corresponding speed and acceleration of the annular valve plug. The use of a valve-plug holder that is under independent control to move the annular valve plug between its open and closed positions is important for achieving controllable fast operation of the valve.

  8. Double acting bit holder

    DOEpatents

    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.

  9. 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.

  10. 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

  11. Softwood hemicellulose-degrading enzymes from Aspergillus niger: purification and properties of a beta-mannanase.

    PubMed

    Ademark, P; Varga, A; Medve, J; Harjunpää, V; Drakenberg, T; Tjerneld, F; Stålbrand, H

    1998-08-27

    The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose. PMID:9803534

  12. Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

    PubMed

    Gómez-Arreaza, Amaranta; Acosta, Hector; Quiñones, Wilfredo; Concepción, Juan Luis; Michels, Paul A M; Avilán, Luisana

    2014-02-01

    In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. PMID:24602601

  13. Pullulan degrading enzymes of bacterial origin.

    PubMed

    Domań-Pytka, Monika; Bardowski, Jacek

    2004-01-01

    Pullulan degrading enzymes belong to a group of glycosylhydrolases that are widely distributed in nature and are produced by an extremely wide variety of species. Among them the thermophilic and mesophilic bacteria are a rich source of these enzymes. There are many biotechnological applications for these enzymes and a rapidly growing amount of information about their diversity, genetic as well as biochemical and biophysical characteristics. The properties of these enzymes vary and are somewhat linked to the natural environment inhabited by the producing organisms. Genes for these enzymes have been cloned from several strains and their amino acid sequences show highly conserved regions common to the enzymes of the amylase family. Molecular studies have greatly extended our knowledge on pullulan degrading enzymes and their biosynthesis. However, enzyme production levels have usually not been as high as had been assumed possible, and the properties of some enzymes are less than optimal for their industrial applications. Some of these problems can be overcome with the use of good producer organisms, optimized expression/secretion vectors, and site-directed mutagenesis. The molecular biology of pullulan degrading enzymes has been and continues to be a valuable system for studying basic questions of cell biology, such as mechanisms of gene regulation and secretion, and the structure-function relationships of proteins. PMID:15239382

  14. Immobilization of enzymes: a literature survey.

    PubMed

    Brena, Beatriz; González-Pombo, Paula; Batista-Viera, Francisco

    2013-01-01

    The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding. PMID:23934795

  15. Evolving hardware as model of enzyme evolution.

    PubMed

    Lahoz-Beltra, R

    2001-06-01

    Organism growth and survival is based on thousands of enzymes organized in networks. The motivation to understand how a large number of enzymes evolved so fast inside cells may be relevant to explaining the origin and maintenance of life on Earth. This paper presents electronic circuits called 'electronic enzymes' that model the catalytic function performed by biological enzymes. Electronic enzymes are the hardware realization of enzymes defined as molecular automata with a finite number of internal conformational states and a set of Boolean operators modelling the active groups of the active site. One of the main features of electronic enzymes is the possibility of evolution finding the proper active site by means of a genetic algorithm yielding a metabolic ring or k-cycle that bears a resemblance to Krebs (k=7) or Calvin (k=4) cycles present in organisms. The simulations are consistent with those results obtained in vitro evolving enzymes based on polymerase chain reaction (PCR) as well as with the general view that suggests the main role of recombination during enzyme evolution. The proposed methodology shows how molecular automata with evolvable features that model enzymes or other processing molecules provide an experimental framework for simulation of the principles governing metabolic pathways evolution and self-organization. PMID:11448522

  16. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

    We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)- coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K{sub m} obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner.

  17. Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies.

    PubMed

    Kitatani, Kazuyuki; Taniguchi, Makoto; Okazaki, Toshiro

    2015-06-01

    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

  18. 3D imaging of enzymes working in situ.

    PubMed

    Jamme, F; Bourquin, D; Tawil, G; Viksø-Nielsen, A; Buléon, A; Réfrégiers, M

    2014-06-01

    Today, development of slowly digestible food with positive health impact and production of biofuels is a matter of intense research. The latter is achieved via enzymatic hydrolysis of starch or biomass such as lignocellulose. Free label imaging, using UV autofluorescence, provides a great tool to follow one single enzyme when acting on a non-UV-fluorescent substrate. In this article, we report synchrotron DUV fluorescence in 3-dimensional imaging to visualize in situ the diffusion of enzymes on solid substrate. The degradation pathway of single starch granules by two amylases optimized for biofuel production and industrial starch hydrolysis was followed by tryptophan autofluorescence (excitation at 280 nm, emission filter at 350 nm). The new setup has been specially designed and developed for a 3D representation of the enzyme-substrate interaction during hydrolysis. Thus, this tool is particularly effective for improving knowledge and understanding of enzymatic hydrolysis of solid substrates such as starch and lignocellulosic biomass. It could open up the way to new routes in the field of green chemistry and sustainable development, that is, in biotechnology, biorefining, or biofuels. PMID:24796213

  19. 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

  20. 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

  1. DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

    PubMed Central

    Riazuddin, S; Athar, A; Ahmed, Z; Lali, S M; Sohail, A

    1987-01-01

    Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively. PMID:3628000

  2. 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.

  3. [Euthanasia and medical act].

    PubMed

    2011-05-01

    Right to life -as the prohibition of intentionally and arbitrarily taking life, even with authorization of the concerned one- is an internationally recognized right. In many countries, debate regarding euthanasia is more centered in its convenience, social acceptability and how it is regulated, than in its substantial legitimacy. Some argue that euthanasia should be included as part of clinical practice of health professionals, grounded on individual's autonomy claims-everyone having the liberty to choose how to live and how to die. Against this, others sustain that life has a higher value than autonomy, exercising autonomy without respecting the right to life would become a serious moral and social problem. Likewise, euthanasia supporters some-times claim a 'right to live with dignity', which must be understood as a personal obligation, referred more to the ethical than to the strictly legal sphere. In countries where it is already legalized, euthanasia practice has extended to cases where it is not the patient who requests this but the family or some healthcare professional, or even the legal system-when they think that the patient is living in a condition which is not worthy to live. Generalization of euthanasia possibly will end in affecting those who need more care, such as elder, chronically ill or dying people, damaging severely personal basic rights. Nature, purpose and tradition of medicine rule out the practice of euthanasia, which ought not be considered a medical act or legitimately compulsory for physicians. Today's medicine counts with effective treatments for pain and suffering, such as palliative care, including sedative therapy, which best preserves persons dignity and keeps safe the ethos of the medical profession. PMID:22051717

  4. Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme.

    PubMed

    Wan, Hu; Kang, Tinghao; Zhan, Sha; You, Hong; Zhu, Fuxing; Lee, Kwang Sik; Zhao, Haigang; Jin, Byung Rae; Li, Jianhong

    2014-09-01

    In this study, we describe the cloning and characterization of a Prx from the common cutworm Spodoptera litura (SlPrx5). The SlPrx5 cDNA contains an open reading frame of 477 bp encoding a predicted protein of 159 amino acid residues, 16.902 kDa, and an isoelectric point of 7.68. Furthermore, the deduced amino acid sequence of the SlPrx5 cDNA showed 86% identity to Papilio xuthus Prx5, 72% to Aedes aegypti Prx5, and 64-67% to other insect Prxs. A phylogenetic analysis further revealed that the deduced amino acid sequence of SlPrx5 groups within the atypical 2-Cys Prx cluster. Recombinant SlPrx5 (20 kDa) purified from baculovirus-infected insect cells was found to reduce H2O2 in the presence of electrons donated by dithiothreitol and protect super-coiled DNA from damage by metal-catalyzed oxidation in vitro. During S. litura development, SlPrx5 is constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the fat body and midgut. Additionally, SlPrx5 mRNA expression was up-regulated after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. A disc diffusion assay indicated that recombinant SlPrx5 can play a functional role in protecting cells from oxidative stress in vivo. These results provide insight into the role of SlPrx5 during development and the oxidative stress response of S. litura. PMID:24998343

  5. Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

    PubMed Central

    Fukuda, Wakao; Sari Ismail, Yulia; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2005-01-01

    Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+ rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence. PMID:15876562

  6. Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

    PubMed Central

    van Hijum, Sacha A. F. T.; Kralj, Slavko; Ozimek, Lukasz K.; Dijkhuizen, Lubbert; van Geel-Schutten, Ineke G. H.

    2006-01-01

    Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified. PMID:16524921

  7. Cysteine cathepsins as digestive enzymes in the spider Nephilengys cruentata.

    PubMed

    Fuzita, Felipe J; Pinkse, Martijn W H; Verhaert, Peter D E M; Lopes, Adriana R

    2015-05-01

    Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles. PMID:25818482

  8. Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

    PubMed

    Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

    2015-04-01

    Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. PMID:25641162

  9. Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

    PubMed

    Ge, F; Tenent, R C; Wipf, D O

    2001-01-01

    The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process. PMID:11993673

  10. Enzyme/indicator optrodes for detection of heavy metal ions and pesticides

    NASA Astrophysics Data System (ADS)

    Nabok, Alexei V.; Ray, Asim K.; Starodub, Nickolaj F.; Dowker, Kenneth P.

    2000-12-01

    Composite films containing enzyme and indicator molecules were produced by means of polyelectrolyte self-assembly. These membranes provide two functions: (i) molecular recognition of the substratum by respective enzyme, and (ii) optrode transducing, when the products o the substratum decomposition affect optical spectra of indicator molecules. Apart from direct registration of enzyme reactions, inhibition reactions can also be monitored with this method. Particularly, heavy metal salts and phosphor organic pesticides acting as inhibitors for Urease and Cholinesterase, respectively, were registered. Composite PESA films were deposited onto glass slides and consisted of several layers of poly(alylamine) hydrochloride (PAA) alternated with indicator molecules, either Cyclo-tetra- chromotropylene or Thymol Blue, both containing SO3- Na+ groups. Then a few layers of PAA/enzyme were deposited on top. A typical structure of the samples was (PAA/Indicator)n/(PAA/Enzyme)m/PAA with n equals 1-5. The obtained films were characterized with UV-visible absorption spectroscopy. The effect of the substrate decomposition on the UV-vis spectra of respective indicator molecules was studied. The inhibition of enzymes Urease and Cholinesterase by heavy metal ions and phosphor organic pesticide, respectively was found. The results obtained show the prospects towards development of optical enzyme sensor arrays.

  11. Quantum dot based enzyme activity sensors present deviations from Michaelis-Menten kinetic model

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Brown, Carl W.; Malanoski, Anthony P.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2016-03-01

    Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.

  12. On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme

    PubMed Central

    Kuhn, Claus-D.; Wilusz, Jeremy E.; Zheng, Yuxuan; Beal, Peter A.; Joshua-Tor, Leemor

    2015-01-01

    SUMMARY Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menβ, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates. PMID:25640237

  13. Enzymes in Fish and Seafood Processing.

    PubMed

    Fernandes, Pedro

    2016-01-01

    Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

  14. Enzymes in Fish and Seafood Processing

    PubMed Central

    Fernandes, Pedro

    2016-01-01

    Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

  15. 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

  16. The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

    PubMed Central

    Gangoiti, Joana; Pijning, Tjaard

    2015-01-01

    The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes. PMID:26590275

  17. Extracellular enzymes produced by marine eukaryotes, thraustochytrids.

    PubMed

    Taoka, Yousuke; Nagano, Naoki; Okita, Yuji; Izumida, Hitoshi; Sugimoto, Shinichi; Hayashi, Masahiro

    2009-01-01

    Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes, depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes. PMID:19129663

  18. 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.

  19. 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.

  20. Isolation of Cytoplasmic Enzymes from Pollen 1

    PubMed Central

    Weeden, Norman F.; Gottlieb, Leslie D.

    1980-01-01

    The cytoplasmic isozyme of many cytoplasmic-organelle isozyme pairs, as well as other cytoplasmic enzymes in plants, can be readily obtained from pollen by soaking it in an appropriate buffer for 4 hours. Enzymes localized in subcellular organelles appear not to be released during the soaking period, although they are released if the pollen is crushed. The technique is a useful initial step in studies of subcellular localization of enzymes or for obtaining small quantities of cytoplasmic enzymes free of organellar contaminants. Images PMID:16661444

  1. Finding Sequences for over 270 Orphan Enzymes

    PubMed Central

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

    2014-01-01

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

  2. Liver enzyme alteration: a guide for clinicians

    PubMed Central

    Giannini, Edoardo G.; Testa, Roberto; Savarino, Vincenzo

    2005-01-01

    ISOLATED ALTERATIONS OF BIOCHEMICAL MARKERS OF LIVER DAMAGE in a seemingly healthy patient can present a challenge for the clinician. In this review we provide a guide to interpreting alterations to liver enzyme levels. The functional anatomy of the liver and pathophysiology of liver enzyme alteration are briefly reviewed. Using a schematic approach that classifies enzyme alterations as predominantly hepatocellular or predominantly cholestatic, we review abnormal enzymatic activity within the 2 subgroups, the most common causes of enzyme alteration and suggested initial investigations. PMID:15684121

  3. Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids.

    PubMed

    Oohora, K; Hayashi, T

    2016-01-01

    An important strategy used in engineering of hemoproteins to generate artificial enzymes involves replacement of heme with an artificial cofactor after removal of the native heme cofactor under acidic conditions. Replacement of heme in an enzyme with a nonnatural metalloporphyrinoid can significantly alter the reactivity of the enzyme. This chapter describes the design and synthesis of three types of artificial metalloporphyrinoid cofactors consisting of mono-, di-, and tri-anionic ligands (tetradehydrocorrin, porphycene, and corrole, respectively). In addition, practical procedures for the preparation of apo-hemoproteins, incorporation of artificial cofactors, and characterization techniques are presented. Furthermore, the representative catalytic activities of artificial enzymes generated by reconstitution of hemoproteins are summarized. PMID:27586344

  4. 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.

  5. Designing Artificial Enzymes by Intuition and Computation

    PubMed Central

    Nanda, Vikas; Koder, Ronald L.

    2012-01-01

    The rational design of artificial enzymes either by applying physio-chemical intuition of protein structure and function or with the aid of computation methods is a promising area of research with the potential to tremendously impact medicine, industrial chemistry and energy production. Designed proteins also provide a powerful platform for dissecting enzyme mechanisms of natural systems. Artificial enzymes have come a long way, from simple α-helical peptide catalysts to proteins that facilitate multi-step chemical reactions designed by state-of-the-art computational methods. Looking forward, we examine strategies employed by natural enzymes which could be used to improve the speed and selectivity of artificial catalysts. PMID:21124375

  6. Androgen-metabolizing enzymes: A structural perspective.

    PubMed

    Manenda, Mahder Seifu; Hamel, Charles Jérémie; Masselot-Joubert, Loreleï; Picard, Marie-Ève; Shi, Rong

    2016-07-01

    Androgen-metabolizing enzymes convert cholesterol, a relatively inert molecule, into some of the most potent chemical messengers in vertebrates. This conversion involves thermodynamically challenging reactions catalyzed by P450 enzymes and redox reactions catalyzed by Aldo-Keto Reductases (AKRs). This review covers the structures of these enzymes with a focus on active site interactions and proposed mechanisms. Due to their role in a number of diseases, particularly in cancer, androgen-metabolizing enzymes have been targets of drug design. Hence we will also highlight how existing knowledge of structure is being used to this end. PMID:26924584

  7. Bicontinuous Nanoporous Frameworks: Caged Longevity for Enzymes.

    PubMed

    Bae, Jae-Sung; Jeon, Eunkyung; Moon, Su-Young; Oh, Wangsuk; Han, Sun-Young; Lee, Jeong Hun; Yang, Sung Yun; Kim, Dong-Myung; Park, Ji-Woong

    2016-09-12

    The preparation of bicontinuous nanoporous covalent frameworks, which are promising for caging active enzymes, is demonstrated. The frameworks have three- dimensionally continuous, hydrophilic pores with widths varying between 5 and 30 nm. Enzymes were infiltrated into the bicontinuous pore by applying a pressured enzyme solution. The new materials and methods allowed the amount of caged proteins to be controlled precisely. The resulting enzyme-loaded framework films could be recycled many times with nearly no loss of catalytic activity. Entropic trapping of proteins by a bicontinuous pore with the right size distribution is an unprecedented strategy toward facile in vitro utilization of biocatalysts. PMID:27513827

  8. Relation between Enzymic Catalysis and Energy Coupling

    NASA Astrophysics Data System (ADS)

    Fry, Mitchell; Blondin, George A.; Green, David E.

    1980-10-01

    The principles that underlie enzyme catalysis also apply to energy coupling processes. A comparison is made between a kinase system that mediates the phosphorylation of glucose by ATP (hexokinase), as the prototype for enzymic catalysis, and the mitochondrial electron-transfer complexes, as the prototypes for energy coupling systems. Induced polarization of chemical bonds and charge separation and elimination are common component events of both enzyme catalysis and energy coupling. Thus, definite limits can be imposed on models of energy coupling; they must comply with the basic principles of enzymic catalysis.

  9. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

    PubMed

    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides. PMID:26791508

  10. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER... Development Act, 7 U.S.C. 2008j, as amended by section 11009 of the Food, Conservation, and Energy Act of...

  11. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER... Development Act, 7 U.S.C. 2008j, as amended by section 11009 of the Food, Conservation, and Energy Act of...

  12. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER... Development Act, 7 U.S.C. 2008j, as amended by section 11009 of the Food, Conservation, and Energy Act of...

  13. 7 CFR 63.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) NATIONAL SHEEP INDUSTRY IMPROVEMENT CENTER... Development Act, 7 U.S.C. 2008j, as amended by section 11009 of the Food, Conservation, and Energy Act of...

  14. The Privacy Act of 1974.

    ERIC Educational Resources Information Center

    O'Reilly, James T.

    This report describes the possible impact of the comprehensive Privacy Act of 1974, which went into effect on 27 September 1975. Specifically, the implications of the act for limitation of disclosure, federal information collection, individual access, private suits; criminal provisions; and exceptions to the provisions of the law are detailed. In…

  15. 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…

  16. 76 FR 59073 - Privacy Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-23

    ... From the Federal Register Online via the Government Publishing 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...

  17. 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.

  18. Education Leaders Applaud ATTAIN Act

    ERIC Educational Resources Information Center

    Curriculum Review, 2007

    2007-01-01

    This article talks about Achievement Through Technology and Innovation (ATTAIN) Act, a bill introduced by Senators Bingaman (D-NM), Burr (R-NC), and Murray (D-WA) and applauded by a coalition of education and industry groups. The proposed ATTAIN Act is similar to its companion in the House (HR 2449), and builds upon the Enhancing Education Through…

  19. Act of 13 March 1989.

    PubMed

    1989-01-01

    This document contains the text of a 1989 Act of Utah prohibiting surrogate parenthood agreements of any type. Surrogate mothers are the legal mothers of their children, and their husbands are the legal fathers. This Act has no impact on adoption laws. PMID:12344464

  20. Implementing the Amended FOI Act.

    ERIC Educational Resources Information Center

    McClain, Wallis

    The Freedom of Information Act amendments, which became effective in February 1975, have so far yielded mixed results. This report provides an account of how different federal agencies are implementing this amended statute. Among the topics discussed are modifications of the original 1966 Freedom of Information Act, which were made in the attempt…

  1. 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

  2. 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

    , 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

  3. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

    PubMed Central

    Henderson, G B; Ulrich, P; Fairlamb, A H; Rosenberg, I; Pereira, M; Sela, M; Cerami, A

    1988-01-01

    The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase. PMID:3135548

  4. 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...

  5. 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.

  6. Spectroscopic studies of copper enzymes

    SciTech Connect

    Dooley, D.M.; Moog, R.; Zumft, W.; Koenig, S.H.; Scott, R.A.; Cote, C.E.; McGuirl, M.

    1986-05-01

    Several spectroscopic methods, including absorption, circular dichroism (CD), magnetic CD (MCD), X-ray absorption, resonance Raman, EPR, NMR, and quasi-elastic light-scattering spectroscopy, have been used to probe the structures of copper-containing amine oxidases, nitrite reductase, and nitrous oxide reductase. The basic goals are to determine the copper site structure, electronic properties, and to generate structure-reactivity correlations. Collectively, the results on the amine oxidases permit a detailed model for the Cu(II) sites in these enzymes to be constructed that, in turn, rationalizes the ligand-binding chemistry. Resonance Raman spectra of the phenylhydrazine and 2,4-dinitrophenyl-hydrazine derivatives of bovine plasma amine oxidase and models for its organic cofactor, e.g. pyridoxal, methoxatin, are most consistent with methoxatin being the intrinsic cofactor. The structure of the Cu(I) forms of the amine oxidases have been investigated by X-ray absorption spectroscopy (XAS); the copper coordination geometry is significantly different in the oxidized and reduced forms. Some anomalous properties of the amine oxidases in solution are explicable in terms of their reversible aggregation, which the authors have characterized via light scattering. Nitrite and nitrous oxide reductases display several novel spectral properties. The data suggest that new types of copper sites are present.

  7. Enzymes of glucose metabolism in Frankia sp.

    PubMed

    Lopez, M F; Torrey, J G

    1985-04-01

    Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures. PMID:3980434

  8. Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

    PubMed Central

    Xavier, Karina B.; Peist, Ralf; Kossmann, Marina; Boos, Winfried; Santos, Helena

    1999-01-01

    Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis

  9. Characterization of Soil Samples of Enzyme Activity

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1977-01-01

    Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

  10. Cobalamin- and Corrinoid-Dependent Enzymes

    PubMed Central

    Matthews, Rowena G.

    2011-01-01

    This chapter will review the literature on cobalamin- and corrinoid-containing enzymes. These enzymes fall into two broad classes, those using methylcobalamin or related methylcorrinoids as prosthetic groups and catalyzing methyltransfer reactions, and those using adenosylcobalamin as the prosthetic group and catalyzing the generation of substrate radicals that in turn undergo rearrangements and/or eliminations. PMID:20877792

  11. 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)

  12. 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.)

  13. 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.

  14. 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…

  15. KINEXP: Computer Simulation in Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Gelpi, Josep Lluis; Domenech, Carlos

    1988-01-01

    Describes a program which allows students to identify and characterize several kinetic inhibitory mechanisms. Uses the generic model of reversible inhibition of a monosubstrate enzyme but can be easily modified to run other models such as bisubstrate enzymes. Uses MS-DOS BASIC. (MVL)

  16. Screening assays for biomass-degrading enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymes that break down the components of lignocellulosic biomass play an important role in the production of value-added chemical feedstocks and biofuels. In order to accommodate the variety of substrates and industrial process conditions, enzymes with diverse activity profiles are required. Th...

  17. 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)

  18. The evolution of enzyme kinetic power.

    PubMed Central

    Keleti, T; Welch, G R

    1984-01-01

    Evolution of the kinetic potential of enzyme reactions is discussed. Quantitative assessment of the evolution of enzyme action has usually focused on optimization of the parametric ratio kcat./Km, which is the apparent second-order rate constant for the reaction of free substrate with free enzyme to give product. We propose that the general form kcat.[E]T/Km (where [E]T is total enzyme concentration), which is designated the 'kinetic power', is the real measure of kinetic/catalytic potential in situ. The standard paradigm of 'perfection' dictates the evolutionary maximum of 'kinetic power' to be k+s[E]T/2, where k+s is the diffusion-controlled rate constant for formation of the ES complex (and, hence, for the overall enzyme reaction). We discuss the role of protein conformational mobility in determining this state of 'perfection', via gating of substrate binding and determination of the catalytic configuration. Going beyond the level of the individual enzyme, we indicate the manner by which the organizational features of enzyme action in vivo may enhance the 'kinetic power'. Through evolutionary 'perfection' of the microenvironment, one finds that the 'kinetic power' of enzymes can be affected by alteration of [E]T as well as the unitary rate constants. At this level of complexity, we begin to realize that the 'kinetic' description of cell metabolism must be supplemented with thermodynamic concepts. PMID:6497848

  19. 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)

  20. 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…

  1. 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...

  2. 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.

  3. Imaging of enzyme replacement therapy using PET

    PubMed Central

    Phenix, Christopher P.; Rempel, Brian P.; Colobong, Karen; Doudet, Doris J.; Adam, Michael J.; Clarke, Lorne A.; Withers, Stephen G.

    2010-01-01

    Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid β-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme® used to treat Gaucher disease, using an 18F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue 18F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to 18F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs. PMID:20534487

  4. 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...

  5. 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

  6. Assessment & Commitment Tracking System (ACTS)

    SciTech Connect

    Bryant, Robert A.; Childs, Teresa A.; Miller, Michael A.; Sellars, Kevin J.

    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

  7. ACTS and OLYMPUS propagation experiments

    NASA Technical Reports Server (NTRS)

    Bostian, Charles W.; Baker, Kenneth R.

    1988-01-01

    The OLYMPUS and ACTS satellites both provide opportunities for 10 to 30 GHz propagation measurements. The spacecraft are sufficiently alike that OLYMPUS can be used to test some prototype ACTS equipment and experiments. Data are particularly needed on short term signal behavior and in support of uplink power control and adaptive forward error correction (FEC) techniques. The Virginia Tech Satellite Communications Group has proposed a set of OLYMPUS experiments including attenuation and fade rate measurements, data communications, uplink power control, rain scatter interference, and small-scale site diversity operation. A digital signal processing receiver for the OLYMPUS and ACTS beacon signals is being developed.

  8. Directed Evolution of Enzymes for Industrial Biocatalysis.

    PubMed

    Porter, Joanne L; Rusli, Rukhairul A; Ollis, David L

    2016-02-01

    Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment. PMID:26661585

  9. Evaluation of selected enzymes for thiamine determination.

    PubMed

    Defibaugh, P W

    1987-01-01

    Seven commercially available enzymes were studied for suitability as substitutes in the AOAC thiamine determination, because the enzyme Takadiastase used in the method has not been available since 1976, and alternative enzymes were likewise unavailable or unsuitable for releasing thiamine from its phosphate esters. Four factors (substrate ester, enzyme level, time/temperature, and pH) at 2 levels were studied in a 2(4) factorial arrangement of treatments. Data were expressed in terms of mean percentage conversion (MPC) and were statistically evaluated by analysis of variance. Significant main effects and any interactions among treatments were calculated. Takadiastase and alpha-amylase (Miles) with MPCs of 101 and 102, respectively, appeared effective in dephosphorylation within method parameters. Potato phosphatase appeared marginally suitable. Wheat germ phosphatase, alpha-amylase (Sigma), Mylase 100, and Clarase 40,000 were judged unacceptable as enzyme substitutes. PMID:3610965

  10. 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

  11. Tyrosinase immobilized enzyme reactor: development and evaluation.

    PubMed

    de Oliveira, Karina Bora; Mischiatti, Keylla Lençone; Fontana, José Domingos; de Oliveira, Brás Heleno

    2014-01-15

    Immobilized enzyme reactors of tyrosinase (tyr-IMERs) for use on-line in HPLC system were prepared by different procedures and then compared. The enzyme, obtained from Agaricus bisporus, was immobilized on epoxy-silica which was prepared using different conditions. Enzyme immobilization was conducted by both in situ and in batch techniques. The different procedures were compared in terms of protein and activity retention, IMERs activity, kinetics and stability. The influence of immobilization procedure on enzyme activity and the behavior of the IMERs against a standard inhibitor were also investigated. In situ immobilization on epoxy-silica, synthesized using microwave assistance, provided the best conditions to prepare tyrosinase IMERs. The tyr-IMERs were successfully tested with known and potential inhibitors of tyrosinase, and the results showed that they can be used for the screening of inhibitors of that enzyme. PMID:24317418

  12. Enzymatic bioremediation: from enzyme discovery to applications.

    PubMed

    Sutherland, T D; Horne, I; Weir, K M; Coppin, C W; Williams, M R; Selleck, M; Russell, R J; Oakeshott, J G

    2004-11-01

    1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors. 2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range. 3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications. 4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses. PMID:15566400

  13. Highly efficient self-replicating RNA enzymes.

    PubMed

    Robertson, Michael P; Joyce, Gerald F

    2014-02-20

    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

  14. Enzyme stabilization: state of the art.

    PubMed

    Gianfreda, L; Scarfi, M R

    1991-02-01

    'Enzyme stabilization' is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies. PMID:2008180

  15. Model Occupational Therapy Practice Act

    ERIC Educational Resources Information Center

    American Journal of Occupational Therapy, 1975

    1975-01-01

    The Model Occupational Therapy Practice Act has been assembled by the Government Affairs Department, American Occupational Therapy Association, for use as a guide for affiliate organizations concerned with developing legislation to regulate the practice of occupational therapy. (Author/JA)

  16. 78 FR 73466 - Privacy Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-06

    ... or qualifications for Federal civilian employment, military service, Federal contracts or access to... From the Federal Register Online via the Government Publishing Office OVERSEAS PRIVATE INVESTMENT CORPORATION 22 CFR Part 707 Privacy Act AGENCY: Overseas Private Investment Corporation. ACTION: Notice...

  17. Prelinguistic Vocalizations Distinguish Pointing Acts

    ERIC Educational Resources Information Center

    Grunloh, Thomas; Liszkowski, Ulf

    2015-01-01

    The current study investigated whether point-accompanying characteristics, like vocalizations and hand shape, differentiate infants' underlying motives of prelinguistic pointing. We elicited imperative (requestive) and declarative (expressive and informative) pointing acts in experimentally controlled situations, and analyzed accompanying…

  18. 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.

  19. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic.

    PubMed

    Schoonen, Lise; Nolte, Roeland J M; van Hest, Jan C M

    2016-08-14

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. PMID:27407020

  20. Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

    PubMed

    Correro, M Rita; Moridi, Negar; Schützinger, Hansjörg; Sykora, Sabine; Ammann, Erik M; Peters, E Henrik; Dudal, Yves; Corvini, Philippe F-X; Shahgaldian, Patrick

    2016-05-17

    The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses. PMID:27062137

  1. Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments.

    PubMed

    Zanaroli, Giulio; Negroni, Andrea; Calisti, Cecilia; Ruzzi, Maurizio; Fava, Fabio

    2011-12-10

    In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings. PMID:22142734

  2. 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

    ... the Sunshine Act, Freedom of Information Act (``FOIA''), and Federal Election Campaign Act (``FECA... of Information Act of 1966, as amended, 5 U.S.C. 552, and the Federal Election Campaign Act (``FECA... requirements. 11 CFR Part 104 Campaign funds, Political committees and parties, Reporting and...

  3. 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

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

    PubMed Central

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

    2014-01-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. PMID:25239895

  5. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    PubMed

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase. PMID:26621459

  6. Main factors providing specificity of repair enzymes.

    PubMed

    Nevinsky, G A

    2011-01-01

    Specific and nonspecific DNA complex formation with human uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and apurine/apyrimidine endonuclease, as well as with E. coli 8-oxoguanine-DNA glycosylase and RecA protein was analyzed using the method of stepwise increase in DNA-ligand complexity. It is shown that high affinity of these enzymes to any DNA (10(-4)-10(-8) M) is provided by a large number of weak additive contacts mainly with DNA internucleoside phosphate groups and in a less degree with bases of nucleotide links "covered" by protein globules. Enzyme interactions with specific DNA links are comparable in efficiency with weak unspecific contacts and provide only for one-two orders of affinity (10(-1)-10(-2) M), but these contacts are extremely important at stages of DNA and enzyme structural adaptation and catalysis proper. Only in the case of specific DNA individual for each enzyme alterations in DNA structure provide for efficient adjustment of reacting enzyme atoms and DNA orbitals with accuracy up to 10-15° and, as a result, for high reaction rate. Upon transition from nonspecific to specific DNA, reaction rate (k(cat)) increases by 4-8 orders of magnitude. Thus, stages of DNA and enzyme structural adaptation as well as catalysis proper are the basis of specificity of repair enzymes. PMID:21568843

  7. Enzyme immobilization by means of ultrafiltration techniques.

    PubMed

    Scardi, V; Cantarella, M; Gianfreda, L; Palescandolo, R; Alfani, F; Greco, G

    1980-01-01

    Unstirred, plane membrane, ultrafiltration cells have been used as enzymatic reactor units. Because of the concentration polarization phenomena which take place in the system, at steady-state the enzyme is confined (dynamically immobilized) within an extremely narrow region upstream the ultrafiltration membrane. Correspondingly its concentration attains fairly high values. Kinetic studies have been therefore performed under quite unusual experimental conditions in order to better approximate local enzyme concentration levels in immobilized enzyme systems. Studies have been also carried out on the kinetics of enzyme deactivation in the continuous presence of substrate and reaction products. Once the enzyme concentration profile is completely developed, further injection into the system of suitable amounts of an inert proteic macromolecule (albumin polymers) gives rise to the formation of a gel layer onto the ultrafiltration membrane within which the enzyme is entrapped (statically immobilized). The effect of this immobilization technique has been studied as far as the kinetics of the main reaction, the substrate mass transfer resistances and the enzyme stability are concerned. The rejective properties of such gel layers towards enzymatic molecules have been exploited in producing multilayer, multi-enzymatic reactors. PMID:7417597

  8. Extracorporeal delivery of a therapeutic enzyme

    PubMed Central

    Zhang, Chun; Pu, Jun; Yang, Xiaolan; Feng, Tao; Liu, Fang; Wang, Deqiang; Hu, Xiaolei; Gao, Ang; Liu, Hongbo; Zhan, Chang-Guo; Liao, Fei

    2016-01-01

    To remove circulating harmful small biochemical(s)/substrates causing/deteriorating certain chronic disease, therapeutic enzyme(s) delivered via vein injection/infusion suffer(s) from immunoresponse after repeated administration at proper intervals for a long time and short half-lives since delivery. Accordingly, a novel, generally-applicable extracorporeal delivery of a therapeutic enzyme is proposed, by refitting a conventional hemodialysis device bearing a dialyzer, two pumps and connecting tubes, to build a routine extracorporeal blood circuit but a minimal dialysate circuit closed to circulate the therapeutic enzyme in dialysate. A special quantitative index was derived to reflect pharmacological action and thus pharmacodynamics of the delivered enzyme. With hyperuricemic blood in vitro and hyperuricemic geese, a native uricase via extracorporeal delivery was active in the dialysate for periods much longer than that in vivo through vein injection, and exhibited the expected pharmacodynamics to remove uric acid in hyperuricemic blood in vitro and multiple forms of uric acid in hyperuricemic geese. Therefore, the extracorporeal delivery approach of therapeutic enzymes was effective to remove unwanted circulating small biochemical(s)/substrates, and was expected to avoid immunogenicity problems of therapeutic enzymes after repeated administration at proper intervals for a long time due to no contacts with macromolecules and cells in the body. PMID:27477538

  9. Extracorporeal delivery of a therapeutic enzyme.

    PubMed

    Zhang, Chun; Pu, Jun; Yang, Xiaolan; Feng, Tao; Liu, Fang; Wang, Deqiang; Hu, Xiaolei; Gao, Ang; Liu, Hongbo; Zhan, Chang-Guo; Liao, Fei

    2016-01-01

    To remove circulating harmful small biochemical(s)/substrates causing/deteriorating certain chronic disease, therapeutic enzyme(s) delivered via vein injection/infusion suffer(s) from immunoresponse after repeated administration at proper intervals for a long time and short half-lives since delivery. Accordingly, a novel, generally-applicable extracorporeal delivery of a therapeutic enzyme is proposed, by refitting a conventional hemodialysis device bearing a dialyzer, two pumps and connecting tubes, to build a routine extracorporeal blood circuit but a minimal dialysate circuit closed to circulate the therapeutic enzyme in dialysate. A special quantitative index was derived to reflect pharmacological action and thus pharmacodynamics of the delivered enzyme. With hyperuricemic blood in vitro and hyperuricemic geese, a native uricase via extracorporeal delivery was active in the dialysate for periods much longer than that in vivo through vein injection, and exhibited the expected pharmacodynamics to remove uric acid in hyperuricemic blood in vitro and multiple forms of uric acid in hyperuricemic geese. Therefore, the extracorporeal delivery approach of therapeutic enzymes was effective to remove unwanted circulating small biochemical(s)/substrates, and was expected to avoid immunogenicity problems of therapeutic enzymes after repeated administration at proper intervals for a long time due to no contacts with macromolecules and cells in the body. PMID:27477538

  10. Irc3 is a mitochondrial DNA branch migration enzyme

    PubMed Central

    Gaidutšik, Ilja; Sedman, Tiina; Sillamaa, Sirelin; Sedman, Juhan

    2016-01-01

    Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules. PMID:27194389

  11. Irc3 is a mitochondrial DNA branch migration enzyme.

    PubMed

    Gaidutšik, Ilja; Sedman, Tiina; Sillamaa, Sirelin; Sedman, Juhan

    2016-01-01

    Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules. PMID:27194389

  12. Nutritional significance of lectins and enzyme inhibitors from legumes.

    PubMed

    Lajolo, Franco M; Genovese, Maria Inés

    2002-10-23

    Legumes have natural components, such as lectins, amylase, and trypsin inhibitors, that may adversely affect their nutritional properties. Much information has already been obtained on their antinutritional significance and how to inactivate them by proper processing. Chronic ingestion of residual levels is unlikely to pose risks to human health. On the other hand, the ability of these molecules to inhibit some enzymes such as trypsin, chymotrypsin, disaccharidases, and alpha-amylases, to selectively bind to glycoconjugates, and to enter the circulatory system may be a useful tool in nutrition and pharmacology. Trypsin inhibitors have also been studied as cancer risk reducing factors. These components seem to act as plant defense substances. However, increased contents may represent an impairment of the nutritional quality of legumes because these glycoproteins and the sulfur-rich protease inhibitors have been shown to be poorly digested and to participate in chemical reactions during processing reducing protein digestibility, a still unsolved question. PMID:12381157

  13. Immobilized Enzymes and Cells as Practical Catalysts

    NASA Astrophysics Data System (ADS)

    Klibanov, Alexander M.

    1983-02-01

    Performance of enzymes and whole cells in commercial applications can often be dramatically improved by immobilization of the biocatalysts, for instance, by their covalent attachment to or adsorption on solid supports, entrapment in polymeric gels, encapsulation, and cross-linking. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. Applications of immobilized enzymes and cells in the chemical, pharmaceutical, and food industries, in clinical and chemical analyses, and in medicine, as well as probable future trends in enzyme technology are discussed.

  14. Immobilized enzymes and cells as practical catalysts

    SciTech Connect

    Klibanov, A.M.

    1983-02-11

    Performance of enzymes and whole cells in commercial applications can often be dramatically improved by immobilization of the biocatalysts, for instance, by their covalent attachment to or adsorption on solid supports, entrapment in polymeric gels, encapsulation, and cross-linking. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. Applications of immobilized enzymes and cells in the chemical, pharmaceutical, and food industries, in clinical and chemical analyses, and in medicine, as well as probable future trends in enzyme technology are discussed.

  15. Steroid promiscuity: Diversity of enzyme action. Preface.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri; Mason, J Ian

    2015-07-01

    This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity, dwells on the growing realization that the 'one ligand, one binding site' and 'one enzyme, one reaction' concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled 'Steroid/Sterol Signaling' . PMID:25596328

  16. Enzyme Activities in Polarized Cell Membranes

    PubMed Central

    Bass, L.; McIlroy, D. K.

    1968-01-01

    The theoretical pH dependence of enzyme activities in membranes of low dielectric constant is estimated. It is shown that in biological membranes some types of enzymes may attain a limiting pH sensitivity such that an increment of only 0.2 pH unit (sufficient to induce action potentials in squid axons) causes a relative activity change of over 25%. The transients of enzyme activity generated by membrane depolarization and by pH increments in the bathing solution are discussed in relation to the transients of nervous excitation. PMID:5641405

  17. Process for preparing multilayer enzyme coating on a fiber

    DOEpatents

    Kim, Jungbae; Kwak, Ja Hun; Grate, Jay W.

    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.

  18. Beta-lactamase-free penicillin amidase from Alcaligenes sp.: isolation strategy, strain characteristics, and enzyme immobilization.

    PubMed

    Pal, A; Samanta, T B

    1999-11-01

    Isolation and characterization of a beta-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1. 11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme. PMID:10489431

  19. 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... 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...

  20. 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... 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...

  1. 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... 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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... 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...

  7. 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...

  8. 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)

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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.

  15. 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.

  16. Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

    PubMed Central

    Hartley, Meredith D.; Schneggenburger, Philipp E.; Imperiali, Barbara

    2013-01-01

    Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting protein–protein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly. PMID:24302767

  17. Dietary modulation of thymic enzymes.

    PubMed

    Susana, Feliu María; Paula, Perris; Slobodianik, Nora

    2014-01-01

    Malnutrition is a complex syndrome caused by an inadequate intake of energy, protein, minerals and vitamins which affects the immune system. Nutritional imbalances, present in children with energy-protein malnutrition and infections, make defining the specific effects of each of them on the thymus difficult. For this reason, it is necessary to design an experimental model in animals that could define a single variable. As the thymus atrophy described in humans is similar to that observed in murines, a rat experimental model makes the extrapolation to man possible. Some authors suggest that the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP)--involved in purine metabolism--have an influence on T lymphocyte development and the immune system, due to intracellular accumulation of toxic levels of deoxynucleotides. Studies in our group, performed in an experimental model on Wistar growing rats, have demonstrated that protein deficiency or imbalance in the profile of essential amino acids in the diet, produce loss of thymus weight, reduction in the number of thymocytes, a diminished proportion of T cells presenting the W3/13 antigenic determinant and DNA content with concomitant increase in cell size, and the proportion of immature T cells and activity of ADA and PNP, without modifying the activity of 5´Nucleotidase in the thymus. It is important to point out that there were neither differences in energy intake between experimental groups and their controls, nor clinical symptoms of deficiency of other nutrients. The increase in these thymic enzyme activities was an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. On the other hand, the administration of a recovery diet, with a high amount of high quality protein, was able to reverse the mentioned effects. The quick reply of Adenosine Deaminase to nutritional disorders and the following nutritional recovery, points

  18. Differential effects of progestins on breast tissue enzymes.

    PubMed

    Pasqualini, J R

    2003-12-10

    There is substantial evidence that mammary cancer tissue contains all the enzymes responsible for the local biosynthesis of estradiol (E2) from circulating precursors. Two principal pathways are implicated in the final steps of E2 formation in breast cancer tissue: the 'aromatase pathway' that transforms androgens into estrogens and the 'sulfatase pathway' that converts estrone sulfate (E1S) into estrone (E1) via estrone sulfatase. The final step is the conversion of weak E1 to potent biologically active E2 via reductive 17beta-hydroxysteroid dehydrogenase type 1 activity. It is also well established that steroid sulfotransferases, which convert estrogens into their sulfates, are present in breast cancer tissues. One of the possible means of blocking E2 effects in breast cancer is to use anti-estrogens, which act by binding to the estrogen receptor (ER). Another option is to block E2 using anti-enzymes (anti-sulfatase, anti-aromatase, or anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD). Various progestins (e.g. promegestone, nomegestrol acetate, medrogestone, 17-deacetyl norgestimate, dydrogesterone and its 20-dihydro derivative), as well as tibolone and its metabolites, have been shown to inhibit estrone sulfatase and 17beta-hydroxysteroid dehydrogenase. Some progestins and tibolone can also stimulate sulfotransferase activity. These various progestins may therefore provide a new option for the treatment of breast cancer. PMID:14670645

  19. Angiotensin I converting enzyme inhibitory peptide extracted from freshwater zooplankton.

    PubMed

    Lee, Jung Kwon; Lee, Min-Su; Park, Heum Gi; Kim, Se-Kwon; Byun, Hee-Guk

    2010-04-01

    In this study, hydrolysates obtained from the freshwater rotifer Brachionus calyciflonus were investigated for angiotensin I converting enzyme (ACE) inhibitory peptides. Freshwater rotifer protein was hydrolyzed using six separate enzymes in a batch reactor. The peptic hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The highest ACE inhibitory peptide was separated using Sephadex G-25 column chromatography and high-performance liquid chromatography on a C18 column. The 50% inhibitory concentration (IC(50)) value of purified ACE inhibitory peptide was 40.01 microg/mL. ACE inhibitory peptide was identified as being seven amino acid residues of Ala-Gln-Gly-Glu-Arg-His-Arg by N-terminal amino acid sequence analysis. The IC(50) value of purified ACE inhibitory peptide was 47.1 microM, and Lineweaver-Burk plots suggested that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. The results of this study suggest that peptides derived from freshwater rotifers may be beneficial as antihypertension compounds in functional foods or as pharmaceuticals. PMID:20170338

  20. 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.

  1. Substrate Tunnels in Enzymes: Structure-Function Relationships and Computational Methodology

    PubMed Central

    Kingsley, Laura J.; Lill, Markus A.

    2015-01-01

    In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein and in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years due to implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with tunnels and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. PMID:25663659

  2. ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor.

    PubMed

    Doria, Margherita; Tomaselli, Sara; Neri, Francesca; Ciafrè, Silvia Anna; Farace, Maria Giulia; Michienzi, Alessandro; Gallo, Angela

    2011-05-01

    The adenosine deaminases acting on RNA (ADAR) enzymes catalyse conversion of adenosine to inosine in dsRNA. A positive effect of ADAR1 on human immunodeficiency virus type 1 (HIV-1) replication has recently been reported. Here, we show that another ADAR enzyme, ADAR2, positively affects the replication process of HIV-1. We found that, analogously to ADAR1, ADAR2 enhances the release of progeny virions by an editing-dependent mechanism. However, differently from the ADAR1 enzyme, ADAR2 does not increase the infectious potential of the virus. Importantly, downregulation of ADAR2 in Jurkat cells significantly impairs viral replication. Therefore, ADAR2 shares some but not all proviral functions of ADAR1. These results suggest a novel role of ADAR2 as a viral regulator. PMID:21289159

  3. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    NASA Astrophysics Data System (ADS)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

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

    PubMed

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

    2013-01-01

    Of the two independent processes by which enzymes lose activity with increasing temperature, irreversible thermal inactivation and rapid reversible equilibration with an inactive form, the latter is only describable by the Equilibrium Model. Any investigation of the effect of temperature upon enzymes, a mandatory step in rational enzyme engineering and study of enzyme temperature adaptation, thus requires determining the enzymes' thermodynamic parameters as defined by the Equilibrium Model. The necessary data for this procedure can be collected by carrying out multiple isothermal enzyme assays at 3-5°C intervals over a suitable temperature range. If the collected data meet requirements for V max determination (i.e., if the enzyme kinetics are "ideal"), then the enzyme's Equilibrium Model parameters (ΔH eq, T eq, ΔG (‡) cat, and ΔG (‡) inact) can be determined using a freely available iterative model-fitting software package designed for this purpose.Although "ideal" enzyme reactions are required for determination of all four Equilibrium Model parameters, ΔH eq, T eq, and ΔG (‡) cat can be determined from initial (zero-time) rates for most nonideal enzyme reactions, with substrate saturation being the only requirement. PMID:23504427

  5. Enzyme mechanisms: Flexibility leads to function

    NASA Astrophysics Data System (ADS)

    Zhao, Jianhua; Rubinstein, John L.

    2014-03-01

    ATP synthase is an important enzyme for the storage and release of energy in cells. Ion-mobility mass spectrometry has now been used to study its structure, revealing important mechanistic details about its operation and regulation.

  6. 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).

  7. Impulsive Enzymes: A New Force in Mechanobiology

    PubMed Central

    Butler, Peter J.; Dey, Krishna K.; Sen, Ayusman

    2015-01-01

    We review studies that quantify newly discovered forces from single enzymatic reactions. These forces arise from the conversion of chemical energy to kinetic energy, which can be harnessed to direct diffusion of the enzyme up a concentration gradient of substrate, a novel phenomenon of molecular chemotaxis. When immobilized, enzymes can move fluid around them and perform directional pumping in microfluidic chambers. Because of the extensive array of enzymes in biological cells, we also develop three new hypotheses: that enzymatic self diffusion can assist in organizing signaling pathways in cells, can assist in pumping of fluid in cells, and can impose biologically significant forces on organelles, which will be manifested as stochastic motion not explained by thermal forces or myosin II. Such mechanochemical phenomena open up new directions in research in mechanobiology in which all enzymes, in addition to their primary function as catalysts for reactions, may have secondary functions as initiators of mechanosensitive transduction pathways. PMID:26019728

  8. Enzyme mimics: Halogen and chalcogen team up

    NASA Astrophysics Data System (ADS)

    Metrangolo, Pierangelo; Resnati, Giuseppe

    2012-06-01

    The behaviour of di-selenol enzyme mimics indicates that a halogen bond between selenium and iodine, and a chalcogen interaction between the two selenium atoms, play an important role in the activation of thyroid hormones.

  9. Practical Enzyme Kinetics: A Biochemical Laboratory Experiment.

    ERIC Educational Resources Information Center

    Rowe, H. Alan; Brown, Morris

    1988-01-01

    Describes an experiment that provides a fundamental understanding of the kinetics of the enzyme papain. Discusses background, materials, procedures and results. Mentions analogous experiments that can be conducted with enzymatic contact-lens cleaning solutions. (CW)

  10. 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.

  11. Enzymes as useful tools for environmental purposes.

    PubMed

    Rao, M A; Scelza, R; Acevedo, F; Diez, M C; Gianfreda, L

    2014-07-01

    In the environment enzymes may play important and different roles at least in three cases: as main agents (as isolated, cell-bound or immobilized enzymes) in charge of either the transformation and/or degradation of compounds polluting the environment and the restoration of the polluted environment; as reliable and sensitive tools to detect and measure the amount and concentration of pollutants before, during and after the restoration process; as reliable, easy and sensitive indicators of quality and health status of the environment subjected to the restoration process. To our knowledge papers or reviews integrating findings on these three functions of enzymes are missing in literature. Therefore the main scope of the present paper is to briefly encompass general and specific concepts about roles of enzymes as decontaminating agents, pollutant assaying agents and indicators of environment safety. Examples chosen among those published very recently, supporting and confirming peculiarities, features, and performance of enzymatic agents will be illustrated. PMID:24411841

  12. 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

  13. 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.

  14. Detoxification of aromatic pollutants by fungal enzymes

    SciTech Connect

    Bollag, J.M.; Dec, J.

    1995-12-31

    Fungal enzymes, such as laccase, peroxidase, and tyrosinase, play a prominent role in catalyzing the transformation of various aromatic compounds in the environment. The enzyme-mediated oxidative coupling reaction results in covalent binding of chlorinated phenols and anilines to soil organic matter or polymerization of the substrates in aquatic systems. Both of these processes are accompanied by a detoxification effect. Therefore, it has been postulated that they be exploited for the treatment of polluted soil and water. The mechanism and efficiency of oxidative coupling in pollutant removal were studied by incubation of chlorinated phenols and anilines with various humic substances or soil and analysis of the reaction products by chromatography and mass and {sup 13}C nuclear magnetic resonance (NMR) spectrometry. The decontamination effect could be enhanced by optimization of the reaction conditions and immobilization of enzymes on solid materials. The results obtained strongly support the concept of using enzymes for control of environmental pollution.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Purification of cytochrome P-450 enzymes.

    PubMed

    Bell-Parikh, L C; Hosea, N A; Martin, M V; Guengerich, F P

    2002-01-01

    Among the liver P-450 xenobiotic-metabolizing enzymes, P450-2E1 is of interest because of its activation of potent carcinogens, and P-450 1A2 is of interest because of its role in oxidation of drugs and carcinogens. This unit describes column chromatography protocols for purification of recombinant forms of these enzymes expressed in a bacterial expression system. PMID:23045082

  20. Semisupervised Gaussian Process for Automated Enzyme Search.

    PubMed

    Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

    2016-06-17

    Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM

  1. Purification and Properties of an Enzyme Capable of Degrading the Sheath of Sphaerotilus natans

    PubMed Central

    Takeda, Minoru; Iohara, Keishi; Shinmaru, Sachie; Suzuki, Ichiro; Koizumi, Jun-Ichi

    2000-01-01

    Microorganisms which can degrade and grow on the purified sheath of a sheathed bacterium Sphaerotilus natans were collected from soil and river water. Two bacterial strains were isolated from the soil and designated strains TB and TK. Both strains are rod shaped, negatively stained by gram staining, facultatively anaerobic, and formed ellipsoidal endospores. These characteristics suggested that the isolates belong to the genus Paenibacillus, according to Ash et al. (C. Ash, F. G. Priest, and M. D. Collins, Antonie Leeuwenhoek 64:253–260, 1993). Phylogenetic analysis based on the 16S rDNA supported this possibility. Purification of the sheath-degrading enzyme was carried out from the culture broth of strain TB. The molecular weight of the enzyme was calculated to be 78,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis and gel filtration chromatography, respectively. Enzyme activity was optimized at pH 6.5 to 7.0 and 30 to 40°C. The reaction was accelerated by the addition of Mg2+, Ca2+, Fe3+, and iodoacetamide, whereas it was inhibited by the addition of Cu2+, Mn2+, and dithiothreitol. The enzyme acted on the polysaccharide moiety of the sheath, producing an oligosaccharide the size of which was between the sizes of maltopentaose and maltohexaose. As the reaction proceeded, the absorbance at 235 nm of the reaction mixture increased, suggesting the generation of unsaturated sugars. Incorporation of unsaturated sugars was also suggested by the thiobarbituric acid reaction. It is possible that the enzyme is not a hydrolytic enzyme but a kind of polysaccharide eliminase which acts on the basic polysaccharide. PMID:11055955

  2. Ruminant Nutrition Symposium: Improving cell wall digestion and animal performance with fibrolytic enzymes.

    PubMed

    Adesogan, A T; Ma, Z X; Romero, J J; Arriola, K G

    2014-04-01

    This paper aimed to summarize published responses to treatment of cattle diets with exogenous fibrolytic enzymes (EFE), to discuss reasons for variable EFE efficacy in animal trials, to recommend strategies for improving enzyme testing and EFE efficacy in ruminant diets, and to identify proteomic differences between effective and ineffective EFE. A meta-analysis of 20 dairy cow studies with 30 experiments revealed that only a few increased lactational performance and the response was inconsistent. This variability is attributable to several enzyme, feed, animal, and management factors that were discussed in this paper. The variability reflects our limited understanding of the synergistic and sequential interactions between exogenous glycosyl hydrolases, autochthonous ruminal microbes, and endogenous fibrolytic enzymes that are necessary to optimize ruminal fiber digestion. An added complication is that many of the standard methods of assaying EFE activities may over- or underestimate their potential effects because they are based on pure substrate saccharification and do not simulate ruminal conditions. Our recent evaluation of 18 commercial EFE showed that 78 and 83% of them exhibited optimal endoglucanase and xylanase activities, respectively, at 50 °C, and 77 and 61% had optimal activities at pH 4 to 5, respectively, indicating that most would likely act suboptimally in the rumen. Of the many fibrolytic activities that act synergistically to degrade forage fiber, the few usually assayed, typically endoglucanase and xylanase, cannot hydrolyze the recalcitrant phenolic acid-lignin linkages that are the main constraints to ruminal fiber degradation. These factors highlight the futility of random addition of EFE to diets. This paper discusses reasons for the variable animal responses to dietary addition of fibrolytic enzymes, advances explanations for the inconsistency, suggests a strategy to improve enzyme efficacy in ruminant diets, and describes differences

  3. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal.

    PubMed

    Cronan, John E

    2014-06-01

    ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4'-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP-enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4'-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping. PMID:24825445

  4. 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 (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-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.

  5. Immobilization of enzyme on a polymer surface

    NASA Astrophysics Data System (ADS)

    Shen, Lei; Cheng, Kenneth Chun Kuen; Schroeder, McKenna; Yang, Pei; Marsh, E. Neil G.; Lahann, Joerg; Chen, Zhan

    2016-06-01

    We successfully immobilized enzymes onto polymer surfaces via covalent bonds between cysteine groups of the enzyme and dibromomaleimide functionalities present at the polymer surface. In this work, we used nitroreductase (NfsB) as a model enzyme molecule. The polymers were prepared by chemical vapor deposition (CVD) polymerization, resulting in surfaces with dibromomaleimide groups. NfsB variants were engineered so that each NfsB molecule only has one cysteine group on the enzyme surface. Two different NfsB constructs were studied, with cysteines at the positions of H360 and V424, respectively. A combination of sum frequency generation (SFG) vibrational and attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopies were used to deduce the orientation of the immobilized enzymes on the surface. It was found that the orientation of the immobilized enzymes is controlled by the position of the cysteine residue in the protein. The NfsB H360C construct exhibited a similar orientational behavior on the polymer surface as compared to that on the self-assembled monolayer surface, but the NsfB V424C construct showed markedly different orientations on the two surfaces.

  6. Enzyme catalysis: Cleaner, safer, energy efficient

    SciTech Connect

    Lalonde, J.

    1997-09-01

    Protein catalysts, more commonly referred to as enzymes, are the driving force behind the myriad of chemical reactions occurring in living organisms. By using their ability to distinguish between similar biochemical compounds and optical isomers (enantiomers), with virtually complete discrimination, enzymes are efficient catalysts, making them an attractive alternative for synthetic ones. Tapping into the natural abilities of enzymes, the chemical process industries (CPI) are beginning to realize that enzymes are not only effective for catalyzing reactions of natural compounds within living systems, but that they can also be used to catalyze reactions of unnatural compounds. Enzymes are novel among catalysts in that they are capable of directing asymmetric transformations with complete activity under ambient conditions. As a result, bioconversions, such as the hydroxylation of unactivated hydrocarbon centers, to give alcohols in high optical purity, have few counterparts in traditional chemical catalysis. And unlike most chemical manufacturing catalysts, enzymes work in water, at ambient temperature and near neutral pH. Also, they are easy to dispose of, since they are composed of biodegradable protein. Thus, biocatalysts are the ideal green catalyst, producing less waste and consuming less energy.

  7. Nanodevices for the immobilization of therapeutic enzymes.

    PubMed

    Bosio, Valeria E; Islan, Germán A; Martínez, Yanina N; Durán, Nelson; Castro, Guillermo R

    2016-06-01

    Therapeutic enzymes are one of the most promising applications of this century in the field of pharmaceutics. Biocatalyst properties can be improved by enzyme immobilization on nano-objects, thereby increasing stability and reusability and also enhancing the targeting to specific tissues and cells. Therapeutic biocatalyst-nanodevice complexes will provide new tools for the diagnosis and treatment of old and newly emerging pathologies. Among the advantages of this approach are the wide span and diverse range of possible materials and biocatalysts that promise to make the matrix-enzyme combination a unique modality for therapeutic delivery. This review focuses on the most significant techniques and nanomaterials used for enzyme immobilization such as metallic superparamagnetic, silica, and polymeric and single-enzyme nanoparticles. Finally, a review of the application of these nanodevices to different pathologies and modes of administration is presented. In short, since therapeutic enzymes constitute a highly promising alternative for treating a variety of pathologies more effectively, this review is aimed at providing the comprehensive summary needed to understand and improve this burgeoning area. PMID:25641329

  8. Crystal structure of methane oxidation enzyme determined

    SciTech Connect

    Baum, R.

    1994-01-10

    A team of chemists has determined to 2.2-[angstrom] resolution the crystal structure of the hydroxylase protein of methane monooxygenase, the enzyme system responsible for the biological oxidation of methane. The hydroxylase, at a molecular weight of 251,000 daltons, if by far the largest component of methane monooxygenase. Although the crystal structure of the hydroxylase did not reveal any startling surprises about the enzyme-many features of the hydroxylase had been inferred previously from modeling and spectroscopic studies -- obtaining it is a significant achievement. For one thing, the crystal structure unambiguously confirms aspects of the enzyme structure that been at least somewhat speculative. The three-dimensional structure of the enzyme, the chemist say, also provides important insight into biological methane oxidation, including how methane, a relatively inert gas, might diffuse to and bind near the active site of the enzyme. The structure points to particular amino acid residues that are likely to participate in catalysis, and clarifies the structure of the dinuclear iron core of the enzyme.

  9. Tryptophan biosynthetic enzymes of Staphylococcus aureus.

    PubMed

    Proctor, A R; Kloos, W E

    1973-04-01

    Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway. PMID:4698207

  10. Effects of Cellulytic Enzymes on Phytophthora cinnamomi.

    PubMed

    Downer, A J; Menge, J A; Pond, E

    2001-09-01

    ABSTRACT Two enzyme systems, cellulase (beta-1,4-glucanase) and laminarinase (beta-1,3-glucanase), were added to soil extracts to simulate (in vitro) lytic components found in mulches suppressive to Phytophthora cinnamomi. Concentration ranges of each enzyme were incubated with Phytophthora cinnamomi mycelium, zoospores, zoospores cysts, and zoospore-infected excised roots to evaluate the roles of each enzyme in potential control of avocado root rot disease. Cellulase significantly retarded the development of zoosporangia and chlamydospores when mycelia were incubated in soil extract containing the enzyme at concentrations greater than 10 units/ml. Zoospore production was also reduced by cellulase but not by laminarinase. Laminarinase had little effect on zoosporangia or chlamydospore formation. At high concentrations, laminarinase was consistently more effective at preventing encystment than cellulase. Chlamydospores preformed in root tips were immune to the lytic effects of all treatments except cellulase at 100 units/ml. Zoospores placed in enzyme solutions and plated on a selective medium survived high cellulase concentrations and formed colonies, but there were fewer surviving zoospores when laminarinase was present at greater than 10 units/ml. Low concentrations of cellulase stimulated infection of excised roots, however, low concentrations of laminarinase prevented infection. Cellulase and laminarinase have different effects on the structures of the Phytophthora cinnamomi life history, however, each enzyme may have a role in reduction of inoculum. PMID:18944229

  11. Long-acting muscarinic antagonists.

    PubMed

    Melani, Andrea S

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is a major cause of death and disability worldwide. Inhaled bronchodilators are the mainstay of COPD pharmacological treatment. Long-acting muscarinic antagonists (LAMAs) are a major class of inhaled bronchodilators. Some LAMA/device systems with different characteristics and dosing schedules are currently approved for maintenance therapy of COPD and a range of other products are being developed. They improve lung function and patient-reported outcomes and reduce acute bronchial exacerbations with good safety. LAMAs are used either alone or associated with long-acting β₂-agonists, eventually in fixed dose combinations. Long-acting β₂-agonist/LAMA combinations assure additional benefits over the individual components alone. The reader will obtain a view of the safety and efficacy of the different LAMA/device systems in COPD patients. PMID:26109098

  12. 7 CFR 1150.101 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.101 Act. Act means Title I, Subtitle B, of the Dairy and Tobacco Adjustment Act of...

  13. 7 CFR 1150.101 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ORDERS; MILK), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.101 Act. Act means Title I, Subtitle B, of the Dairy and Tobacco Adjustment Act of...

  14. 7 CFR 1150.101 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.101 Act. Act means Title I, Subtitle B, of the Dairy and Tobacco Adjustment Act of...

  15. 7 CFR 1150.101 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ORDERS; MILK), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.101 Act. Act means Title I, Subtitle B, of the Dairy and Tobacco Adjustment Act of...

  16. Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a Type C feruloyl esterase, which acted on methyl ferulate, methyl p-coumarate, methyl sinapinate, methyl caffeate, but not diferul...

  17. [Suicide, a philosophical act or an act of depression?].

    PubMed

    Heslon, Christian

    2016-01-01

    Suicide is more frequent in people aged over 75 than in the rest of the population. Is it the fact of feeling too old or of being alone? Is the person fully lucid? The question of responsibility is raised, as is the meaning of the act: the ultimate living gesture or capitulation in the face of death? PMID:27173624

  18. Get Your ACT Together: A Resource Guide for ACT Preparation.

    ERIC Educational Resources Information Center

    Oklahoma State Dept. of Education, Oklahoma City.

    This guide is designed as a resource for (1) Oklahoma teachers, counselors, and parents, to help them help their students prepare for the ACT assessment; and (2) students, to help dispel some common myths and misunderstandings as they prepare for the test. Section A, for administrators, teachers, counselors, Indian program staffs, and…

  19. Advanced Communications Technology Satellite (ACTS)

    NASA Astrophysics Data System (ADS)

    Gedney, Richard T.; Schertler, Ronald J.

    1989-06-01

    The NASA Advanced Communications Technology Satellite (ACTS) was conceived to help maintain U.S. leadership in the world's communications-satellite market. This experimental satellite is expected to be launched by NASA in 1992 and to furnish the technology necessary for establishing very small aperture terminal digital networks which provide on-demand full-mesh connectivity, and 1.544-MBPS services with only a single hop. Utilizing on-board switching and processing, each individual voice or data circuit can be separately routed to any location in the network. This paper provides an overview of the ACTS and discusses the value of the technology for future communications systems.

  20. Advanced Communications Technology Satellite (ACTS)

    NASA Technical Reports Server (NTRS)

    Gedney, Richard T.; Schertler, Ronald J.

    1989-01-01

    The NASA Advanced Communications Technology Satellite (ACTS) was conceived to help maintain U.S. leadership in the world's communications-satellite market. This experimental satellite is expected to be launched by NASA in 1992 and to furnish the technology necessary for establishing very small aperture terminal digital networks which provide on-demand full-mesh connectivity, and 1.544-MBPS services with only a single hop. Utilizing on-board switching and processing, each individual voice or data circuit can be separately routed to any location in the network. This paper provides an overview of the ACTS and discusses the value of the technology for future communications systems.

  1. Self-acting shaft seals

    NASA Technical Reports Server (NTRS)

    Ludwig, L. P.

    1978-01-01

    Self-acting seals are described in detail. The mathematical models for obtaining a seal force balance and the equilibrium operating film thickness are outlined. Particular attention is given to primary ring response (seal vibration) to rotating seat face runout. This response analysis reveals three different vibration models with secondary seal friction being an important parameter. Leakage flow inlet pressure drop and affects of axisymmetric sealing face deformations are discussed. Experimental data on self-acting face seals operating under simulated gas turbine conditions are given. Also a spiral groove seal design operated to 244 m/sec (800 ft/sec) is described.

  2. 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.

  3. Sucrose:Fructan 6-Fructosyltransferase, a Key Enzyme for Diverting Carbon from Sucrose to Fructan in Barley Leaves.

    PubMed Central

    Duchateau, N.; Bortlik, K.; Simmen, U.; Wiemken, A.; Bancal, P.

    1995-01-01

    Sucrose:sucrose 6-fructosyltransferase, an enzyme activity recently identified in fructan-accumulating barley (Hordeum vulgare) leaves, was further characterized. The purified enzyme catalyzed the transfer of a fructosyl group from sucrose to various acceptors. It displayed some [beta]-fructosidase (invertase) activity, indicating that water could act as fructosyl acceptor. Moreover, it transferred the fructosyl residue of unlabeled sucrose to [U-14C]Glc, producing [U-14C]sucrose and unlabeled glucose. Most significantly for fructan synthesis, the enzyme used as acceptors but not as donors a variety of oligofructans containing [beta](2->1)- and [beta](2->6)-linked fructosyl moieties. Thus, it acted as a general sucrose:fructan fructosyltransferase. The products formed by the enzyme from sucrose and various purified, structurally characterized oligofructans were analyzed by liquid chromatography and identified by comparison with structurally characterized standards. The results showed that the enzyme formed exclusively [beta](2->6) fructosyl-fructose linkages, either initiating or elongating a fructan chain of the phlein type. We propose, therefore, to rename the purified enzyme sucrose:fructan 6-fructosyltransferase. PMID:12228431

  4. 7 CFR 1220.600 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Procedures To Request a Referendum Definitions § 1220.600 Act. Act means the...

  5. 7 CFR 1220.600 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Procedures To Request a Referendum Definitions § 1220.600 Act. Act means the...

  6. 7 CFR 1220.600 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Procedures To Request a Referendum Definitions § 1220.600 Act. Act means the...

  7. 7 CFR 1220.600 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Procedures To Request a Referendum Definitions § 1220.600 Act. Act means the...

  8. 7 CFR 1220.600 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SOYBEAN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Procedures To Request a Referendum Definitions § 1220.600 Act. Act means the...

  9. 7 CFR 1207.302 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Definitions § 1207.302 Act. Act means the Potato Research...

  10. 7 CFR 1207.302 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Definitions § 1207.302 Act. Act means the Potato Research...

  11. 7 CFR 1207.302 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Definitions § 1207.302 Act. Act means the Potato Research...

  12. 7 CFR 1207.302 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Definitions § 1207.302 Act. Act means the Potato Research...

  13. 7 CFR 1207.302 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POTATO RESEARCH AND PROMOTION PLAN Potato Research and Promotion Plan Definitions § 1207.302 Act. Act means the Potato Research...

  14. 7 CFR 1218.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.1 Act. Act means...

  15. 7 CFR 1218.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.1 Act. Act means...

  16. 7 CFR 1218.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.1 Act. Act means...

  17. 7 CFR 1218.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.1 Act. Act means...

  18. 7 CFR 1218.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.1 Act. Act means...

  19. 7 CFR 1216.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.1 Act. Act means...

  20. 7 CFR 1216.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.1 Act. Act means...

  1. 7 CFR 1216.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.1 Act. Act means...

  2. 7 CFR 1216.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.1 Act. Act means...

  3. 7 CFR 1216.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.1 Act. Act means...

  4. 7 CFR 1221.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.1 Act. Act means...

  5. 7 CFR 1221.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.1 Act. Act means...

  6. 7 CFR 1221.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.1 Act. Act means...

  7. 7 CFR 1221.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.1 Act. Act means...

  8. 7 CFR 1206.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.1 Act. Act means the...

  9. 7 CFR 1206.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.1 Act. Act means the...

  10. 7 CFR 1206.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.1 Act. Act means the...

  11. 7 CFR 1206.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.1 Act. Act means the...

  12. 7 CFR 1206.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.1 Act. Act means the...

  13. 7 CFR 1221.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE SORGHUM PROMOTION, RESEARCH, AND INFORMATION ORDER Sorghum Promotion, Research, and Information Order Definitions § 1221.1 Act. Act means...

  14. Analysis of a theoretical model for anisotropic enzyme membranes application to enzyme electrodes.

    PubMed

    Pedersen, H; Chotani, G K

    1981-12-01

    A theoretical model of diffusion and reaction in an anisotropic enzyme membrane is presented with particular emphasis on the application of such membranes in enzyme electrodes. The dynamic response of systems in which the kinetics are linear, which comprises the practical operating regime for enzyme electrodes in analysis, is investigated via an analytic solution of the governing differential equations. The response is presented as a function of a single dimensionless group, Μ, that is the membrane modulus. PMID:24233978

  15. Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

    PubMed

    Marden, James H

    2013-12-01

    Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. PMID:24106889

  16. Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.

    PubMed Central

    Sardana, M K; Kappas, A

    1987-01-01

    Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin. Images PMID:3470805

  17. Act To Promote Adult Education.

    ERIC Educational Resources Information Center

    1970

    An act of the German Lower Saxony Parliament to promote adult education is presented. It has 24 general provisions relating to the following: purpose of adult education, principle for promotion, conditions for promotions of establishments, independence of adult education, prerequisites and form of acknowledgement of entitlement to promotion,…

  18. 78 FR 46256 - Privacy Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-31

    ... From the Federal Register Online via the Government Publishing Office FEDERAL ELECTION COMMISSION 11 CFR Part 1 Privacy Act CFR Correction In Title 11 of the Code of Federal Regulations, revised as of January 1, 2012, on page 5, in Sec. 1.2, the words ``95 and 96 of the Internal Revenue Code...

  19. The Indian Child Welfare Act.

    ERIC Educational Resources Information Center

    Steward, Katy Jo

    The Indian Child Welfare Act of 1978 (I.C.W.A.) is federal legislation which preempts state law whenever Indian children may be removed from their families. The I.C.W.A. permits Indian tribal courts to decide the future of Indian children, establishes minimum federal standards for removal of Indian children from their families, requires that…

  20. The Ontogenesis of Speech Acts

    ERIC Educational Resources Information Center

    Bruner, Jerome S.

    1975-01-01

    A speech act approach to the transition from pre-linguistic to linguistic communication is adopted in order to consider language in relation to behavior and to allow for an emphasis on the use, rather than the form, of language. A pilot study of mothers and infants is discussed. (Author/RM)

  1. ACT and General Cognitive Ability

    ERIC Educational Resources Information Center

    Koenig, Katherine A.; Frey, Meredith C.; Detterman, Douglas K.

    2008-01-01

    Research on the SAT has shown a substantial correlation with measures of "g" such as the Armed Services Vocational Aptitude Battery (ASVAB). Another widely administered test for college admission is the American College Test (ACT). Using the National Longitudinal Survey of Youth 1979, measures of "g" were derived from the ASVAB and correlated with…

  2. Clery Act: Road to Compliance

    ERIC Educational Resources Information Center

    McNeal, Laura R.

    2007-01-01

    The purpose of this study was to explore what factors served as impediments to institutional efforts to comply with Clery Act guidelines through the perceptions of campus law administrators. Statistical analyses were performed on data collected from an online survey, which was distributed to members of the International Association of Campus Law…

  3. Dual acting slit control mechanism

    NASA Technical Reports Server (NTRS)

    Struthoff, G. L. (Inventor)

    1980-01-01

    A dual acting control system for mass spectrometers is described, which permits adjustment of the collimating slit width and centering of the collimating slit while using only one vacuum penetration. Coaxial shafts, each with independent vacuum bellows are used to independently move the entire collimating assembly or to adjust the slit dimension through a parallelogram linkage.

  4. Implementing the Reading Excellence Act.

    ERIC Educational Resources Information Center

    Lacy, Laurie; Dole, Janice; Donaldson, Becky; Donaldson, Brady

    This slide presentation outlines one state's (Utah) version of implementation of a model for literacy learning under the Reading Excellence Act (REA). According to the presentation, the model is called "The Utah Reads K-3 Literacy Model." The presentation is divided into the following sections: Utah's Vision: What We've Learned So Far; One…

  5. The Federal Employees' Compensation Act.

    ERIC Educational Resources Information Center

    Nordlund, Willis J.

    1991-01-01

    The 1916 Federal Employees' Compensation Act is still the focal point around which the federal workers compensation program works today. The program has gone through many changes on its way to becoming a modern means of compensating workers for job-related injury, disease, and death. (Author)

  6. Acting White: A Critical Review

    ERIC Educational Resources Information Center

    Sohn, Kitae

    2011-01-01

    The hypothesis of acting White has been heatedly debated and influential over the last 20 years or so in explaining the Black-White test score gap. Recently, economists have joined the debate and started providing new theoretical and empirical analyses of the phenomenon. This paper critically reviews the arguments that have been advanced to…

  7. 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 α-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

  8. 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

  9. Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes

    NASA Astrophysics Data System (ADS)

    Mitsuoka, Hiroshi; Kistler, Erik B.; Schmid-Schönbein, Geert W.

    2000-02-01

    One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

  10. Transepithelial transport and stability in blood serum of angiotensin-I-converting enzyme inhibitory dipeptides.

    PubMed

    Pentzien, Anne-Kathrin; Meisel, Hans

    2008-01-01

    The dipeptides Ala-Trp, Val-Phe, and Val-Tyr inhibit the angiotensin-I-converting enzyme. They are encrypted within the primary sequences of different food proteins, e.g. milk proteins. The angiotensin-I-converting enzyme inhibitory potency of these synthetic dipeptides was quantified using a spectrophotometric assay. The dipeptides showed no adverse effects on differentiated Caco-2 cells (model for human intestinal epithelium), as confirmed by transepithelial electrical resistance, microscopy and the activity of the brush-border enzyme dipeptidyl aminopeptidase IV. Furthermore, the transport of these bioactive dipeptides through intact Caco-2 monolayers and their stability to incubation in human blood serum has been demonstrated for the first time. Low molecular mass peptides represent the minimal structures required for angiotensin-I-converting enzyme inhibition which have a high potential bioavailability. Therefore, they may act as target peptides in enriched hydrolysates for the preparation of an angiotensin-I-converting enzyme inhibitory peptide and for the use in special formulations as functional foods/foods of specified health use. PMID:18669035

  11. Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1.

    PubMed

    Niyonzima, Francois N; More, Sunil S

    2014-01-01

    A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseed oil as an inducer for protease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium. PMID:25477924

  12. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

  13. A multi-enzyme model for Pyrosequencing.

    PubMed

    Agah, Ali; Aghajan, Mariam; Mashayekhi, Foad; Amini, Sasan; Davis, Ronald W; Plummer, James D; Ronaghi, Mostafa; Griffin, Peter B

    2004-01-01

    Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated. PMID:15576673

  14. 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

  15. Actinomycetes: A Source of Lignocellulolytic Enzymes

    PubMed Central

    Saini, Anita; Aggarwal, Neeraj K.; Sharma, Anuja; Yadav, Anita

    2015-01-01

    Lignocellulose is the most abundant biomass on earth. Agricultural, forest, and agroindustrial activities generate tons of lignocellulosic wastes annually, which present readily procurable, economically affordable, and renewable feedstock for various lignocelluloses based applications. Lignocelluloses are the focus of present decade researchers globally, in an attempt to develop technologies based on natural biomass for reducing dependence on expensive and exhaustible substrates. Lignocellulolytic enzymes, that is, cellulases, hemicellulases, and lignolytic enzymes, play very important role in the processing of lignocelluloses which is prerequisite for their utilization in various processes. These enzymes are obtained from microorganisms distributed in both prokaryotic and eukaryotic domains including bacteria, fungi, and actinomycetes. Actinomycetes are an attractive microbial group for production of lignocellulose degrading enzymes. Various studies have evaluated the lignocellulose degrading ability of actinomycetes, which can be potentially implemented in the production of different value added products. This paper is an overview of the diversity of cellulolytic, hemicellulolytic, and lignolytic actinomycetes along with brief discussion of their hydrolytic enzyme systems involved in biomass modification. PMID:26793393

  16. The temperature response of fungal enzyme kinetics

    NASA Astrophysics Data System (ADS)

    Curran, M.; Lu, Y.; Taylor, J.; Allison, S. D.

    2013-12-01

    Extracellular enzymes produced and excreted by microbes mediate the decomposition of carbon (C), nitrogen (N), and phosphorus (P) -containing compounds in their environment. Climate change has the potential to alter the rate of decomposition especially in high latitude regions where stocks of recalcitrant, or long-lived, C are abundant. This project compares extracellular enzyme activity (EEA) across ten fungi strains within the model family Neurospora in order to assess the range of variation in temperature sensitivities of fungal enzyme Vmax and Km. Vmax values of most enzymes tested increased exponentially,which was hypothesized and consistant with thermodynamic principles. We also hypothesized that Neurospora strains would exhibit different EEA temperature sensitivities based on their native climate. We observed strain-dependent variation in enzyme temperature responses consistent with strain-specific adaptation to local conditions. Since fungi are the major decomposers of organic carbon in high-latitude ecosystems, an increase in EEA in-situ would result in higher carbon dioxide emissions. These findings suggest a shift in fungal processing of soil organic carbon and nutrients in response to changing climate.

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

    PubMed

    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

    2013-09-01

    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

  18. Acting Out; Theoretical and Clinical Aspects.

    ERIC Educational Resources Information Center

    Abt, Lawrence Edwin, Ed.; Weissman, Stuart L.

    The beneficial and harmful effects of acting out are studied in a series of short essays by numerous authors. Included are four articles on the theoretical and dynamic considerations of acting out, along with five clinical manifestations of acting out involving suicide and criminality in adolescents and adults. Special forms of harmful acting out…

  19. 76 FR 13550 - Fur Products Labeling Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-14

    ...In December 2010, Congress passed the Truth in Fur Labeling Act (TFLA), which amends the Fur Products Labeling Act (Fur Act) by: (1) Eliminating the Commission's discretion to exempt fur products of relatively small quantity or value from disclosure requirements; and (2) providing that the Fur Act will not apply to certain fur products obtained through trapping or hunting and sold in face to......

  20. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND 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 reputation of American-grown grapes and plums...

  1. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND 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 reputation of American-grown grapes and plums...

  2. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND 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 reputation of American-grown grapes and plums...

  3. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND 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 reputation of American-grown grapes and plums...

  4. 7 CFR 35.1 - Act.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND 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 reputation of American-grown grapes and plums...

  5. 10 CFR 430.61 - Prohibited acts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Prohibited acts. 430.61 Section 430.61 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY CONSERVATION PROGRAM FOR CONSUMER PRODUCTS Certification and Enforcement § 430.61 Prohibited acts. (a) Each of the following is a prohibited act pursuant to section 332 of the Act: (1) Failure to permit access...

  6. 76 FR 25665 - No Fear Act

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-05

    ... COMMISSION No Fear Act AGENCY: American Battle Monuments Commission. ACTION: Notice. SUMMARY: The American... notification obligation under the Notification and Federal Employees Antidiscrimination and Retaliation Act (No... Retaliation Act of 2002,'' which is now known as the No FEAR Act. See Public Law 107-174, codified at 5...

  7. Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

    PubMed Central

    Kholodenko, B N; Brown, G C

    1996-01-01

    It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties. PMID:8615766

  8. ENZVU--An Enzyme Kinetics Computer Simulation Based upon a Conceptual Model of Enzyme Action.

    ERIC Educational Resources Information Center

    Graham, Ian

    1985-01-01

    Discusses a simulation on enzyme kinetics based upon the ability of computers to generate random numbers. The program includes: (1) enzyme catalysis in a restricted two-dimensional grid; (2) visual representation of catalysis; and (3) storage and manipulation of data. Suggested applications and conclusions are also discussed. (DH)

  9. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  10. 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...

  11. 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

  12. Enzyme replacement in Tay-Sachs disease.

    PubMed

    von Specht, B U; Geiger, B; Arnon, R; Passwell, J; Keren, G; Goldman, B; Padeh, B

    1979-06-01

    Enzyme replacement therapy was attempted with two Tay-Sachs-diseased individuals--a 14-month-old child and a 7-week-old infant. Treatment consisted of repeated weekly intrathecal injections of pure hexosaminidase A. Injection of this enzyme resulted in almost complete disappearance of GM2 from the serum, but did not bring about dissolution of the GM2 membranous cytoplasmic bodies in the brain, as detected by electronmicroscopy. Both patients tolerated the treatment without apparent clinical complications, but no clear-cut improvement was noted as a result of prolonged injections of hexosaminidase A. Since this treatment was initiated in both an advanced stage and a very early stage of the disease, we conclude that enzyme replacement treatment by this route is not beneficial for patients with Tay-Sachs disease. PMID:572006

  13. Enzyme activity in dialkyl phosphate ionic liquids.

    PubMed

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea 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. PMID:22001053

  14. Inhibitors of alanine racemase enzyme: a review.

    PubMed

    Azam, Mohammed Afzal; Jayaram, Unni

    2016-08-01

    Alanine racemase is a fold type III PLP-dependent amino acid racemase enzyme catalysing the conversion of l-alanine to d-alanine utilised by bacterial cell wall for peptidoglycan synthesis. As there are no known homologs in humans, it is considered as an excellent antibacterial drug target. The standard inhibitors of this enzyme include O-carbamyl-d-serine, d-cycloserine, chlorovinyl glycine, alaphosphin, etc. d-Cycloserine is indicated for pulmonary and extra pulmonary tuberculosis but therapeutic use of drug is limited due to its severe toxic effects. Toxic effects due to off-target affinities of cycloserine and other substrate analogs have prompted new research efforts to identify alanine racemase inhibitors that are not substrate analogs. In this review, an updated status of known inhibitors of alanine racemase enzyme has been provided which will serve as a rich source of structural information and will be helpful in generating selective and potent inhibitor of alanine racemase. PMID:26024289

  15. 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.

  16. 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

  17. Entrapping Enzyme in a Functionalized Nanoporous Support

    SciTech Connect

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

    2002-09-25

    The enzyme organophosphorus hydrolase (OPH) was spontaneously entrapped in carboxylethyl- or aminopropyl-functionalized mesoporous silica with rigid, uniform open-pore geometry (30 nm). This approach yielded larger amounts of protein loading and much higher specific activity of the enzyme when compared to the unfunctionalized mesoporous silica and normal porous silica with the same pore size. When OPH was incubated with the functionalized mesoporous silica, protein molecules were sequestered in or excluded from the porous material, depending on electrostatic interaction with the charged functional groups. OPH entrapped in the organically functionalized nanopores showed an exceptional high immobilization efficiency of more than 200% and enhanced stability far exceeding that of the free enzyme in solution. The combination of high protein loading, high immobilization efficiency and stability is attributed to the large and uniform pore structure, and to the optimum environment introduced by the functional groups.

  18. Multicarbohydrase Enzymes for Non-ruminants

    PubMed Central

    Masey O’Neill, H. V.; Smith, J. A.; Bedford, M. R.

    2014-01-01

    The first purpose of this review is to outline some of the background information necessary to understand the mechanisms of action of fibre-degrading enzymes in non-ruminants. Secondly, the well-known and understood mechanisms are described, i) eliminating the nutrient encapsulating effect of the cell wall and ii) ameliorating viscosity problems associated with certain Non Starch Polysaccharides, particularly arabinoxylans and β-glucans. A third, indirect mechanism is then discussed: the activity of such enzymes in producing prebiotic oligosaccharides and promoting beneficial cecal fermentation. The literature contains a wealth of information on various non starch polysaccharide degrading enzyme (NSPase) preparations and this review aims to conclude by discussing this body of work, with reference to the above mechanisms. It is suggested that the way in which multi- versus single-component products are compared is often flawed and that some continuity should be employed in methods and terminology. PMID:25049954

  19. Nanoparticles for Use in Enzyme Assays.

    PubMed

    Kim, Young-Pil; Kim, Hak-Sung

    2016-02-01

    Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology. Here we review recent advances in NP-based biosensors and their use in enzyme assays. With fast and specific responses to enzyme-mediated reactions, NPs transduce and amplify the initial responses into various types of signals, such as electrochemical, optical and magnetic ones. Translation of their potential should lead to functionalized NPs finding wide applications in diagnostics, drug development and biotechnology. PMID:26662229

  20. 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.