Homogalacturonan-modifying enzymes: structure, expression, and roles in plants
Sénéchal, Fabien; Wattier, Christopher; Rustérucci, Christine; Pelloux, Jérôme
2014-01-01
Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses. PMID:25056773
Pectin-modifying enzymes and pectin-derived materials: applications and impacts.
Bonnin, Estelle; Garnier, Catherine; Ralet, Marie-Christine
2014-01-01
Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.
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 or...
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 prepared...
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 prepared...
21 CFR 184.1287 - Enzyme-modified fats.
Code of Federal Regulations, 2010 CFR
2010-04-01
... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...
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 prepared...
Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G
2016-12-09
A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5 M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.
Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance.
Ene, Iuliana V; Walker, Louise A; Schiavone, Marion; Lee, Keunsook K; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A R; Munro, Carol A; Brown, Alistair J P
2015-07-28
The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by
Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance
Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.
2015-01-01
ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968
21 CFR 184.1063 - Enzyme-modified lecithin.
Code of Federal Regulations, 2014 CFR
2014-04-01
... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Enzyme-modified lecithin. 184.1063 Section 184... as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified lecithin is prepared by treating... percent of total phosphatides as determined by “Determination of Lysolecithin Content of Enzyme-Modified...
21 CFR 184.1063 - Enzyme-modified lecithin.
Code of Federal Regulations, 2011 CFR
2011-04-01
... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified lecithin. 184.1063 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...
21 CFR 184.1063 - Enzyme-modified lecithin.
Code of Federal Regulations, 2010 CFR
2010-04-01
... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified lecithin. 184.1063 Section 184.1063... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...
Allosteric regulation of epigenetic modifying enzymes.
Zucconi, Beth E; Cole, Philip A
2017-08-01
Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.
Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.
Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A
2015-09-23
The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.
Wu, Nan; Wang, Siming; Yang, Ye; Song, Jiayi; Su, Ping; Yang, Yi
2018-07-01
A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl- L -arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research. Copyright © 2018. Published by Elsevier B.V.
Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A
2015-01-01
Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.
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.
Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.
Dongowski, G; Sembries, S
2001-09-01
The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.
Huber, Donald J.; Lee, James H.
1988-01-01
Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed. PMID:16666191
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cosgrove, Daniel J.
The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potentialmore » pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.« less
Huberman, Lori B; Murray, Andrew W
2014-01-01
Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.
Huberman, Lori B.; Murray, Andrew W.
2014-01-01
Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559
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.
Cosgrove, Daniel J
2016-01-01
The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
The influence of unstable modified wall squat exercises on the posture of female university students
Lee, Yoonmi
2015-01-01
[Purpose] The purpose of this study was to examine the effect of unstable modified wall squat exercises on the posture of female university students. [Subjects] The subjects of this study were 30 female university students who were equally and randomly allocated to an unstable modified wall squat exercises group the experimental group and a stable modified wall squat exercises group the control group. [Methods] Both groups performed their respective exercises for 30 minutes three times per week over a six-week period. Using BackMapper, trunk inclination, trunk imbalance, pelvic position, pelvic torsion, pelvic rotation, and position of the scapulae were evaluated. [Results] The unstable modified wall squat exercises group obtained significant results for trunk inclination, trunk imbalance, pelvic position, pelvic torsion, position of the scapulae, while the stable modified wall squat exercises group obtained significant results for trunk imbalance and pelvic position. [Conclusion] Unstable modified wall squat exercises may be applied as a method to correct the posture of average adults. PMID:26356770
Enzyme Amplified Detection of Microbial Cell Wall Components
NASA Technical Reports Server (NTRS)
Wainwright, Norman R.
2004-01-01
This proposal is MBL's portion of NASA's Johnson Space Center's Astrobiology Center led by Principal Investigator, Dr. David McKay, entitled: 'Institute for the Study of Biomarkers in Astromaterials.' Dr. Norman Wainwright is the principal investigator at MBL and is responsible for developing methods to detect trace quantities of microbial cell wall chemicals using the enzyme amplification system of Limulus polyphemus and other related methods.
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.
Inhibition of Cell Wall-Associated Enzymes in Vitro and in Vivo with Sugar Analogs
Nagahashi, Gerald; Tu, Shu-I; Fleet, George; Namgoong, Sun K.
1990-01-01
Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with β-glucosidase, β-GIcNAcase, and α-mannosidase, respectively. Swainsonine specifically inhibited α-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited β-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised. PMID:16667291
The roles of ubiquitin modifying enzymes in neoplastic disease.
Kumari, Nishi; Jaynes, Patrick William; Saei, Azad; Iyengar, Prasanna Vasudevan; Richard, John Lalith Charles; Eichhorn, Pieter Johan Adam
2017-12-01
The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFβ, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy. Copyright © 2017 Elsevier B.V. All rights reserved.
Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A
2015-03-18
Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.
Shi, Jiafu; Yang, Chen; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi; Zhang, Wenyan; Song, Xiaokai; Ai, Qinghong; Tian, Chunyong
2013-10-23
Microcapsules with diverse wall structures may exhibit different performance in specific applications. In the present study, three kinds of mussel-inspired polydopamine (PDA) microcapsules with different wall structures have been prepared by a template-mediated method. More specifically, three types of CaCO3 microspheres (poly(allylamine hydrochloride), (PAH)-doped CaCO3; pure-CaCO3; and poly(styrene sulfonate sodium), (PSS)-doped CaCO3) were synthesized as sacrificial templates, which were then treated by dopamine to obtain the corresponding PDA-CaCO3 microspheres. Through treating these microspheres with disodium ethylene diamine tetraacetic acid (EDTA-2Na) to remove CaCO3, three types of PDA microcapsules were acquired: that was (1) PAH-PDA microcapsule with a thick (∼600 nm) and highly porous capsule wall composed of interconnected networks, (2) pure-PDA microcapsule with a thick (∼600 nm) and less porous capsule wall, (3) PSS-PDA microcapsule with a thin (∼70 nm) and dense capsule wall. Several characterizations confirmed that a higher degree in porosity and interconnectivity of the capsule wall would lead to a higher mass transfer coefficient. When serving as the carrier for catalase (CAT) immobilization, these enzyme-encapsulated PDA microcapsules showed distinct structure-related activity and stability. In particular, PAH-PDA microcapsules with a wall of highly interconnected networks displayed several significant advantages, including increases in enzyme encapsulation efficiency and enzyme activity/stability and a decrease in enzyme leaching in comparison with other two types of PDA microcapsules. Besides, this hierarchically structured PAH-PDA microcapsule may find other promising applications in biocatalysis, biosensors, drug delivery, etc.
Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon
2013-06-01
A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.
The plant cell wall in the feeding sites of cyst nematodes.
Bohlmann, Holger; Sobczak, Miroslaw
2014-01-01
Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium.
Park, Sang -Hyuck; Ong, Rebecca Garlock; Sticklen, Mariam
2015-12-02
Microbial cell wall-deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall-deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall-deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yieldsmore » and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue-specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. As a result, we also summarize methods that have been used to access and maintain activity of plant-generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)
Park, Sang -Hyuck; Ong, Rebecca Garlock; Sticklen, Mariam
Microbial cell wall-deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall-deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall-deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yieldsmore » and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue-specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. As a result, we also summarize methods that have been used to access and maintain activity of plant-generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.« less
Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome.
Kattah, Michael G; Malynn, Barbara A; Ma, Averil
2017-11-10
Ubiquitin and ubiquitin-modifying enzymes play critical roles in a wide variety of intracellular signaling pathways. Inflammatory signaling cascades downstream of TNF, TLR agonists, antigen receptor cross-linking, and cytokine receptors, all rely on ubiquitination events to direct subsequent immune responses. In the past several years, inflammasome activation and subsequent signal transduction have emerged as an excellent example of how ubiquitin signals control inflammatory responses. Inflammasomes are multiprotein signaling complexes that ultimately lead to caspase activation and release of the interleukin-1 (IL-1) family members, IL-1β and IL-18. Inflammasome activation is critical for the host's defense against pathogens, but dysregulation of inflammasomes may contribute to the pathogenesis of multiple diseases. Ultimately, understanding how various ubiquitin interacting proteins control inflammatory signaling cascades could provide new pathways for therapeutic intervention. Here we review specific ubiquitin-modifying enzymes and ubiquitination events that orchestrate inflammatory responses, with an emphasis on the NLRP3 inflammasome. Copyright © 2017 Elsevier Ltd. All rights reserved.
Crowe, Jacob D; Zarger, Rachael A; Hodge, David B
2017-10-04
Simultaneous chemical modification and physical reorganization of plant cell walls via alkaline hydrogen peroxide or liquid hot water pretreatment can alter cell wall structural properties impacting nanoscale porosity. Nanoscale porosity was characterized using solute exclusion to assess accessible pore volumes, water retention value as a proxy for accessible water-cell walls surface area, and solute-induced cell wall swelling to measure cell wall rigidity. Key findings concluded that delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity and that the subsequent cell wall swelling resulted increased nanoscale porosity and improved enzyme binding and hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 Å dextran probe within the cell wall was found to be correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields.
Blanco-Ulate, Barbara; Morales-Cruz, Abraham; Amrine, Katherine C. H.; Labavitch, John M.; Powell, Ann L. T.; Cantu, Dario
2014-01-01
Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth), secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes) identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue. PMID:25232357
Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.
Weil, M; Rausch, T
1990-12-01
The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.
Welker, N. E.
1971-01-01
The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings. PMID:4255338
Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.
Caveney, Nathanael A; Li, Franco Kk; Strynadka, Natalie Cj
2018-06-06
The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents. Copyright © 2018 Elsevier Ltd. All rights reserved.
Devaux, Marie-Françoise; Jamme, Frédéric; André, William; Bouchet, Brigitte; Alvarado, Camille; Durand, Sylvie; Robert, Paul; Saulnier, Luc; Bonnin, Estelle; Guillon, Fabienne
2018-01-01
Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. PMID:29515611
Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.; ...
2017-06-09
Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1
DOE Office of Scientific and Technical Information (OSTI.GOV)
Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.
Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1
Lactobacillus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell walls location.
Saad, N; Urdaci, M; Vignoles, C; Chaignepain, S; Tallon, R; Schmitter, J M; Bressollier, P
2009-12-01
The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.
Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.
Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F
2018-04-17
Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.
NASA Astrophysics Data System (ADS)
Shi, Jin; Claussen, Jonathan C.; McLamore, Eric S.; Haque, Aeraj ul; Jaroch, David; Diggs, Alfred R.; Calvo-Marzal, Percy; Rickus, Jenna L.; Porterfield, D. Marshall
2011-09-01
This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM - 1 cm - 2), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H2O2 response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.
Rai, Krishan Mohan; Balasubramanian, Vimal Kumar; Welker, Cassie Marie; Pang, Mingxiong; Hii, Mei Mei; Mendu, Venugopal
2015-08-01
The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database. In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process
Preparation of immobilized glucose oxidase wafer enzyme on calcium-bentonite modified by surfactant
NASA Astrophysics Data System (ADS)
Widi, R. K.; Trisulo, D. C.; Budhyantoro, A.; Chrisnasari, R.
2017-07-01
Wafer glucose oxidase (GOx) enzymes was produced by addition of PAH (Poly-Allyamine Hydrochloride) polymer into immobilized GOx enzyme on modified-Tetramethylammonium Hydroxide (TMAH) 5%-calsium-bentonite. The use of surfactant molecul (TMAH) is to modify the surface properties and pore size distribution of the Ca-bentonite. These properties are very important to ensure GOx molecules can be bound on the Ca-bentonit surface to be immobilized. The addition of the polymer (PAH) is expected to lead the substrates to be adsorbed onto the enzyme. In this study, wafer enzymes were made in various concentration ratio (Ca-bentonite : PAH) which are 1:0, 1:1, 1:2 and 1:3. The effect of PAH (Poly-Allyamine Hydrochloride) polymer added with various ratios of concentrations can be shown from the capacitance value on LCR meter and enzyme activity using DNS method. The addition of the polymer (PAH) showed effect on the activity of GOx, it can be shown from the decreasing of capacitance value by increasing of PAH concentration.
Enzyme-modified electrolyte-gated organic field-effect transistors
NASA Astrophysics Data System (ADS)
Buth, Felix; Donner, Andreas; Stutzmann, Martin; Garrido, Jose A.
2012-10-01
Organic solution-gated field-effect transistors (SGFETs) can be operated at low voltages in aqueous environments, paving the way to the use of organic semiconductors in bio-sensing applications. However, it has been shown that these devices exhibit only a rather weak sensitivity to standard electrolyte parameters such as pH and ionic strength. In order to increase the sensitivity and to add specificity towards a given analyte, the covalent attachment of functional groups and enzymes to the device surface would be desirable. In this contribution we demonstrate that enzyme modified organic SGFETs can be used for the in-situ detection of penicillin in the low μM regime. In a first step, silane molecules with amine terminal groups are grafted to α-sexithiophene-based thin film transistors. Surface characterization techniques like X-ray photoemission confirm the modification of the surface with these functional groups, which are stable in standard aqueous electrolytes. We show that the presence of surface-bound amphoteric groups (e.g. amino or carboxylic moieties) increases the pH-sensitivity of the organic SGFETs. In addition, these groups serve as anchoring sites for the attachment of the enzyme penicillinase. The resulting enzyme-FETs are used for the detection of penicillin, enabling the study of the influence of the buffer strength and the pH of the electrolyte on the enzyme kinetics. The functionalization of the organic FETs shown here can be extended to a large variety of enzymes, allowing the specific detection of different chemical and biochemical analytes.
Wang, Y D; Guo, L; Qian, S J; Meng, G Z; Zhang, S Z
2000-07-01
The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.
Harbuzova, V Iu
2002-01-01
The intensity of the lipid peroxydation (LPO) and the antioxidant enzyme activity (superoxide dismutase, glutathione peroxydase and catalase) on injecting vitamin D in high doses (10,000 U/kg) was examined in the arterial and venous walls of rabbits. The increase in the amount of the intermediate and final LPO products has been found in the vessels of all types. The lowest intensity of LPO was noted in the vena cava. The decrease in the antioxidant activity has been revealed. But vena cava inferior was the exception because the activity of all studied antioxidant enzymes grew in its wall. This increase is likely to be one of the reasons for vena resistance to the action of damaging factors.
Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi
USDA-ARS?s Scientific Manuscript database
Discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly c...
NASA Technical Reports Server (NTRS)
Gibeaut, David M.; Karuppiah, Nadarajah; Chang, S.-R.; Brock, Thomas G.; Vadlamudi, Babu; Kim, Donghern; Ghosheh, Najati S.; Rayle, David L.; Carpita, Nicholas C.; Kaufman, Peter B.
1990-01-01
The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response and asymmetric processes involving degradation of starch and cell wall synthesis. Cellular and biochemical events were studied by investigation of the activities of related enzymes and changes in cell walls and their constituents. It is suggested that an osmotic potential gradient acts as the driving factor for growth, while wall extensibility is a limiting factor in pulvinus growth.
Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.
McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D
2013-01-01
A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.
Brand, Philipp; Lin, Wei; Johnson, Brian R.
2018-01-01
Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379
Jung, Sang-Kyu; Parisutham, Vinuselvi; Jeong, Seong Hun; Lee, Sung Kuk
2012-01-01
A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. PMID:22911272
Cho, Misuk
2013-01-01
[Purpose] The purpose of this study was to compare the effects of bridge exercises applying the abdominal drawing-in method and modified wall squat exercises on deep abdominal muscle thickness and lumbar stability. [Subjects] A total of 30 subjects were equally divided into an experimental group and a control group. [Methods] The experimental group completed modified wall squat exercises, and the control group performed bridge exercises. Both did so for 30 minutes three times per week over a six-week period. Both groups’ transversus abdominis (Tra), internal oblique (IO), and multifidus muscle thickness were measured using ultrasonography, while their static lumbar stability and dynamic lumbar stability were measured using a pressure biofeedback unit. [Results] A comparison of the pre-intervention and post-intervention measures of the experimental group and the control group was made; the Tra and IO thicknesses were significantly different in both groups. [Conclusion] The modified wall squat exercise and bridge exercise affected the thicknesses of the Tra and the IO muscles. While the bridge exercise requirs space and a mattress to lie on, the modified wall squat exercise can be conveniently performed anytime. PMID:24259831
The molecular basis of plant cell wall extension.
Darley, C P; Forrester, A M; McQueen-Mason, S J
2001-09-01
In all terrestrial and aquatic plant species the primary cell wall is a dynamic structure, adjusted to fulfil a diversity of functions. However a universal property is its considerable mechanical and tensile strength, whilst being flexible enough to accommodate turgor and allow for cell elongation. The wall is a composite material consisting of a framework of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides, interlaced with structural proteins and pectic polymers. The assembly and modification of these polymers within the growing cell wall has, until recently, been poorly understood. Advances in cytological and genetic techniques have thrown light on these processes and have led to the discovery of a number of wall-modifying enzymes which, either directly or indirectly, play a role in the molecular basis of cell wall expansion.
Enzyme-modified starch as an oil delivery system for bake-only chicken nuggets.
Purcell, Sarah; Wang, Ya-Jane; Seo, Han-Seok
2014-05-01
This study investigated the effects of enzyme modification on starch as an effective oil delivery system for bake-only chicken nuggets. Various native starches were hydrolyzed by amyloglucosidase to a hydrolysis degree of 20% to 25% and plated with 50% (w/w, starch dry basis) with canola oil to create a starch-oil matrix. This matrix was then blended into a dry ingredient blend for batter and breader components. Nuggets were prepared by coated with predust, hydrated batter, and breader, and the coated nuggets were steam-baked until fully cooked and then frozen until texture and sensory analyses. The enzyme-modified starches showed a significant decrease in pasting viscosities for all starch types. For textural properties of nuggets, no clear relationship was found between peak force and starch source or amylose content. Sensory attributes related to fried foods (for example, crispness and mouth-coating) did not significantly differ between bake-only nuggets formulated using the enzyme-modified starches and the partially fried and baked ones. The present findings suggest that enzyme-modified starches can deliver sufficient quantity of oil to create sensory attributes similar to those of partially fried chicken nuggets. Further study is needed to optimize the coating formulation of bake-only chicken nugget to become close to the fried one in sensory aspects. The food industry has become increasingly focused on healthier items. Frying imparts several critical and desirable product functionalities, such as developing texture and color, and providing mouth-feel and flavor. The food industry has yet to duplicate all of the unique characteristics of fried chicken nuggets with a baking process. This study investigated the application of enzyme-modified starch as an oil delivery system in bake-only chicken nugget formulation in attempts to provide characteristics of fried items. This information is useful to improve the nutritional value of fried food by eliminating the
HIstome--a relational knowledgebase of human histone proteins and histone modifying enzymes.
Khare, Satyajeet P; Habib, Farhat; Sharma, Rahul; Gadewal, Nikhil; Gupta, Sanjay; Galande, Sanjeev
2012-01-01
Histones are abundant nuclear proteins that are essential for the packaging of eukaryotic DNA into chromosomes. Different histone variants, in combination with their modification 'code', control regulation of gene expression in diverse cellular processes. Several enzymes that catalyze the addition and removal of multiple histone modifications have been discovered in the past decade, enabling investigations of their role(s) in normal cellular processes and diverse pathological conditions. This sudden influx of data, however, has resulted in need of an updated knowledgebase that compiles, organizes and presents curated scientific information to the user in an easily accessible format. Here, we present HIstome, a browsable, manually curated, relational database that provides information about human histone proteins, their sites of modifications, variants and modifying enzymes. HIstome is a knowledgebase of 55 human histone proteins, 106 distinct sites of their post-translational modifications (PTMs) and 152 histone-modifying enzymes. Entries have been grouped into 5 types of histones, 8 types of post-translational modifications and 14 types of enzymes that catalyze addition and removal of these modifications. The resource will be useful for epigeneticists, pharmacologists and clinicians. HIstome: The Histone Infobase is available online at http://www.iiserpune.ac.in/∼coee/histome/ and http://www.actrec.gov.in/histome/.
Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.
Ermert, Susanne; Marx, Andreas; Hacker, Stephan M
2017-04-01
Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.
Jin, Lixia; Son, Yowhan; Yoon, Tae Kyung; Kang, Yu Jin; Kim, Woong; Chung, Haegeun
2013-02-01
Nanomaterials such as single-walled carbon nanotubes (SWCNTs) may enter the soil environment with unknown consequences resulting from the development of nanotechnology for a variety of applications. We determined the effects of SWCNTs on soil enzyme activity and microbial biomass through a 3-week incubation of urban soils treated with different concentrations of SWCNTs ranging from 0 to 1000 μg g(-1) soil. The activities of cellobiohydrolase, β-1,4-glucosidase, β-1,4-xylosidase, β-1,4-N-acetylglucosaminidase, L-leucine aminopeptidase, and acid phosphatase and microbial biomass were measured in soils treated with powder and suspended forms of SWCNTs. SWCNTs of concentrations at 300-1000 μg g(-1) soil significantly lowered activities of most enzymes and microbial biomass. It is noteworthy that the SWCNTs showed similar effects to that of multi-walled carbon nanotubes (MWCNTs), but at a concentration approximately 5 times lower; we suggest that this is mainly due to the higher surface area of SWCNTs than that of MWCNTs. Indeed, our results show that surface area of CNTs has significant negative relationship with relative enzyme activity and biomass, which suggests that greater microorganism-CNT interactions could increase the negative effect of CNTs on microorganisms. Current work may contribute to the preparation of a regulatory guideline for the release of CNTs to the soil environment. Copyright © 2012 Elsevier Inc. All rights reserved.
Koch, James L.; Nevins, Donald J.
1989-01-01
Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces β-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the
Brecht, Jeffrey K.; Huber, Donald J.
1988-01-01
Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO2 and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit. PMID:16666417
Anderson, F. B.; Millbank, J. W.
1966-01-01
1. The digestive juice of the snail Helix pomatia was used in the study of the degradation of isolated cell-wall preparations from a strain of Saccharomyces carlsbergensis. 2. The crude enzyme system was fractionated by gel filtration and the activities of the more specific fractions thus obtained were examined. 3. Results are discussed with respect to (a) the nature of various factors that are essential for protoplast formation and cell-wall dissolution and (b) structures envisaged in yeast cell walls that are responsible for the observed variations in susceptibility to attack by snail juice. PMID:5964965
NASA Astrophysics Data System (ADS)
Watanabe, Toshio; Yamada, Yohei; Motonaka, Junko; Yabutani, Tomoki; Sakuraba, Haruhiko; Yasuzawa, Mikito
In this study, electrodeposition of thermostable enzyme Bacillus subtilis CotA, which is a laccase and has a bilirubin oxidase (BOD) activity, was investigated. The electrodeposition was operated in a mixture of Bacillus subtilis CotA in the PBS (pH 8.0) and TritonX-100 under applying potential (1100 mV vs. Ag/AgCl for 5 min.). The current response was measured by linear sweep voltammetry technique (LSV). The thermostable enzyme Bacillus subtilis CotA electrodeposited electrode was compared with a mesophile BOD electrodeposited electrode. As a result, the Bacillus subtilis CotA modified electrode showed better sensitivity and long-term stability than the mesophile BOD modified electrode.
Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino
2015-01-01
Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.
Benjdia, Alhosna; Balty, Clémence; Berteau, Olivier
2017-01-01
Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota.
dbHiMo: a web-based epigenomics platform for histone-modifying enzymes
Choi, Jaeyoung; Kim, Ki-Tae; Huh, Aram; Kwon, Seomun; Hong, Changyoung; Asiegbu, Fred O.; Jeon, Junhyun; Lee, Yong-Hwan
2015-01-01
Over the past two decades, epigenetics has evolved into a key concept for understanding regulation of gene expression. Among many epigenetic mechanisms, covalent modifications such as acetylation and methylation of lysine residues on core histones emerged as a major mechanism in epigenetic regulation. Here, we present the database for histone-modifying enzymes (dbHiMo; http://hme.riceblast.snu.ac.kr/) aimed at facilitating functional and comparative analysis of histone-modifying enzymes (HMEs). HMEs were identified by applying a search pipeline built upon profile hidden Markov model (HMM) to proteomes. The database incorporates 11 576 HMEs identified from 603 proteomes including 483 fungal, 32 plants and 51 metazoan species. The dbHiMo provides users with web-based personalized data browsing and analysis tools, supporting comparative and evolutionary genomics. With comprehensive data entries and associated web-based tools, our database will be a valuable resource for future epigenetics/epigenomics studies. Database URL: http://hme.riceblast.snu.ac.kr/ PMID:26055100
dbHiMo: a web-based epigenomics platform for histone-modifying enzymes.
Choi, Jaeyoung; Kim, Ki-Tae; Huh, Aram; Kwon, Seomun; Hong, Changyoung; Asiegbu, Fred O; Jeon, Junhyun; Lee, Yong-Hwan
2015-01-01
Over the past two decades, epigenetics has evolved into a key concept for understanding regulation of gene expression. Among many epigenetic mechanisms, covalent modifications such as acetylation and methylation of lysine residues on core histones emerged as a major mechanism in epigenetic regulation. Here, we present the database for histone-modifying enzymes (dbHiMo; http://hme.riceblast.snu.ac.kr/) aimed at facilitating functional and comparative analysis of histone-modifying enzymes (HMEs). HMEs were identified by applying a search pipeline built upon profile hidden Markov model (HMM) to proteomes. The database incorporates 11,576 HMEs identified from 603 proteomes including 483 fungal, 32 plants and 51 metazoan species. The dbHiMo provides users with web-based personalized data browsing and analysis tools, supporting comparative and evolutionary genomics. With comprehensive data entries and associated web-based tools, our database will be a valuable resource for future epigenetics/epigenomics studies. © The Author(s) 2015. Published by Oxford University Press.
Benjdia, Alhosna; Balty, Clémence; Berteau, Olivier
2017-01-01
Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota. PMID:29167789
Chateigner-Boutin, Anne-Laure; Suliman, Muhtadi; Bouchet, Brigitte; Alvarado, Camille; Lollier, Virginie; Rogniaux, Hélène; Guillon, Fabienne; Larré, Colette
2015-01-01
Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called ‘the bran’ is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel). PMID:25769308
NASA Astrophysics Data System (ADS)
Shinohara, Naoki; Sunagawa, Naoki; Tamura, Satoru; Yokoyama, Ryusuke; Ueda, Minoru; Igarashi, Kiyohiko; Nishitani, Kazuhiko
2017-04-01
Cellulose is an economically important material, but routes of its industrial processing have not been fully explored. The plant cell wall - the major source of cellulose - harbours enzymes of the xyloglucan endotransglucosylase/hydrolase (XTH) family. This class of enzymes is unique in that it is capable of elongating polysaccharide chains without the requirement for activated nucleotide sugars (e.g., UDP-glucose) and in seamlessly splitting and reconnecting chains of xyloglucan, a naturally occurring soluble analogue of cellulose. Here, we show that a recombinant version of AtXTH3, a thus far uncharacterized member of the Arabidopsis XTH family, catalysed the transglycosylation between cellulose and cello-oligosaccharide, between cellulose and xyloglucan-oligosaccharide, and between xyloglucan and xyloglucan-oligosaccharide, with the highest reaction rate observed for the latter reaction. In addition, this enzyme formed cellulose-like insoluble material from a soluble cello-oligosaccharide in the absence of additional substrates. This newly found activity (designated “cellulose endotransglucosylase,” or CET) can potentially be involved in the formation of covalent linkages between cellulose microfibrils in the plant cell wall. It can also comprise a new route of industrial cellulose functionalization.
NASA Astrophysics Data System (ADS)
Wati, Elvis; Meukam, Pierre; Damfeu, Jean Claude
2017-12-01
Uninsulated concrete block walls commonly found in tropical region have to be retrofitted to save energy. The thickness of insulation layer used can be reduced with the help of modified laterite based bricks layer (with the considerably lower thermal conductivity than that of concrete block layer) during the retrofit building fabrics. The aim of this study is to determine the optimum location and distribution of different materials. The investigation is carried out under steady periodic conditions under the climatic conditions of Garoua in Cameroon using a Simulink model constructed from H-Tools (the library of Simulink models). Results showed that for the continuous air-conditioned space, the best wall configuration from the maximum time lag, minimum decrement factor and peak cooling transmission load perspective, is dividing the insulation layer into two layers and placing one at the exterior surface and the other layer between the two different massive layers with the modified laterite based bricks layer at the interior surface. For intermittent cooling space, the best wall configuration from the minimum energy consumption depends on total insulation thickness. For the total insulation thickness less than 8 cm approximately, the best wall configuration is placing the half layer of insulation material at the interior surface and the other half between the two different massive layers with the modified earthen material at the exterior surface. Results also showed that, the optimum insulation thickness calculated from the yearly cooling transmission (estimated only during the occupied period) and some economic considerations slightly depends on the location of that insulation.
Roles of tRNA in cell wall biosynthesis
Dare, Kiley; Ibba, Michael
2013-01-01
Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. PMID:22262511
Ramos, Araceli M; Gally, Marcela; Szapiro, Gala; Itzcovich, Tatiana; Carabajal, Maira; Levin, Laura
Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.
Budnik, Lygia T; Scheer, Edwin; Burge, P Sherwood; Baur, Xaver
2017-01-01
The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays. Published by the
Dean, R A; Timberlake, W E
1989-01-01
The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity. PMID:2535501
Corcoran, Callan C.; Grady, Cameron R.; Pisitkun, Trairak; Parulekar, Jaya
2017-01-01
The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. PMID:27974320
Mulchandani, A; Bassi, A S
1996-01-01
Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.
Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility
2014-01-01
Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274
DOE Office of Scientific and Technical Information (OSTI.GOV)
Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.
2009-08-28
The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal endmore » of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.« less
Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.
Okubo-Kurihara, Emiko; Matsui, Minami
2018-01-01
The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.
Corcoran, Callan C; Grady, Cameron R; Pisitkun, Trairak; Parulekar, Jaya; Knepper, Mark A
2017-03-01
The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1,647 genes to these pathways, representing ~7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database ( Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct. Copyright © 2017 the American Physiological Society.
Pérez-Donoso, Alonso G.; Sun, Qiang; Roper, M. Caroline; Greve, L. Carl; Kirkpatrick, Bruce; Labavitch, John M.
2010-01-01
The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- β -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease. PMID:20107028
Enzyme release of phenolics from muscadine grape (Vitis rotundifolia Michx.) skins and seeds.
Xu, Changmou; Yagiz, Yavuz; Borejsza-Wysocki, Wlodzimierz; Lu, Jiang; Gu, Liwei; Ramírez-Rodrigues, Milena M; Marshall, Maurice R
2014-08-15
Enzyme degradation of plant cell wall polysaccharides can potentially enhance the release of bioactive phenolics. The aim of this study was to evaluate various combinations of solvent and enzyme, enzyme type (cellulase, pectinase, ß-glucosidase), and hydrolysis time (1, 4, 8, 24 h) on the release of muscadine grape skin and seed phenolics, and their antioxidant activities. Results showed that pre-treated muscadine skins and seeds with enzymes decreased total phenolic yield compared with solvent (50% ethanol) alone. Enzyme release of phenolics from skins of different muscadine varieties was significantly different while release from seeds was similar. Enzyme hydrolysis was found to shorten extraction time. Most importantly, enzyme hydrolysis modified the galloylated form of polyphenols to low molecular weight phenolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity. Copyright © 2014 Elsevier Ltd. All rights reserved.
Enzyme-modified nanoporous gold-based electrochemical biosensors.
Qiu, Huajun; Xue, Luyan; Ji, Guanglei; Zhou, Guiping; Huang, Xirong; Qu, Yinbo; Gao, Peiji
2009-06-15
On the basis of the unique physical and chemical properties of nanoporous gold (NPG), which was obtained simply by dealloying Ag from Au/Ag alloy, an attempt was made in the present study to develop NPG-based electrochemical biosensors. The NPG-modified glassy carbon electrode (NPG/GCE) exhibited high-electrocatalytic activity toward the oxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)), which resulted in a remarkable decrease in the overpotential of NADH and H(2)O(2) electro-oxidation when compared with the gold sheet electrode. The high density of edge-plane-like defective sites and large specific surface area of NPG should be responsible for the electrocatalytic behavior. Such electrocatalytic behavior of the NPG/GCE permitted effective low-potential amperometric biosensing of ethanol or glucose via the incorporation of alcohol dehydrogenase (ADH) or glucose oxidase (GOD) within the three-dimensional matrix of NPG. The ADH- and GOD-modified NPG-based biosensors showed good analytical performance for biosensing ethanol and glucose due to the clean, reproducible and uniformly distributed microstructure of NPG. The stabilization effect of NPG on the incorporated enzymes also made the constructed biosensors very stable. After 1 month storage at 4 degrees C, the ADH- and GOD-based biosensors lost only 5.0% and 4.2% of the original current response. All these indicated that NPG was a promising electrode material for biosensors construction.
2011-01-01
One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500 μA cm−2 at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning. PMID:21417322
Tasca, Federico; Harreither, Wolfgang; Ludwig, Roland; Gooding, John Justin; Gorton, Lo
2011-04-15
One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500 μA cm(-2) at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning. © 2011 American Chemical Society
Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression
Chaffin, W. Lajean; López-Ribot, José Luis; Casanova, Manuel; Gozalbo, Daniel; Martínez, José P.
1998-01-01
The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis. PMID:9529890
Shivakumara, Tagginahalli N; Chaudhary, Sonam; Kamaraju, Divya; Dutta, Tushar K; Papolu, Pradeep K; Banakar, Prakash; Sreevathsa, Rohini; Singh, Bhupinder; Manjaiah, K M; Rao, Uma
2017-01-01
The complex parasitic strategy of Meloidogyne incognita appears to involve simultaneous expression of its pharyngeal gland-specific effector genes in order to colonize the host plants. Research reports related to effector crosstalk in phytonematodes for successful parasitism of the host tissue is yet underexplored. In view of this, we have used in planta effector screening approach to understand the possible interaction of pioneer genes ( msp-18 and msp-20 , putatively involved in late and early stage of M. incognita parasitism, respectively) with other unrelated effectors such as cell-wall modifying enzymes (CWMEs) in M. incognita . Host-induced gene silencing (HIGS) strategy was used to generate the transgenic eggplants expressing msp-18 and msp-20 , independently. Putative transformants were characterized via qRT-PCR and Southern hybridization assay. SiRNAs specific to msp-18 and msp - 20 were also detected in the transformants via Northern hybridization assay. Transgenic expression of the RNAi constructs of msp-18 and msp-20 genes resulted in 43.64-69.68% and 41.74-67.30% reduction in M. incognita multiplication encompassing 6 and 10 events, respectively. Additionally, transcriptional oscillation of CWMEs documented in the penetrating and developing nematodes suggested the possible interaction among CWMEs and pioneer genes. The rapid assimilation of plant-derived carbon by invading nematodes was also demonstrated using 14 C isotope probing approach. Our data suggests that HIGS of msp-18 and msp-20 , improves nematode resistance in eggplant by affecting the steady-state transcription level of CWME genes in invading nematodes, and safeguard the plant against nematode invasion at very early stage because nematodes may become the recipient of bioactive RNA species during the process of penetration into the plant root.
Lin, Sansan; Fischl, Anthony S; Bi, Xiahui; Parce, Wally
2003-03-01
Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.
NASA Astrophysics Data System (ADS)
Kim, Byoung Chan; Lee, Inseon; Kwon, Seok-Joon; Wee, Youngho; Kwon, Ki Young; Jeon, Chulmin; An, Hyo Jin; Jung, Hee-Tae; Ha, Su; Dordick, Jonathan S.; Kim, Jungbae
2017-01-01
CNTs need to be dispersed in aqueous solution for their successful use, and most methods to disperse CNTs rely on tedious and time-consuming acid-based oxidation. Here, we report the simple dispersion of intact multi-walled carbon nanotubes (CNTs) by adding them directly into an aqueous solution of glucose oxidase (GOx), resulting in simultaneous CNT dispersion and facile enzyme immobilization through sequential enzyme adsorption, precipitation, and crosslinking (EAPC). The EAPC achieved high enzyme loading and stability because of crosslinked enzyme coatings on intact CNTs, while obviating the chemical pretreatment that can seriously damage the electron conductivity of CNTs. EAPC-driven GOx activity was 4.5- and 11-times higher than those of covalently-attached GOx (CA) on acid-treated CNTs and simply-adsorbed GOx (ADS) on intact CNTs, respectively. EAPC showed no decrease of GOx activity for 270 days. EAPC was employed to prepare the enzyme anodes for biofuel cells, and the EAPC anode produced 7.5-times higher power output than the CA anode. Even with a higher amount of bound non-conductive enzymes, the EAPC anode showed 1.7-fold higher electron transfer rate than the CA anode. The EAPC on intact CNTs can improve enzyme loading and stability with key routes of improved electron transfer in various biosensing and bioelectronics devices.
Nutrient depletion modifies cell wall adsorption activity of wine yeast.
Sidari, R; Caridi, A
2016-06-01
Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions-early, mid, 'adjusted' for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in 'adjusted' for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters.
Bindon, Keren A; Li, Sijing; Kassara, Stella; Smith, Paul A
2016-11-09
For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.
Dai, Hong; Chi, Yuwu; Wu, Xiaoping; Wang, Youmei; Wei, Mingdeng; Chen, Guonan
2010-02-15
A new biocompatible ECL biosensor based on enzyme/titanate nanotubes/chitosan composite film was developed for the determination of analytes in biological samples. In the fabrication of the new ECL biosensor, biocompatible titanate nanotubes (TNTs) and a model enzyme, i.e., choline oxidase (ChOX), were immobilized on a chitosan modified glassy carbon electrode (GCE) via electrostatic adsorption and covalent interaction, respectively. By this ECL biosensor, choline was enzymatically oxidized to hydrogen peroxide and detected by a sensitive luminol ECL system. The use of TNTs not only provided a biocompatible microenvironment for the immobilized enzyme, which resulted in an excellent stability and long lifetime of the ECL biosensor, but also exhibited great enhancement towards luminol ECL and thus led to a significant improvement in sensitivity of ECL biosensor. Satisfactory results were obtained when employing this biosensor in assaying the total choline in milk samples. The work would provide a common platform to develop various sensitive, selective and biocompatible ECL biosensors based on using enzyme/TNTs/CHIT composite films. Copyright 2009 Elsevier B.V. All rights reserved.
Genetic engineering of grass cell wall polysaccharides for biorefining.
Bhatia, Rakesh; Gallagher, Joe A; Gomez, Leonardo D; Bosch, Maurice
2017-09-01
Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass-derived lignocellulose processing industry. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Zhang, Susu; He, Ping; Zhang, Guangli; Lei, Wen; He, Huichao
2015-01-01
Graphite nanosheets prepared by thermal expansion and successive sonication were utilized for the construction of a multi-walled carbon nanotubes/graphite nanosheets based amperometric sensing platform to simultaneously determine acetaminophen and dopamine in the presence of ascorbic acid in physiological conditions. The synergistic effect of multi-walled carbon nanotubes and graphite nanosheets catalyzed the electrooxidation of acetaminophen and dopamine, leading to a remarkable potential difference up to 200 mV. The as-prepared modified electrode exhibited linear responses to acetaminophen and dopamine in the concentration ranges of 2.0 × 10(-6) - 2.4 × 10(-4) M (R = 0.999) and 2.0 × 10(-6) - 2.0 × 10(-4) M (R = 0.998), respectively. The detection limits were down to 2.3 × 10(-7) M for acetaminophen and 3.5 × 10(-7) M for dopamine (S/N = 3). Based on the simple preparation and prominent electrochemical properties, the obtained multi-walled carbon nanotubes/graphite nanosheets modified electrode would be a good candidate for the determination of acetaminophen and dopamine without the interference of ascorbic acid.
Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael
2016-01-01
ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a
Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth
Mollet, Jean-Claude; Leroux, Christelle; Dardelle, Flavien; Lehner, Arnaud
2013-01-01
The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed. PMID:27137369
Poonsrisawat, Aphisit; Paemanee, Atchara; Wanlapatit, Sittichoke; Piyachomkwan, Kuakoon; Eurwilaichitr, Lily; Champreda, Verawat
2017-10-01
In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from Aspergillus aculeatus BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45 Pa s with the enzyme dosage of 2.19 mg/g on a dried weight basis within the first 2 h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4 mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96 h at 40 °C. Simultaneous saccharification and fermentation of cassava pulp by Saccharomyces cerevisiae with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.
Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4
NASA Astrophysics Data System (ADS)
El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt
2016-02-01
We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.
Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4
El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt
2016-01-01
We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743
NASA Astrophysics Data System (ADS)
Dong, Qi; Wan, Guoshun; Xu, Yongzheng; Guo, Yunli; Du, Tianxiang; Yi, Xiaosu; Jia, Yuxi
2017-12-01
The numerical model of carbon fiber reinforced polymer (CFRP) laminates with electrically modified interlayers subjected to lightning strike is constructed through finite element simulation, in which both intra-laminar and inter-laminar lightning damages are considered by means of coupled electrical-thermal-pyrolytic analysis method. Then the lightning damage extents including the damage volume and maximum damage depth are investigated. The results reveal that the simulated lightning damages could be qualitatively compared to the experimental counterparts of CFRP laminates with interlayers modified by nickel-coated multi-walled carbon nanotubes (Ni-MWCNTs). With higher electrical conductivity of modified interlayer and more amount of modified interlayers, both damage volume and maximum damage depth are reduced. This work provides an effective guidance to the anti-lightning optimization of CFRP laminates.
Autolysis and extension of isolated walls from growing cucumber hypocotyls
NASA Technical Reports Server (NTRS)
Cosgrove, D. J.; Durachko, D. M.
1994-01-01
Walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, we treated the walls in various ways to enhance or inhibit long-term wall extension ('creep') and measured autolysis as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, we found no correlation between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. We also found that exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). Our results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.
Berg Miller, Margret E.; Antonopoulos, Dionysios A.; Rincon, Marco T.; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M.; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J.; Bayer, Edward A.; White, Bryan A.
2009-01-01
Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. Methodology/Principal Findings The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. Conclusions/Significance The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has
Berg Miller, Margret E; Antonopoulos, Dionysios A; Rincon, Marco T; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J; Bayer, Edward A; White, Bryan A
2009-08-14
Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes
Use of yeast spores for microencapsulation of enzymes.
Shi, Libing; Li, Zijie; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki
2014-08-01
Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.
NASA Astrophysics Data System (ADS)
Özcan, Selçuk; Açıkbaş, Gökhan; Çalış Açıkbaş, Nurcan
2018-04-01
Hydrophobic surfaces are also known to have antimicrobial effect by restricting the adherence of microorganisms. However, ceramic products are produced by high temperature processes resulting in a hydrophilic surface. In this study, an industrial ceramic wall tile glaze composition was modified by the inclusion of metallic zinc powder in the glaze suspension applied on the pre-sintered wall tile bodies by spraying. The glazed tiles were gloss fired at industrially applicable peak temperatures ranging from 980 °C to 1100 °C. The fired tile surfaces were coated with a commercial fluoropolymer avoiding water absorption. The surfaces were characterized with SEM, EDS, XRD techniques, roughness, sessile water drop contact angle, surface energy measurements, and standard antimicrobial tests. The surface hydrophobicity and the antimicrobial activity results were compared with that of unmodified, uncoated gloss fired wall tiles. A superhydrophobic contact angle of 150° was achieved at 1000 °C peak temperature due to the formation of micro-structured nanocrystalline zinc oxide granules providing a specific surface topography. At higher peak temperatures the hydrophobicity was lost as the specific granular surface topography deteriorated with the conversion of zinc oxide granules to the ubiquitous willemite crystals embedded in the glassy matrix. The antimicrobial efficacy also correlated with the hydrophobic character.
Grenzebach, Ulrike H; Schnorbus, Ulrike; Büchner, Thomas; Busse, Holger; Stoll, Wolfgang
2003-05-01
Permanent visual damage due to an increase in volume of the orbital contents may be the result of the failure of conservative therapeutic concepts in the treatment of endocrine orbitopathy. Considerable progress has been achieved in developing successful orbital decompression techniques with regard to functional and cosmetic outcome. Decompression techniques with resection of the bony orbital walls are adequate tools in restoring visual acuity and reducing exophthalmus. A considerable degree of deterioration of motility disorders has been described in the literature depending on the techniques being used. The purpose of this study was to investigate whether a modified technique of 3-wall orbital decompression with preservation of a medial part of the periorbital tissue to support the medial rectus muscle, is able to reduce the postoperative risk of diplopia. A modified technique of orbital 3-wall decompression with resection of the medial orbital wall, the medial orbital floor and the floor of the frontal sinus has been used in patients with compressive optic neuropathy (n = 20) and for cosmetic reasons (n = 7) in cases of uni- or bilateral proptosis. Analysis of the results was performed concerning visual outcome, exophthalmus reduction and development of horizontal and vertical motility changes. In all cases of optic neuropathy improvement of visual function at an average of 4.63 +/- 4.5 lines could be achieved. Exophthalmus reduction was 3.2 +/- 2.4 mm in the functional group and 3.9 +/- 1.7 mm in the rehabilitative group. In this group motility of the medial rectus muscle remained unaffected except in one eye. In the functional group motility deterioration was observed in 62 %. The modified 3-wall decompression technique with preservation of a medial periorbital tissue strip is an adequate alternative technique in the therapy of optic neuropathy and exophthalmus reduction in endocrine orbitopathy with a low risk of postoperative motility disorders.
Salazar-Iribe, Alexis; Zúñiga-Sánchez, Esther; Mejía, Emma Zavaleta; Gamboa-deBuen, Alicia
2017-01-01
The root-knot nematode Meloidogyne incognita infects a variety of plants, including Arabidopsis thaliana. During migration, root-knot nematodes secrete different proteins to modify cell walls, which include pectolytic enzymes. However, the contribution of host cell wall proteins has not been described during this process. The function of two DUF642 cell wall proteins, BIIDXI (BDX, At4g32460) and TEEBE (TEB, At2g41800), in plant development could be related to the regulation of pectin methyl esterification status in the cell walls of different tissues. Accordingly, the expression of these two genes is up-regulated by auxin. BDX and TEB were highly induced during early M. incognita inoculation. Moreover, cell wall localization of the proteins was also induced. The cell wall localization of BDX and TEB DUF642 proteins during M. incognita early inoculation suggested that these two proteins could be involved in the regulation of the degree of pectin methylation during cell separation. PMID:29238286
Wang, Youdan; Joshi, Pratixa P; Hobbs, Kevin L; Johnson, Matthew B; Schmidtke, David W
2006-11-07
In this study, we describe the construction of glucose biosensors based on an electrostatic layer-by-layer (LBL) technique. Gold electrodes were initially functionalized with negatively charged 11-mercaptoundecanoic acid followed by alternate immersion in solutions of a positively charged redox polymer, poly[(vinylpyridine)Os(bipyridyl)2Cl(2+/3+)], and a negatively charged enzyme, glucose oxidase (GOX), or a GOX solution containing single-walled carbon nanotubes (SWNTs). The LBL assembly of the multilayer films were characterized by UV-vis spectroscopy, ellipsometry, and cyclic voltammetry, while characterization of the single-walled nanotubes was performed with transmission electron microscopy, Raman spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. When the GOX solution contained single-walled carbon nanotubes (GOX-SWNTs), the oxidation peak currents during cyclic voltammetry increased 1.4-4.0 times, as compared to films without SWNTs. Similarly the glucose electro-oxidation current also increased (6-17 times) when SWNTs were present. By varying the number of multilayers, the sensitivity of the sensors could be controlled.
Cao, Jianglin; Wang, Ying; Chen, Chunyang; Yu, Fei; Ma, Jie
2018-05-15
Capacitive deionization (CDI) is a technology used to remove salt from brackish water, and it is an energy-saving, low-cost method compared with other methods, such as reverse osmosis, multi-stage ash distillation and electrodialysis. In this paper, three-dimensional (3D) graphene hydrogels modified with single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs) were synthesized by a one-step water bath method to increase the conductivity of materials and reduce the aggregation of the graphene sheets. The CDI performance differences between the two materials were compared and discussed. The results suggested that SWCNTs/rGO had a higher electrosorption capacity (48.73 mg/g) than MWCNTs/rGO, and this was attributed to its high specific surface area (308.37 m 2 /g), specific capacity (36.35 F/g), and smaller charge transfer resistance compared with those of the MWCNTs/rGO electrode. The results indicate SWCNTs/rGO is a promising and suitable material for CDI technology and we provide basic guidance for further CNTs/graphene composite research. Copyright © 2018 Elsevier Inc. All rights reserved.
Yang, Jiawei; Cheng, Shaoan; Sun, Yi; Li, Chaochao
2017-10-01
To increase the power generation of microbial fuel cells (MFCs), anode modification with carbon materials (activated carbon, carbon nanotubes, and carbon nanohorns) was investigated. Maximum power densities of a stainless-steel anode MFC with a non-modified electrode (SS-MFC), an activated carbon-modified electrode (AC-MFC), a carbon nanotube-modified electrode (CNT-MFC) and a carbon nanohorn-modified electrode (CNH-MFC) were 72, 244, 261 and 327 mW m -2 , respectively. The total polarization resistance measured by electrochemical impedance spectroscopy were 3610 Ω for SS-MFC, 283 Ω for AC-MFC, 231 Ω for CNTs-MFC, and 136 Ω for CNHs-MFC, consistent with the anode resistances obtained by fitting the anode polarization curves. Single-wall carbon nanohorns are better than activated carbon and carbon nanotubes as a new anode modification material for improving anode performance.
Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad
2014-01-01
Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.
Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad
2014-01-01
Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes. PMID:25763058
Disruption of cell walls for enhanced lipid recovery
Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose
2015-03-24
Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.
Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping
2013-01-01
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174
Norman, C; Vidal, S; Palva, E T
1999-07-01
Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.
Zhang, Wei-Ya; Sun, Yin; Wang, Cheng-Ming; Wu, Cai-Ying
2011-09-01
Based on polyethylene glycol modified single-walled carbon nanotubes, a novel sol-gel fiber coating was prepared and applied to the headspace microextraction of chlorinated organic carriers (COCs) in textiles by gas chromatography-electron capture detection. The preparation of polyethylene glycol modified single-walled carbon nanotubes and the sol-gel fiber coating process was stated and confirmed by infrared spectra, Raman spectroscopy, and scanning electron microscopy. Several parameters affecting headspace microextraction, including extraction temperature, extraction time, salting-out effect, and desorption time, were optimized by detecting 11 COCs in simulative sweat samples. Compared with the commercial solid-phase microextraction fibers, the sol-gel polyethylene glycol modified single-walled carbon nanotubes fiber showed higher extraction efficiency, better thermal stability, and longer life span. The method detection limits for COCs were in the range from 0.02 to 7.5 ng L(-1) (S/N = 3). The linearity of the developed method varied from 0.001 to 50 μg L(-1) for all analytes, with coefficients of correlation greater than 0.974. The developed method was successfully applied to the analysis of trace COCs in textiles, the recoveries of the analytes indicated that the developed method was considerably useful for the determination of COCs in ecological textile samples.
Vanholme, Bartel; Vanholme, Ruben; Turumtay, Halbay; Goeminne, Geert; Cesarino, Igor; Goubet, Florence; Morreel, Kris; Rencoret, Jorge; Bulone, Vincent; Hooijmaijers, Cortwa; De Rycke, Riet; Gheysen, Godelieve; Ralph, John; De Block, Marc; Meulewaeter, Frank; Boerjan, Wout
2014-01-01
To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane. PMID:24664205
Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.
2013-01-01
The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. PMID:23686272
Singh, Padmanabh; Konar, Arpita; Kumar, Ashish; Srivas, Sweta; Thakur, Mahendra K
2015-08-01
The amnesic potential of scopolamine is well manifested through synaptic plasticity gene expression changes and behavioral paradigms of memory impairment. However, the underlying mechanism remains obscure and consequently ideal therapeutic target is lacking. In this context, chromatin-modifying enzymes, which regulate memory gene expression changes, deserve major attention. Therefore, we analyzed the expression of chromatin-modifying enzymes and recovery potential of enzyme modulators in scopolamine-induced amnesia. Scopolamine administration drastically up-regulated DNA methyltransferases (DNMT1) and HDAC2 expression while CREB-binding protein (CBP), DNMT3a and DNMT3b remained unaffected. HDAC inhibitor sodium butyrate and DNMT inhibitor Aza-2'deoxycytidine recovered scopolamine-impaired hippocampal-dependent memory consolidation with concomitant increase in the expression of synaptic plasticity genes Brain-derived neurotrophic factor (BDNF) and Arc and level of histone H3K9 and H3K14 acetylation and decrease in DNA methylation level. Sodium butyrate showed more pronounced effect than Aza-2'deoxycytidine and their co-administration did not exhibit synergistic effect on gene expression. Taken together, we showed for the first time that scopolamine-induced up-regulation of chromatin-modifying enzymes, HDAC2 and DNMT1, leads to gene expression changes and consequent decline in memory consolidation. Our findings on the action of scopolamine as an epigenetic modulator can pave a path for ideal therapeutic targets. We propose the following putative pathway for scopolamine-mediated memory impairment; scopolamine up-regulates hippocampal DNMT1 and HDAC2 expression, induces methylation and deacetylation of BDNF and Arc promoter, represses gene expression and eventually impairs memory consolidation. On the other hand, Aza-2 and NaB inhibit DNMT1 and HDAC2 respectively, up-regulate BDNF and Arc expression and recover memory consolidation. We elucidate the action of
NASA Astrophysics Data System (ADS)
Furutaka, Hajime; Nemoto, Kentaro; Inoue, Yuki; Hidaka, Hiroki; Muguruma, Hitoshi; Inoue, Hitoshi; Ohsawa, Tatsuya
2016-05-01
An amperometric biosensor based on a glassy carbon electrode modified with long-length multiwalled carbon nanotubes (MWCNTs) and enzyme nicotinamide-adenine-dinucleotide-dependent glucose dehydrogenase (GDH) is presented. We demonstrate the effect of the MWCNT length on the amperometric response of the enzyme biosensor. The long length of MWCNT is 200 µm (average), whereas the normal length of MWCNT is 1 µm (average). The response of the long MWCNT-GDH electrode is 2 times more sensitive than that of the normal-length MWCNT-GDH electrode in the concentration range from 0.25-35 mM. The result of electrochemical impedance spectroscopy measurements suggest that the long-length MWCNT-GDH electrode formed a better electron transfer network than the normal-length one.
Anderson, Nickolas A.; Tobimatsu, Yuki; Ciesielski, Peter N.; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S.; Ladisch, Michael; Chapple, Clint
2015-01-01
Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. PMID:26265762
Anderson, Nickolas A; Tobimatsu, Yuki; Ciesielski, Peter N; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint
2015-08-01
Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. © 2015 American Society of Plant Biologists. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ullah, Sami, E-mail: samichemist1@gmail.com, E-mail: azmibustam@petronas.com.my, E-mail: azmish@petronas.com.my, E-mail: lkhlfh@gmail.com, E-mail: hmurshid@gmail.com, E-mail: nadiariazz@gmail.com; Bustam, M. A., E-mail: samichemist1@gmail.com, E-mail: azmibustam@petronas.com.my, E-mail: azmish@petronas.com.my, E-mail: lkhlfh@gmail.com, E-mail: hmurshid@gmail.com, E-mail: nadiariazz@gmail.com; Shariff, A. M., E-mail: samichemist1@gmail.com, E-mail: azmibustam@petronas.com.my, E-mail: azmish@petronas.com.my, E-mail: lkhlfh@gmail.com, E-mail: hmurshid@gmail.com, E-mail: nadiariazz@gmail.com
2014-10-24
MOF-5 was synthesized by solvothermal method and its reactivation under anhydrous conditions. This research is conducted to investigate the effect of MOF-5 and MOF-5 modified with multi-wall carbon nanotubes (MWCNTs) and expandable graphite (EG) on the performance of CO{sub 2} adsorption. The synthesized MOFs were characterized using Field emission scanning electron microscopy (FESEM) for surface morphology, Thermogravimetric analysis (TGA) for thermal stability, X-ray diffraction (XRD) for crystals plane, Brunauer-Emmet-Teller (BET) for surface area and CO{sub 2} adsorption. The result had showed that the modified MOF-5 enhanced the CO{sub 2} adsorption compared to the pure MOF-5. The increment in the CO{submore » 2} uptake capacities of MOF materials was attributed to the decrease in the pore size and enhancement of micropore volume of MOF-5 by multi-walled carbon nanotube and EG incorporation. The BET surface area of the synthesized MOF-5@MWCNTs is more than MOF-5. The CO{sub 2} sorption capacities of MOF-5 and MOF-5@MWCNTs were observed to increase from 0.00008 to 0.00048 mol g-1 at 298 K and 1 bar. The modified MOF-5@MWCNTs resulted in the highest CO{sub 2} adsorption followed by the modified MOF-5@ EG and lastly, MOF-5.« less
Hong, Xiao-ping; Zhu, Yan; Zhang, Yan-zhen
2012-01-01
A highly sensitive amperometric sulfadiazine sensor fabricated by electrochemical deposition of poly(cobalt tetraaminophthalocyanine) (poly(CoIITAPc)) on the surface of a multi-walled carbon nanotubes-Nafion (MWCNTs-Nafion) modified electrode is described. This electrode showed a very attractive performance by combining the advantages of CoIITAPc, MWCNTs, and Nafion. Compared with the bare glassy carbon electrode (GCE) and the MWCNTs-Nafion modified electrode, the electrocatalytic activity of poly(CoIITAPc)-coated MWCNTs-Nafion GCE generated greatly improved electrochemical detections toward sulfadiazine including low oxidation potential, high current responses, and good anti-fouling performance. The oxidation peak currents of sulfadiazine obtained on the new modified electrode increased linearly while increasing the concentration of sulfadiazine from 0.5 to 43.5 μmol/L with the detection limit of 0.17 μmol/L. PMID:22661213
NASA Astrophysics Data System (ADS)
Li, Juan; Li, Minjie; Tang, Jieli; Li, Xiaozhou; Zhang, Hanqi; Zhang, Yihua
2008-08-01
This paper described a novel assay of enzyme based on the measurement of enhanced resonance light-scattering (RLS) signals resulting from the electrostatic and coordination interaction of functionalized CdTe nanoparticles with enzyme. The CdTe nanoparticles which were modified with 3-mercaptocarboxylic acid (MPA) have abundant carboxylic groups ( sbnd COOH). So the nanoparticles are water-soluble, stable and biocompatible. At pH 8.3 phosphate buffered saline (PBS), the RLS signals of functionalized nano-CdTe are greatly enhanced by bromelain and papain in the region of 220-800 nm characterized by the peak around 318-314 nm, respectively. The optimization conditions of the reaction were also examined and selected. Under the selected conditions, the enhanced RLS intensity is linearly proportional to the concentration of bromelain and papain. The liner range is (0.09-0.9) × 10 -6 mol/L for bromelain and (0.048-0.702) × 10 -6 mol/L for papain. The influences of some foreign substances were also examined. This method can be applied to the determination of enzyme.
Falk, Shaun P; Weisblum, Bernard
2013-03-01
Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chen, Gang; Xu, Xuejiao; Lin, Yuehe
2007-07-27
A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was 27.4° compared with 66.3° for the pure PMMA. In addition, the electro-osmoticmore » flow increased from 2.13×10-4 cm2 V-1 s-1 for the native-PMMA channel to 4.86×10-4 cm2 V-1 s-1 for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74 882.3 m-1 compared with 14 730.5 m-1 for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.« less
Lanzi, Cinzia; Zaffaroni, Nadia; Cassinelli, Giuliana
2017-01-01
Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
How do plant cell walls extend?
NASA Technical Reports Server (NTRS)
Cosgrove, D. J.
1993-01-01
This article briefly summarizes recent work that identifies the biophysical and biochemical processes that give rise to the extension of plant cell walls. I begin with the biophysical notion of stress relaxation of the wall and follow with recent studies of wall enzymes thought to catalyze wall extension and relaxation. Readers should refer to detailed reviews for more comprehensive discussion of earlier literature (Taiz, 1984; Carpita and Gibeaut, 1993; Cosgrove, 1993).
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Bo; Wei, Xian-ding; Zhao, Yu-tian
2014-07-01
To investigate the dosimetric characteristics of irradiation of the chest wall and supraclavicular region as an integrated volume with intensity-modulated radiation therapy (IMRT) after modified radical mastectomy. This study included 246 patients who received modified radical mastectomy. The patients were scanned with computed tomography, and the chest wall (with or without the internal mammary lymph nodes) and supraclavicular region were delineated. For 143 patients, the chest wall and supraclavicular region were combined as an integrated planning volume and treated with IMRT. For 103 patients, conventional treatments were employed with 2 tangential fields for the chest wall, abutting a mixed fieldmore » of 6-MV x-rays (16 Gy) and 9-MeV electrons (34 Gy) for the upper supraclavicular region. The common prescription dose was 50 Gy/25 Fx/5 W to 90% of the target volume. The dosimetric characteristics of the chest wall, the supraclavicular region, and normal organs were compared. For the chest wall target, compared with conventional treatments, the integrated IMRT plans lowered the maximum dose, increased the minimum dose, and resulted in better conformity and uniformity of the target volume. There was an increase in minimum, average, and 95% prescription dose for the integrated IMRT plans in the supraclavicular region, and conformity and uniformity were improved. The V{sub 30} of the ipsilateral lung and V{sub 10}, V{sub 30}, and mean dose of the heart on the integrated IMRT plans were lower than those of the conventional plans. The V{sub 5} and V{sub 10} of the ipsilateral lung and V{sub 5} of the heart were higher on the integrated IMRT plans (p < 0.05) than on conventional plans. Without an increase in the radiation dose to organs at risk, the integrated IMRT treatment plans improved the dose distribution of the supraclavicular region and showed better dose conformity and uniformity of the integrated target volume of the chest wall and supraclavicular region.« less
Tran, Tu O; Lammert, Emily G; Chen, Jie; Merchant, Stephen A; Brunski, Daniel B; Keay, Joel C; Johnson, Matthew B; Glatzhofer, Daniel T; Schmidtke, David W
2011-05-17
In this study, we describe the effects of incorporating single-walled carbon nanotubes (SWNTs) into redox polymer-enzyme hydrogels. The hydrogels were constructed by combining the enzyme glucose oxidase with a redox polymer (Fc-C(6)-LPEI) in which ferrocene was attached to linear poly(ethylenimine) by a six-carbon spacer. Incorporation of SWNTs into these films changed their morphology and resulted in a significant increase in the enzymatic response at saturating glucose concentrations (3 mA/cm(2)) as compared to films without SWNTs (0.6 mA/cm(2)). Likewise, the sensitivity at 5 mM glucose was significantly increased in the presence of SWNTs (74 μA/cm(2)·mM) as compared to control films (26 μA/cm(2)·mM). We demonstrate that the increase in the electrochemical and enzymatic response of these films depends on the amount of SWNTs incorporated and the method of SWNT incorporation. Furthermore, we report that the presence of SWNTs in thick films allows for more of the ferrocene redox centers to become accessible. The high current densities of the hydrogels should allow for the construction of miniature biosensors and enzymatic biofuel cells.
Choudhary, Bharti; Nagpure, Anand; Gupta, Rajinder K
2014-12-01
An antifungal actinomycete strain MT9 was isolated from Loktak Lake, Manipur, India and its cultural characteristics, fatty acid methyl ester, 16S rRNA gene analysis suggests that strain MT9 is identical to Streptomyces exfoliatus. Strain MT9 displayed strong and broad-spectrum antagonism towards several fruit-rotting fungi by mycelial growth suppression. Crude fungal cell-wall lytic enzymes, i.e., chitinase, β-1,3-glucanase, and protease produced by S. exfoliatus MT9 were optimally active at pH 8.0 and 50 °C, pH 5.0 and 60 °C, pH 9.0 and 70 °C, respectively. All three mycolytic enzymes had good stability over a wide pH range of 5.0-10.0, with protease being more thermostable than both chitinase and β-1,3-glucanase. Interestingly zymogram analysis revealed that S. exfoliatus MT9 secretes six distinct chitinase isoenzymes with approximate molecular weights of 9.42, 13.93, 27.87, 36.43, 54.95, 103.27 kDa, six active protease isoenzymes with apparent molecular weights of 12.45, 30.20, 37.45, 46.32, 52.46, 131.46 kDa, and an active band of 119.39 kDa as β-1,3-glucanase enzyme. Extracellular fluid and its organic solvent extracts also exhibited inhibitory activity to various fruit-rotting fungi. The MIC value of n-butanol extract was 2-25 µg/ml against tested fruit-rotting fungi. Antifungal secondary metabolite(s) was found to be polyene in nature. To the best of our knowledge, this is the first report on extracellular production of fungal cell-wall lytic enzymes and antifungal metabolites by bioactive S. exfoliatus MT9 under submerged fermentation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition.
Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena
2017-01-01
Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO 3 - ) and ammonium (NH 4 + ). However, the composition of the N source is important, because excess of NH 4 + promotes morphological disorders. Plants cultured on NH 4 + as the sole N source exhibit serious growth inhibition, commonly referred to as "ammonium toxicity syndrome." NH 4 + -mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH 4 + nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH 4 + as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH 4 + toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH 4 + -mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia , a receptor-like kinase involved in the control of cell wall extension.
Andriukonis, Eivydas; Stirke, Arunas; Garbaras, Andrius; Mikoliunaite, Lina; Ramanaviciene, Almira; Remeikis, Vidmantas; Thornton, Barry; Ramanavicius, Arunas
2018-04-01
In this study, the metabolism of yeast cells (Saccharomyces cerevisiae) was utilized for the synthesis of the conducting polymer - polypyrrole (Ppy).Yeast cells were modified in situ by synthesized Ppy. The Ppy was formed in the cell wall by redox-cycling of [Fe(CN) 6 ] 3-/4- , performed by the yeast cells. Fluorescence microscopy, enzymatic digestions, atomic force microscopy and isotope ratio mass spectroscopy were applied to determine both the polymerization reaction itself and the polymer location in yeast cells. Ppy formation resulted in enhanced resistance to lytic enzymes, significant increase of elasticity and alteration of other mechanical cell wall properties evaluated by atomic force microscopy (AFM). The suggested method of polymer synthesis allows the introduction of polypyrrole structures within the cell wall, which is build up from polymers consisting of carbohydrates. This cell wall modification strategy could increase the usefulness of yeast as an alternative energy source in biofuel cells, and in cell based biosensors. Copyright © 2018 Elsevier B.V. All rights reserved.
Mizutani, F; Yabuki, S; Sato, Y
1997-01-01
A voltammetric urea-sensing electrode was prepared by combining a lipid-attached urease layer with a 2,5-dihydroxythiophenol-modified gold electrode. A self-assembled monolayer of dihydroxythiophenol was prepared on the gold surface by soaking the electrode into an ethanolic solution containing the modifier. A layer of the lipid-attached enzyme and that of acetyl cellulose overcoat were successively made on the dihydroxythiophenol-modified electrode by applying a dip-coating procedure. The addition of urea in a test solution (10 mM phosphate buffer, pH 7.0) brought about an increase of pH near the urease layer. The pH shift accompanied a negative shift of the anodic peak, which corresponded to the electro-oxidation of dihydroxyphenol moiety to form quinone, on the linear sweep voltammograms for the urease/dihydroxythiophenol electrode. The concentration of urea (0.2-5 mM) could be determined by measuring the electrode current at -0.05 V versus Ag/AgCl from the voltammogram. The electrode was applied to the determination of urea in human urine; the measurement of electrode current at such a low potential provided the urea determination without any electrochemical interference from L-ascorbic acid and uric acid.
Wall characterization for through-the-wall radar applications
NASA Astrophysics Data System (ADS)
Greneker, Gene; Rausch, E. O.
2008-04-01
There has been continuing interest in the penetration of multilayer building materials, such as wood walls with air gaps and concrete hollow core block, using through-the-wall (TTW) radar systems. TTW operational techniques and signal propagation paths vary depending on how the TTW system is intended to be operated. For example, the operator of a TTW radar may be required to place the radar against the intervening wall of interest while collecting data. Other operational doctrines allow the radar to be operated in a stand-off mode from the wall. The stand-off distances can vary from feet to hundreds of feet, depending on the type of radar being used. When a signal is propagated through a multilayer wall with air gaps between the material and the wall construction uses materials of radically different dielectric constants, attenuation may not be the only effect that the probing signal experiences passing through the wall. This paper presents measurements of a hollow core concrete block wall and the measurement of a standard wall constructed of siding and wallboard. Both types of walls are typically found in most U.S. homes. These limited measurements demonstrate that the type of wall being penetrated by a wideband signal can modify the probing signal.
Impact assessment of bisphenol A on lignin-modifying enzymes by basidiomycete Trametes versicolor.
Takamiya, Minako; Magan, Naresh; Warner, Philip J
2008-06-15
The impact of different concentrations of bisphenol A (BPA) was evaluated on growth of the white-rot basidiomycete, Trametes versicolor, and on the expression of genes encoding lignin-modifying enzyme (LME) activities. Effective doses (EDs) were obtained from fungal growth rate to monitor LME activities and the expression levels of their encoding genes. The fungus showed mycelial growth at concentrations of up to 300 microg ml(-1) of BPA with an ED50 value of 185 microg ml(-1). The LME activities were stimulated by BPA concentrations up to 300 microg ml(-1). The lignin peroxidase (LIP) encoding gene may be sensitive to BPA stress.
Laothanachareon, Thanaporn; Khonzue, Parichart; Rattanaphan, Nakul; Tinnasulanon, Phungjai; Apawasin, Saowanee; Paemanee, Atchara; Ruanglek, Vasimon; Tanapongpipat, Sutipa; Champreda, Verawat; Eurwilaichitr, Lily
2011-01-01
Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.
Laasik, Eve; Ojarand, Merli; Pajunen, Maria; Savilahti, Harri; Mäe, Andres
2005-02-01
As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.
He, Yin; Ming, Yue; Li, Wei; Li, Yafang; Wu, Maoqi; Song, Jinzhong; Li, Xiaojiu; Liu, Hao
2018-04-26
A facile method for preparing an easy processing, repeatable and flexible pressure sensor was presented via the synthesis of modified multi-walled carbon nanotubes (m-MWNTs) and polyurethane (PU) films. The surface modification of multi-walled carbon nanotubes (MWNTs) simultaneously used a silane coupling agent (KH550) and sodium dodecyl benzene sulfonate (SDBS) to improve the dispersibility and compatibility of the MWNTs in a polymer matrix. The electrical property and piezoresistive behavior of the m-MWNT/PU composites were compared with raw multi-walled carbon nanotube (raw MWNT)/PU composites. Under linear uniaxial pressure, the m-MWNT/PU composite exhibited 4.282%kPa −1 sensitivity within the pressure of 1 kPa. The nonlinear error, hysteresis error and repeatability error of the piezoresistivity of m-MWNT/PU decreased 9%, 16.72% and 54.95% relative to raw MWNT/PU respectively. Therefore, the piezoresistive response of m-MWNT/PU had better stability than that of raw MWNT/PU composites. The m-MWNT/PU sensors could be utilized in wearable devices for body movement detection, monitoring of respiration and pressure detection in garments.
Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.
2015-01-01
RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1
Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R
2015-01-01
RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1
NASA Technical Reports Server (NTRS)
Nakamura, Yukiko; Wakabayashi, Kazuyuki; Hoson, Takayuki
2003-01-01
The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice (Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10-50 degrees C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30-40 degrees C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40 degrees C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30-40 degrees C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40 degrees C were substantially higher than those grown at 10, 20 and 50 degrees C. Furthermore, the activities of (1-->3),(1-->4)-beta-glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1-->3),(1-->4)-beta-glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30-40 degrees C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of beta -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of beta-glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.
Kim, Ah-Na; Lee, Kyo-Yeon; Kim, Hyun-Jin; Chun, Jiyeon; Kerr, William L; Choi, Sung-Gil
2018-01-01
This study evaluated the effects of grinding at atmospheric pressure (control), under vacuum (∼2.67 kPa), or with modified atmosphere (N 2 and CO 2 ) on the browning, antioxidant activity, phenolics, and oxidative enzyme activity of apples as a function of time. The control group was affected most, showing distinct browning and losing most of the antioxidant activity and concentrations of the main phenolic compounds. The modified atmosphere groups retained color, antioxidant activity, and phenolic compounds better than the control group. Least changes were obtained with vacuum grinding, particularly in terms of preventing enzymatic browning and oxidation of antioxidants apples. At 12 h after grinding, vacuum-ground apples retained total phenolic contents 5.32, 1.54, and 1.49 times higher than control, nitrogen gas, and carbon dioxide gas-ground samples, respectively. The oxidative enzyme activity, including that of polyphenol oxidase and peroxidase, decreased in the control and modified atmosphere group, but they were maintained in the samples ground under the vacuum. In this study, we found that grinding with modified atmosphere or vacuum conditions could effectively prevent browning as well as loss of phenolic compounds and antioxidant activity of ground apples. These results can help scientists and engineers build better grinding systems for retaining nutrient and quality factors of ground apples. In addition, these results may be useful to other fruit and vegetable industries that wish to retain fresh-like quality and nutritional value during grinding and storage. © 2017 Institute of Food Technologists®.
ERIC Educational Resources Information Center
Field, Christopher Ryan
2009-01-01
Developments in analytical chemistry were made using acoustically levitated small volumes of liquid to study enzyme reaction kinetics and by detecting volatile organic compounds in the gas phase using single-walled carbon nanotubes. Experience gained in engineering, electronics, automation, and software development from the design and…
Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition
Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena
2017-01-01
Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO3–) and ammonium (NH4+). However, the composition of the N source is important, because excess of NH4+ promotes morphological disorders. Plants cultured on NH4+ as the sole N source exhibit serious growth inhibition, commonly referred to as “ammonium toxicity syndrome.” NH4+-mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH4+ nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH4+ as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH4+ toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH4+-mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia, a receptor-like kinase involved in the control of cell wall extension. PMID:28848567
Goodell, Barry; Zhu, Yuan; Kim, Seong; Kafle, Kabindra; Eastwood, Daniel; Daniel, Geoffrey; Jellison, Jody; Yoshida, Makoto; Groom, Leslie; Pingali, Sai Venkatesh; O'Neill, Hugh
2017-01-01
Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a "pretreatment" used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action
Drag reduction at a plane wall
NASA Technical Reports Server (NTRS)
Hill, D. C.
1993-01-01
The objective is to determine by analytical means how drag on a plane wall may be modified favorably using a minimal amount of flow information - preferably only information at the wall. What quantities should be measured? How should that information be assimilated in order to arrive at effective control? As a prototypical problem, incompressible, viscous flow, governed by the Navier-Stokes equations, past a plane wall at which the no-slip condition was modified was considered. The streamwise and spanwise velocity components are required to be zero, but the normal component is to be specified according to some control law. The challenge is to choose the wall-normal velocity component based on flow conditions at the wall so that the mean drag is as small as possible. There can be no net mass flux through the wall, and the total available control energy is constrained. A turbulent flow is highly unsteady and has detailed spatial structure. The mean drag on the wall is the integral over the wall of the local shear forces exerted by the fluid, which is then averaged in time; it is a 'macroscopic' property of the flow. It is not obvious how unsteady boundary control is to be applied in order to modify the mean flow most effectively, especially in view of the non- self-adjoint nature of the governing equations. An approximate analytical solution to the suboptimal scheme is pursued.
Melin, Frederic; Noor, Mohamed R.; Pardieu, Elodie; Boulmedais, Fouzia; Banhart, Florian; Cecchini, Gary; Soulimane, Tewfik
2015-01-01
Succinate Quinone reductases (SQRs) are the enzymes which couple the oxidation of succinate and the reduction of quinones in the respiratory chain of prokaryotes and eukaryotes. We compare herein the temperature-dependent activity and structural stability of two SQRs, the first one from the mesophilic bacterium E. coli and the second one from the thermophilic bacterium T. thermophilus by a combined electrochemical and infrared spectroscopy approach. Direct electron transfer was achieved with the full membrane protein complexes at SWNTs-modified electrodes. The possible structural factors which contribute to the temperature-dependent activity of the enzymes and to the thermostability of the T. thermophiles SQR in particular, are discussed. PMID:25139263
Sterol glycosyltransferases--the enzymes that modify sterols.
Chaturvedi, Pankaj; Misra, Pratibha; Tuli, Rakesh
2011-09-01
Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.
Selective Internal Heat Distribution in Modified Trombe Wall
NASA Astrophysics Data System (ADS)
Szyszka, Jerzy; Kogut, Janusz; Skrzypczak, Izabela; Kokoszka, Wanda
2017-12-01
At present, the requirements for thermal insulation of the external walls in buildings are being increased. There is a need to reduce energy consumption for heating rooms during the winter season. This may be achieved by increasing the thermal resistance of the outer partitions, using solutions that utilize either recuperation or solar radiation. The most popular systems include either solar collectors, or heat pump links or ground exchangers. Trombe walls (TW) are a very promising passive heating system, which requires little or no effort to operate, and may be very convenient in different climate conditions. A typical TW consists of a masonry wall painted a dark, heat absorbing paint colour and faced with a single or double layer of glass. The principle of operation is based on the photothermal conversion of solar radiation. There are various modifications of TW. They may improve the energy efficiency in relation to the climate conditions in which they operate. The hybrid solutions are also known. The efficiency of walls is related to the use of proper materials. In TW, the compromise should be sought between the thermal resistance and the ability to distribute heat from the absorbed energy of solar radiation. The paper presents an overview of the most commonly used solutions and discusses its own concept dedicated to the climate conditions of Central Europe.
Gao, Yu; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A; Moore, John P
2016-11-05
The effectiveness of enzyme-mediated-maceration in red winemaking relies on the use of an optimum combination of specific enzymes. A lack of information on the relevant enzyme activities and the corresponding polysaccharide-rich berry cell wall structure is a major limitation. This study used different combinations of purified recombinant pectinases with cell wall profiling tools to follow the deconstruction process during winemaking. Multivariate data analysis of the glycan microarray (CoMPP) and gas chromatography (GC) results revealed that pectin lyase performed almost as effectively in de-pectination as certain commercial enzyme mixtures. Surprisingly the combination of endo-polygalacturonase and pectin-methyl-esterase only unraveled the cell walls without de-pectination. Datasets from the various combinations used confirmed pectin-rich and xyloglucan-rich layers within the grape pomace. These data support a proposed grape cell wall model which can serve as a foundation to evaluate testable hypotheses in future studies aimed at developing tailor-made enzymes for winemaking scenarios. Copyright © 2016 Elsevier Ltd. All rights reserved.
Yin, Peng; Wang, Yuhua; Li, Yan; Deng, Chunhui; Zhang, Xiangmin; Yang, Pengyuan
2012-09-01
In this study, sandwich-structured graphene/mesoporous silica composites (C8-modified graphene@mSiO(2)) were synthesized by coating mesoporous silica onto hydrophilic graphene nanosheets through a surfactant-mediated cocondensation sol-gel process. The newly prepared C8-modified graphene@mSiO(2) nanocomposites possess unique properties of extended plate-like morphology, good water dispersibility, highly open pore structure, uniform pore size (2.8 nm), high surface area (632 m(2)/g), and C8-modified-interior pore walls. The unique structure of the C8-modified graphene@mSiO(2) composite nanosheets not only provide extended planes with hydrophilic surface that prevents aggregation in solution, but also offer a huge number of C8-modified mesopores with high surface area that can ensure an efficient adsorption of peptides through hydrophobic-hydrophobic interaction between C8-moified pore walls and target molecules. The obtained C8-modified graphene@mSiO(2) materials were utilized for size selectively and specifically enriching peptides in standard peptide mixtures and endogenous peptides in real biological samples (mouse brain tissue). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Modified locking thread form for fastener
NASA Technical Reports Server (NTRS)
Roopnarine, (Inventor); Vranish, John D. (Inventor)
1998-01-01
A threaded fastener has a standard part with a standard thread form characterized by thread walls with a standard included angle, and a modified part complementary to the standard part having a modified thread form characterized by thread walls which are symmetrically inclined with a modified included angle that is different from the standard included angle of the standard part's thread walls, such that the threads of one part make pre-loaded edge contact with the thread walls of the other part. The thread form of the modified part can have an included angle that is greater, less, or compound as compared to the included angle of the standard part. The standard part may be a bolt and the modified part a nut, or vice versa. The modified thread form holds securely even under large vibrational forces, it permits bi-directional use of standard mating threads, is impervious to the build up of tolerances and can be manufactured with a wider range of tolerances without loss of functionality, and distributes loading stresses (per thread) in a manner that decreases the possibility of single thread failure.
Liu, Daojun; Perdue, Robbyn K; Sun, Li; Crooks, Richard M
2004-07-06
This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.
Johnsen, Hanne R; Striberny, Bernd; Olsen, Stian; Vidal-Melgosa, Silvia; Fangel, Jonatan U; Willats, William G T; Rose, Jocelyn K C; Krause, Kirsten
2015-08-01
Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
Thomas, Tony; Mascarenhas, Ronald J; D' Souza, Ozma J; Detriche, Simon; Mekhalif, Zineb; Martis, Praveen
2014-07-01
An amperometric sensor for the determination of epinephrine (EP) was fabricated by modifying the carbon paste electrode (CPE) with pristine multi-walled carbon nanotubes (pMWCNTs) using bulk modification followed by drop casting of sodium dodecyl sulfate (SDS) onto the surface for its optimal potential application. The modified electrode showed an excellent electrocatalytic activity towards EP by decreasing the overpotential and greatly enhancing the current sensitivity. FE-SEM images confirmed the dispersion of pMWCNTs in the CPE matrix. EDX analysis ensured the surface coverage of SDS. A comparative study of pMWCNTs with those of oxidized MWCNTs (MWCNTsOX) modified electrodes reveals that the former is the best base material for the construction of the sensor with advantages of lower oxidation overpotential and the least background current. The performance of the modified electrode was impressive in terms of the least charge transfer resistance (Rct), highest values for diffusion coefficient (DEP) and standard heterogeneous electron transfer rate constant (k°). Analytical characterization of the modified electrode exhibited two linear dynamic ranges from 1.0×10(-7) to 1.0×10(-6)M and 1.0×10(-6) to 1.0×10(-4)M with a detection limit of (4.5±0.18)×10(-8)M. A 100-fold excess of serotonin, acetaminophen, folic acid, uric acid, tryptophan, tyrosine and cysteine, 10-fold excess of ascorbic acid and twofold excess of dopamine do not interfere in the quantification of EP at this electrode. The analytical applications of the modified electrode were demonstrated by determining EP in spiked blood serum and adrenaline tartrate injection. The modified electrode involves a simple fabrication procedure, minimum usage of the modifier, quick response, excellent stability, reproducibility and anti-fouling effects. Copyright © 2014 Elsevier B.V. All rights reserved.
Roda, A; Mirasoli, M; Guardigli, M; Michelini, E; Simoni, P; Magliulo, M
2006-03-01
Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.
Probing the Intermediacy of Covalent RNA Enzyme Complexes in RNA Modification Enzymes
Chervin, Stephanie M.; Kittendorf, Jeffrey D.; Garcia, George A.
2009-01-01
Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT). PMID:17673081
2012-01-01
Background Fatty acid modifying enzyme (FAME) has been shown to modify free fatty acids to alleviate their bactericidal effect by esterifying fatty acids to cholesterol or alcohols. Although it has been shown in previous studies that FAME is required for Staphylococcus aureus survival in skin abscesses, FAME is poorly studied compared to other virulence factors. FAME activity had also been detected in coagulase-negative staphylococci (CNS). However, FAME activity was only surveyed after a bacterial culture was grown for 24 h. Therefore if FAME activity was earlier in the growth phase, it would not have been detected by the assay and those strains would have been labeled as FAME negative. Results Fifty CNS bovine mastitis isolates and several S. aureus, Escherichia coli, and Streptococcus uberis strains were assayed for FAME activity over 24 h. FAME activity was detected in 54% of CNS and 80% S. aureus strains surveyed but none in E. coli or S. uberis. While some CNS strains produced FAME activity comparable to the lab strain of S. aureus, the pattern of FAME activity varied among strains and across species of staphylococci. All CNS that produced FAME activity also exhibited lipase activity. Lipase activity relative to colony forming units of these CNS decreased over the 24 h growth period. No relationship was observed between somatic cell count in the milk and FAME activity in CNS. Conclusions Some staphylococcal species surveyed produced FAME activity, but E. coli and S. uberis strains did not. All FAME producing CNS exhibited lipase activity which may indicate that both these enzymes work in concert to alter fatty acids in the bacterial environment. PMID:22726316
He, Yin; Ming, Yue; Li, Wei; Li, Yafang; Wu, Maoqi; Song, Jinzhong; Li, Xiaojiu; Liu, Hao
2018-01-01
A facile method for preparing an easy processing, repeatable and flexible pressure sensor was presented via the synthesis of modified multi-walled carbon nanotubes (m-MWNTs) and polyurethane (PU) films. The surface modification of multi-walled carbon nanotubes (MWNTs) simultaneously used a silane coupling agent (KH550) and sodium dodecyl benzene sulfonate (SDBS) to improve the dispersibility and compatibility of the MWNTs in a polymer matrix. The electrical property and piezoresistive behavior of the m-MWNT/PU composites were compared with raw multi-walled carbon nanotube (raw MWNT)/PU composites. Under linear uniaxial pressure, the m-MWNT/PU composite exhibited 4.282%kPa−1 sensitivity within the pressure of 1 kPa. The nonlinear error, hysteresis error and repeatability error of the piezoresistivity of m-MWNT/PU decreased 9%, 16.72% and 54.95% relative to raw MWNT/PU respectively. Therefore, the piezoresistive response of m-MWNT/PU had better stability than that of raw MWNT/PU composites. The m-MWNT/PU sensors could be utilized in wearable devices for body movement detection, monitoring of respiration and pressure detection in garments. PMID:29701643
NASA Astrophysics Data System (ADS)
Li, Zhou; Hua, Xin; Pei, Hongying; Shen, Yuan; Shen, Guijun
2017-12-01
A glass carbon electrode was prepared that coated with a composite film containing grapheme and multi-walled carbon nanotubes. It was used to study the electrochemical response of terbutaline sulfate. Under the optimized conditions, the oxidation peak current was found to be proportional to its concentration in the range of 0.2-5 μmol·L-1 and 5-40 μmol·L-1).Compared with the bare GC electrode, the GN-MWNTs-modified GC (GN-MWNTs/GC) had many advantages such as relatively high sensitivity, good stability and long life time. The modified electrode was used to determine the TES tablets with satisfactory results.
NASA Technical Reports Server (NTRS)
Santi, L. Michael
1986-01-01
Computational predictions of turbulent flow in sharply curved 180 degree turn around ducts are presented. The CNS2D computer code is used to solve the equations of motion for two-dimensional incompressible flows transformed to a nonorthogonal body-fitted coordinate system. This procedure incorporates the pressure velocity correction algorithm SIMPLE-C to iteratively solve a discretized form of the transformed equations. A multiple scale turbulence model based on simplified spectral partitioning is employed to obtain closure. Flow field predictions utilizing the multiple scale model are compared to features predicted by the traditional single scale k-epsilon model. Tuning parameter sensitivities of the multiple scale model applied to turn around duct flows are also determined. In addition, a wall function approach based on a wall law suitable for incompressible turbulent boundary layers under strong adverse pressure gradients is tested. Turn around duct flow characteristics utilizing this modified wall law are presented and compared to results based on a standard wall treatment.
Plant cell wall: Never too much acetate
Scheller, Henrik V.
2017-03-03
Here, plant cell walls incorporate a variety of acetylated polysaccharides. In addition to enzymes catalysing acetylation (acetyltransferases), plants could produce enzymes to remove acetyl groups (acetylesterases). Previously, pectin acetylesterases were known and now a xylan acetylesterase has been identified — and it has many surprises.
Jiang, Yanping; Duarte, Alexandra Vivas; van den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P; Zhou, Zhihua; Benoit, Isabelle
2016-01-01
To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-D-glucosidase activities of the best mutant were increased from 1.8 IU/ml, 0.1 IU/ml and 0.05 IU/ml to 4.8 IU/ml, 0.4 IU/ml and 0.3 IU/ml, respectively. The sugar yield of wheat straw saccharification by combining enzymes from this mutant and the Aspergillus niger genetically modified strain ΔcreA/xlnR c/araR c was improved up to 7.5 mg/ml, a 229 % increase compared to the combination of wild type strains. Mixing enzymes from T. reesei and A. niger combined with the genetic modification of transcription factors is a promising strategy to increase saccharification efficiency.
Hybrid nanocatalysts containing enzymes and metallic nanoparticles for ethanol/O2 biofuel cell
NASA Astrophysics Data System (ADS)
Aquino Neto, S.; Almeida, T. S.; Palma, L. M.; Minteer, S. D.; de Andrade, A. R.
2014-08-01
We report the preparation of hybrid nanostructured bioanodes containing the enzyme alcohol dehydrogenase (ADH) with either Au, Pt, or Pt0.75Sn0.25 nanoparticles for use in ethanol/O2 hybrid biofuel cells. We describe two different methodologies for the preparation of the bioanodes: in a first case, multi walled carbon nanotubes (MWCNTs) were employed as a support for the metallic nanoparticles and TBAB-modified Nafion® aided enzyme immobilization. In the second case, we immobilized the enzymes using dendrimers-encapsulated nanoparticles as the agent for enzyme anchoring. The biofuel cell tests showed that the addition of metallic nanoparticles to the bioanode structure enhanced the overall biofuel cell performance. The bioelectrode containing Au nanoparticles displaying the best performance, with an open circuit potential of 0.61 ± 0.05 V and a maximum power density of 155 ± 11 μW cm-2. NADH cyclic voltammetric experiments indicated that Au nanoparticles behaved as a catalyst toward NADH oxidation. Comparing the two protocols we used to synthetized nanoparticles, the sample containing the Au nanoparticles supported on MWCNTs furnished fourfold higher values. Therefore, from the satisfactory results obtained, it can be inferred that the combination of small amounts of metallic nanoparticles with enzymes improve bioanode performance.
Li, Liang; Liang, Bo; Shi, Jianguo; Li, Feng; Mascini, Marco; Liu, Aihua
2012-03-15
A novel Nafion/bacteria-displaying xylose dehydrogenase (XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of d-xylose (INS 967), where the XDH-displayed bacteria (XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion-MWNTs composite dispersion (10 μL) was cast onto the inverted glassy carbon electrode, followed by casting 10-μL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-μL of Nafion solution (0.05 wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD(+) in 0.1M PBS buffer (pH7.4), where NAD(+) (nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50 V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7 V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6-100 μM and a low detection limit of 0.5 μM D-xylose (S/N=3). No interference was observed in the presence of 300-fold excess of other saccharides including D-glucose, D-fructose, D-maltose, D-galactose, D-mannose, D-sucrose, and D-cellbiose as well as 60-fold excess of L-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds
Goggin, Danica E; Powles, Stephen B; Toorop, Peter E; Steadman, Kathryn J
2011-04-15
Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A(4). This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release. Copyright © 2010 Elsevier GmbH. All rights reserved.
Lin, Pingtan; Zhao, Shulin; Lu, Xin; Ye, Fanggui; Wang, Hengshan
2013-08-01
A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Modeling of near-wall turbulence
NASA Technical Reports Server (NTRS)
Shih, T. H.; Mansour, N. N.
1990-01-01
An improved k-epsilon model and a second order closure model is presented for low Reynolds number turbulence near a wall. For the k-epsilon model, a modified form of the eddy viscosity having correct asymptotic near wall behavior is suggested, and a model for the pressure diffusion term in the turbulent kinetic energy equation is proposed. For the second order closure model, the existing models are modified for the Reynolds stress equations to have proper near wall behavior. A dissipation rate equation for the turbulent kinetic energy is also reformulated. The proposed models satisfy realizability and will not produce unphysical behavior. Fully developed channel flows are used for model testing. The calculations are compared with direct numerical simulations. It is shown that the present models, both the k-epsilon model and the second order closure model, perform well in predicting the behavior of the near wall turbulence. Significant improvements over previous models are obtained.
Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin
2015-02-15
Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.
2014-04-21
modified with binary nanoparticles consist of multi-walled carbon nanotubes (MWCNTs) and nanoclays together. First, epoxy SC-15 resin was reinforced...modified with binary nanoparticles consist of multi-walled carbon nanotubes (MWCNTs) and nanoclays together. First, epoxy SC-15 resin was reinforced with...7 2.2.1 Carbon Nanotube
Sethuraman, V; Muthuraja, P; Anandha Raj, J; Manisankar, P
2016-10-15
The fabrication, characterization and analytical performances were investigated for a catechol biosensor, based on the PEDOT-rGO-Fe2O3-PPO composite modified glassy carbon (GC) electrode. The graphene oxide (GO) doped conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) was prepared through electrochemical polymerization by potential cycling. Reduction of PEDOT-GO was carried out by amperometric method. Fe2O3 nanoparticles were synthesized in ethanol by hydrothermal method. The mixture of Fe2O3, PPO and glutaraldehyde was casted on the PEDOT-rGO electrode. The surface morphology of the modified electrodes was studied by FE-SEM and AFM. Cyclic voltammetric studies of catechol on the enzyme modified electrode revealed higher reduction peak current. Determination of catechol was carried out successfully by Differential Pulse Voltammetry (DPV) technique. The fabricated biosensor investigated shows a maximum current response at pH 6.5. The catechol biosensor exhibited wide sensing linear range from 4×10(-8) to 6.20×10(-5)M, lower detection limit of 7×10(-9)M, current maxima (Imax) of 92.55µA and Michaelis-Menten (Km) constant of 30.48µM. The activation energy (Ea) of enzyme electrode is 35.93KJmol(-1) at 50°C. There is no interference from d-glucose and l-glutamic acid, ascorbic acid and o-nitrophenol. The PEDOT-rGO-Fe2O3-PPO biosensor was stable for at least 75 days when stored in a buffer at about 4°C. Copyright © 2015 Elsevier B.V. All rights reserved.
Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.
Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P
2013-01-01
Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
Inhibitors of the bacterial cell wall biosynthesis enzyme MurC.
Reck, F; Marmor, S; Fisher, S; Wuonola, M A
2001-06-04
A series of phosphinate transition-state analogues of the L-alanine adding enzyme (MurC) of bacterial peptidoglycan biosynthesis was prepared and tested as inhibitors of the Escherichia coli enzyme. Compound 4 was identified as a potent inhibitor of MurC from Escherichia coli with an IC(50) of 49nM.
Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar
2012-01-01
Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors. PMID:23163477
Highly sensitive glucose sensors based on enzyme-modified whole-graphene solution-gated transistors
NASA Astrophysics Data System (ADS)
Zhang, Meng; Liao, Caizhi; Mak, Chun Hin; You, Peng; Mak, Chee Leung; Yan, Feng
2015-02-01
Noninvasive glucose detections are convenient techniques for the diagnosis of diabetes mellitus, which require high performance glucose sensors. However, conventional electrochemical glucose sensors are not sensitive enough for these applications. Here, highly sensitive glucose sensors are successfully realized based on whole-graphene solution-gated transistors with the graphene gate electrodes modified with an enzyme glucose oxidase. The sensitivity of the devices is dramatically improved by co-modifying the graphene gates with Pt nanoparticles due to the enhanced electrocatalytic activity of the electrodes. The sensing mechanism is attributed to the reaction of H2O2 generated by the oxidation of glucose near the gate. The optimized glucose sensors show the detection limits down to 0.5 μM and good selectivity, which are sensitive enough for non-invasive glucose detections in body fluids. The devices show the transconductances two orders of magnitude higher than that of a conventional silicon field effect transistor, which is the main reason for their high sensitivity. Moreover, the devices can be conveniently fabricated with low cost. Therefore, the whole-graphene solution-gated transistors are a high-performance sensing platform for not only glucose detections but also many other types of biosensors that may find practical applications in the near future.
Highly sensitive glucose sensors based on enzyme-modified whole-graphene solution-gated transistors
Zhang, Meng; Liao, Caizhi; Mak, Chun Hin; You, Peng; Mak, Chee Leung; Yan, Feng
2015-01-01
Noninvasive glucose detections are convenient techniques for the diagnosis of diabetes mellitus, which require high performance glucose sensors. However, conventional electrochemical glucose sensors are not sensitive enough for these applications. Here, highly sensitive glucose sensors are successfully realized based on whole-graphene solution-gated transistors with the graphene gate electrodes modified with an enzyme glucose oxidase. The sensitivity of the devices is dramatically improved by co-modifying the graphene gates with Pt nanoparticles due to the enhanced electrocatalytic activity of the electrodes. The sensing mechanism is attributed to the reaction of H2O2 generated by the oxidation of glucose near the gate. The optimized glucose sensors show the detection limits down to 0.5 μM and good selectivity, which are sensitive enough for non-invasive glucose detections in body fluids. The devices show the transconductances two orders of magnitude higher than that of a conventional silicon field effect transistor, which is the main reason for their high sensitivity. Moreover, the devices can be conveniently fabricated with low cost. Therefore, the whole-graphene solution-gated transistors are a high-performance sensing platform for not only glucose detections but also many other types of biosensors that may find practical applications in the near future. PMID:25655666
(Hydroxyproline-rich glycoproteins of the plant cell wall)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Varner, J.E.
1990-01-01
We are studying the chemistry and architecture of plant cells walls, the extracellular matrices that taken together shape the plant and provide mechanical support for the plant. Cell walls are dynamic structures that regulate, or are the site of, many physiological processes, in addition to being the cells' first line of defense against invading pathogens. In the past year we have examined the role of the cell wall enzyme ascorbic acid oxidase as related to the structure of the wall and its possible interactions with hydroxyproline-rich glycoproteins of the wall.
Directed evolution: an approach to engineer enzymes.
Kaur, Jasjeet; Sharma, Rohit
2006-01-01
Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.
Wang, Xiangming; Liu, Jianhong; Zhu, Zhiwen; Ou, Guangshuo
2015-03-15
Directional cell migration is fundamental for neural development, and extracellular factors are pivotal for this process. Heparan sulfate proteoglycans (HSPGs) that carry long chains of differentially modified sugar residues contribute to extracellular matrix; however, the functions of HSPG in guiding cell migration remain elusive. Here, we used the Caenorhabditis elegans mutant pool from the Million Mutation Project and isolated a mutant allele of the heparan sulfate-modifying enzyme glucuronyl C5-epimerase HSE-5. Loss-of-function of this enzyme resulted in defective Q neuroblast migration. We showed that hse-5 controlled Q cell migration in a cell non-autonomous manner. By performing live cell imaging in hse-5 mutant animals, we found that hse-5 controlled initial polarization during Q neuroblast migration. Furthermore, our genetic epistasis analysis demonstrated that lon-2 might act downstream of hse-5. Finally, rescue of the hse-5 mutant phenotype by expression of human and mouse hse-5 homologs suggested a conserved function for this gene in neural development. Taken together, our results indicated that proper HSPG modification in the extracellular matrix by HSE-5 is essential for neuroblast polarity during migration. Copyright © 2015 Elsevier Inc. All rights reserved.
[Potentialization of antibiotics by lytic enzymes].
Brisou, J; Babin, P; Babin, R
1975-01-01
Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.
Polymer mobility in cell walls of cucumber hypocotyls
NASA Technical Reports Server (NTRS)
Fenwick, K. M.; Apperley, D. C.; Cosgrove, D. J.; Jarvis, M. C.
1999-01-01
Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.
Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls
NASA Technical Reports Server (NTRS)
Cosgrove, D. J.
1989-01-01
Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such "creep" is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q10 (3.8) between 20 degrees and 30 degrees C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q10 (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu2+, Hg2+ and Al3+ were strongly inhibitory whereas most other cations, including Ca2+, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu2+, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.
Regulation of Ubiquitin Enzymes in the TGF-β Pathway.
Iyengar, Prasanna Vasudevan
2017-04-20
The transforming growth factor-β (TGF-β) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-β pathway and how they are regulated.
Davis, Katherine M; Schramma, Kelsey R; Hansen, William A; Bacik, John P; Khare, Sagar D; Seyedsayamdost, Mohammad R; Ando, Nozomi
2017-09-26
Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.
Willis, Jonathan D.; Mazarei, Mitra; Stewart, C. Neal
2016-01-01
Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review. PMID:27303411
Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes
Koch, Claudia; Wabbel, Katrin; Eber, Fabian J.; Krolla-Sidenstein, Peter; Azucena, Carlos; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania; Wege, Christina
2015-01-01
Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins. PMID:26734040
Identification of Genes Coding Aminoglycoside Modifying Enzymes in E. coli of UTI Patients in India.
Mir, Abdul Rouf; Bashir, Yasir; Dar, Firdous Ahmad; Sekhar, M
This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR). Out of 98 isolates, 71 (72.45%) isolates were identified as E. coli and the remaining 27 (27.55%) as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.
Fluorescent probes for exploring plant cell wall deconstruction: a review.
Paës, Gabriel
2014-07-03
Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.
Levitskiĭ, V Iu; Melik-Nubarov, N S; Slepnev, V I; Shikshnis, V A; Mozhaev, V V
1990-01-01
Stabilizing effect of denaturing salts on irreversible thermoinactivation of native and modified alpha-chymotrypsin at elevated temperatures is observed. The effect is caused by a shift of conformational equilibrium, at the primary step of reversible unfolding in the course of thermoinactivation, to a more unfolded form which is not able to refold "incorrectly". The stability of alpha-chymotrypsin is regulated within a wide range by medium alteration: the stabilizing effects are similar to those achieved by multipoint attachment of the enzyme to a support or by hydrophilization of protein by covalent modification.
Enhancement of fruit shelf life by suppressing N-glycan processing enzymes.
Meli, Vijaykumar S; Ghosh, Sumit; Prabha, T N; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis
2010-02-09
In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have been made to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall-degrading proteins. However, these have met with very limited success. N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specific N-glycoprotein modifying enzymes, alpha-mannosidase (alpha-Man) and beta-D-N-acetylhexosaminidase (beta-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed approximately 2.5- and approximately 2-fold firmer fruits in the alpha-Man and beta-Hex RNAi lines, respectively, and approximately 30 days of enhanced shelf life. Overexpression of alpha-Man or beta-Hex resulted in excessive fruit softening. Expression of alpha-Man and beta-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening, rin (ripening inhibitor). Furthermore, transcriptomic comparative studies demonstrate the down-regulation of cell wall degradation- and ripening-related genes in RNAi fruits. It is evident from these results that N-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation of N-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hahn, Michael G.
The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plantmore » cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.« less
Swamy, Prashant S.; Hu, Hao; Pattathil, Sivakumar; ...
2015-08-05
Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues duringmore » regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. In conclusion, taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.« less
Modifying the inlet characteristics of a Turbulent Coanda Wall Jet
NASA Astrophysics Data System (ADS)
Pandey, Anshuman; Gregory, James W.
2017-11-01
The Coanda effect has been ingeniously used over the past century for augmentation of lift. More recently, NOTAR helicopters have employed the use of the Coanda effect for producing the stabilizing side force with quieter and safer configurations. A manifestation of the Coanda effect in its simplest form is a wall jet issuing tangentially to a cylinder that tends to stay attached to the cylinder over turning angles as large as 180 degrees. An experimental study on such a configuration has been performed in this work to understand the effect of inlet characteristics on the evolution of the wall jet. In previous studies, it has been found that the radial momentum influx is maximum near the inlet and it provides the necessary centrifugal force for the jet to stay attached. So it can be hypothesized that a protrusion of the upper wall of the nozzle that issues the jet would lead to an earlier separation and decreased efficiency. On the other hand, the predisposition of the jet to stay attached to the flat surface of the protrusion could create a separation bubble between the jet and the curved wall and lead to increased suction. These competing phenomena have been explored using Particle Image Velocimetry performed in the streamwise direction at mid-span location of a 3 ft long cylinder. The effect of varying the length of the protrusion for different combinations of initial jet width and flow velocity has been studied to understand how changing the inlet affects the efficiency of the wall jet.
Measuring the Density of States of the Inner and Outer Wall of Double-Walled Carbon Nanotubes.
Chambers, Benjamin A; Shearer, Cameron J; Yu, LePing; Gibson, Christopher T; Andersson, Gunther G
2018-06-19
The combination of ultraviolet photoelectron spectroscopy and metastable helium induced electron spectroscopy is used to determine the density of states of the inner and outer coaxial carbon nanotubes. Ultraviolet photoelectron spectroscopy typically measures the density of states across the entire carbon nanotube, while metastable helium induced electron spectroscopy measures the density of states of the outermost layer alone. The use of double-walled carbon nanotubes in electronic devices allows for the outer wall to be functionalised whilst the inner wall remains defect free and the density of states is kept intact for electron transport. Separating the information of the inner and outer walls enables development of double-walled carbon nanotubes to be independent, such that the charge transport of the inner wall is maintained and confirmed whilst the outer wall is modified for functional purposes.
Phage lytic enzymes targeting streptococci
USDA-ARS?s Scientific Manuscript database
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...
Exploring accessibility of pretreated poplar cell walls by measuring dynamics of fluorescent probes.
Paës, Gabriel; Habrant, Anouck; Ossemond, Jordane; Chabbert, Brigitte
2017-01-01
The lignocellulosic cell wall network is resistant to enzymatic degradation due to the complex chemical and structural features. Pretreatments are thus commonly used to overcome natural recalcitrance of lignocellulose. Characterization of their impact on architecture requires combinatory approaches. However, the accessibility of the lignocellulosic cell walls still needs further insights to provide relevant information. Poplar specimens were pretreated using different conditions. Chemical, spectral, microscopic and immunolabeling analysis revealed that poplar cell walls were more altered by sodium chlorite-acetic acid and hydrothermal pretreatments but weakly modified by soaking in aqueous ammonium. In order to evaluate the accessibility of the pretreated poplar samples, two fluorescent probes (rhodamine B-isothiocyanate-dextrans of 20 and 70 kDa) were selected, and their mobility was measured by using the fluorescence recovery after photobleaching (FRAP) technique in a full factorial experiment. The mobility of the probes was dependent on the pretreatment type, the cell wall localization (secondary cell wall and cell corner middle lamella) and the probe size. Overall, combinatory analysis of pretreated poplar samples showed that even the partial removal of hemicellulose contributed to facilitate the accessibility to the fluorescent probes. On the contrary, nearly complete removal of lignin was detrimental to accessibility due to the possible cellulose-hemicellulose collapse. Evaluation of plant cell wall accessibility through FRAP measurement brings further insights into the impact of physicochemical pretreatments on lignocellulosic samples in combination with chemical and histochemical analysis. This technique thus represents a relevant approach to better understand the effect of pretreatments on lignocellulose architecture, while considering different limitations as non-specific interactions and enzyme efficiency.
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)
NASA Astrophysics Data System (ADS)
Irena, Gancarz; Jolanta, Bryjak; Karolina, Zynek
2009-07-01
Poly(ethylene terephthalate) (PET) film was modified by reaction with hydrazine (HD), ethylenediamine (EDA), 1,2-diaminopropane (1,2-DAP) and 1,3-diaminopropane (1,3-DAP). The maximal amount of amine functionalities introduced in the chosen conditions on the surface was found as 0.07, 3.35, 0.76 and 1.99 nmol cm -2 for HD, EDA, 1,2-DAP and 1,3-DAP respectively. During the modification process etching of the sample and an increase of stiffness takes place. FTIR-ATR spectra prove that the surface chemistry after modification in amine solution is very complex. The lack of clear correlation between the surface tension and surface concentration of amine functionalities seems to confirm that. For immobilization purpose invertase, laccase and tyrosinase were used. The amount of covalently attached proteins at first increases with the increase of surface concentration of amine groups but after reaching a certain level of amine groups, decrease of the immobilization level was observed. All enzymes tested showed highest activity for a moderate level of aminolysis and this activity had the highest values for EDA-modified PET.
Froestl, Wolfgang; Pfeifer, Andrea; Muhs, Andreas
2014-01-01
Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers, are very productive. The review "Drugs interacting with Targets other than Receptors or Enzymes. Disease-modifying Drugs" was accepted in October 2012. In the last 20 months, new targets for the potential treatment of Alzheimer's disease were identified. Enormous progress was realized in the pharmacological characterization of natural products with cognitive enhancing properties. This review covers the evolution of research in this field through May 2014.
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.
Zhang, Xiaoyang; Rogowski, Artur; Zhao, Lei; Hahn, Michael G; Avci, Utku; Knox, J Paul; Gilbert, Harry J
2014-01-24
Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.
NASA Astrophysics Data System (ADS)
Ganesh, V.; Muthurasu, A.
2012-04-01
In this paper, we propose various strategies for an enzyme immobilization on electrodes (both metal and semiconductor electrodes). In general, the proposed methodology involves two critical steps viz., (1) chemical modification of substrates using functional monolayers [Langmuir - Blodgett (LB) films and/or self-assembled monolayers (SAMs)] and (2) anchoring of a target enzyme using specific chemical and physical interactions by attacking the terminal functionality of the modified films. Basically there are three ways to immobilize an enzyme on chemically modified electrodes. First method consists of an electrostatic interaction between the enzyme and terminal functional groups present within the chemically modified films. Second and third methods involve the introduction of nanomaterials followed by an enzyme immobilization using both the physical and chemical adsorption processes. As a proof of principle, in this work we demonstrate the sensing and catalytic activity of horseradish peroxidase (HRP) anchored onto SAM modified indium tin oxide (ITO) electrodes towards hydrogen peroxide (H2O2). Structural characterization of such modified electrodes is performed using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle measurements. The binding events and the enzymatic reactions are monitored using electrochemical techniques mainly cyclic voltammetry (CV).
Daku, Rhys M.; Rabbi, Fazle; Buttigieg, Josef; Coulson, Ian M.; Horne, Derrick; Martens, Garnet; Ashton, Neil W.; Suh, Dae-Yeon
2016-01-01
Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores. PMID:26752629
Monolignol 4-O-methyltransferases and uses thereof
Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei
2014-11-18
Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...
2015-09-15
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling
NASA Astrophysics Data System (ADS)
Ma, Xuedan; Doorn, Stephen; Htoon, Han; Brener, Igal
Oxygen dopants in single-walled carbon nanotubes (SWCNTs) have recently been discovered as a novel single photon source enabling single photon generation up to room temperature in the telecom wavelength range. While they are promising for quantum information processing, it is fundamentally important to be able to manipulate their photoluminescence (PL) properties. All-dielectric metasurfaces made from arrays of high index nanoparticles have emerged as an attractive alternative to plasmonic metasurfaces due to their support of both electric and magnetic modes. Their low intrinsic losses at optical frequencies compared to that of plasmonic nanostructures provide a novel setting for tailoring emission from quantum emitters. We couple PL from single oxygen dopants in SWCNTs to the magnetic mode of silicon metasurfaces. Aside from the observation of a PL enhancement due to the Purcell effect, more interestingly, we find that the presence of the silicon metasurfaces significantly modifies the PL polarization of the dopants, which we attribute to near-field polarization modification caused by the silicon metasurfaces. Our finding presents dielectric metasurfaces as potential building blocks of photonic circuits for controlling PL intensity and polarization of single photon sources.
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.
Ye, Yongkang; Ju, Huangxian
2005-11-15
A method for rapid sensitive detection of DNA or RNA was designed using a composite screen-printed carbon electrode modified with multi-walled carbon nanotubes (MWNTs). MWNTs showed catalytic characteristics for the direct electrochemical oxidation of guanine or adenine residues of signal strand DNA (ssDNA) and adenine residues of RNA, leading to indicator-free detection of ssDNA and RNA concentrations. With an accumulation time of 5 min, the proposed method could be used for detection of calf thymus ssDNA ranging from 17.0 to 345 microg ml(-1) with a detection limit of 2.0 microg ml(-1) at 3 sigma and yeast tRNA ranging from 8.2 microg ml(-1) to 4.1 mg ml(-1). AC impedance was employed to characterize the surface of modified electrodes. The advantages of convenient fabrication, low-cost detection, short analysis time and combination with nanotechnology for increasing the sensitivity made the subject worthy of special emphasis in the research programs and sources of new commercial products.
Corrections to the thin wall approximation in general relativity
NASA Technical Reports Server (NTRS)
Garfinkle, David; Gregory, Ruth
1989-01-01
The question is considered whether the thin wall formalism of Israel applies to the gravitating domain walls of a lambda phi(exp 4) theory. The coupled Einstein-scalar equations that describe the thick gravitating wall are expanded in powers of the thickness of the wall. The solutions of the zeroth order equations reproduce the results of the usual Israel thin wall approximation for domain walls. The solutions of the first order equations provide corrections to the expressions for the stress-energy of the wall and to the Israel thin wall equations. The modified thin wall equations are then used to treat the motion of spherical and planar domain walls.
Alhans, Ruby; Singh, Anukriti; Singhal, Chaitali; Narang, Jagriti; Wadhwa, Shikha; Mathur, Ashish
2018-09-01
In the present work, a comparative study was performed between single-walled carbon nanotubes and multi-walled carbon nanotubes coated gold printed circuit board electrodes for glucose detection. Various characterization techniques were demonstrated in order to compare the modified electrodes viz. cyclic voltammetry, electrochemical impedance spectroscopy and chrono-amperometry. Results revealed that single-walled carbon nanotubes outperformed multi-walled carbon nanotubes and proved to be a better sensing interface for glucose detection. The single-walled carbon nanotubes coated gold printed circuit board electrodes showed a wide linear sensing range (1 mM to 100 mM) with detection limit of 0.1 mM with response time of 5 s while multi-walled carbon nanotubes coated printed circuit board gold electrodes showed linear sensing range (1 mM to 100 mM) with detection limit of 0.1 mM with response time of 5 s. This work provided low cost sensors with enhanced sensitivity, fast response time and reliable results for glucose detection which increased the affordability of such tests in remote areas. In addition, the comparative results confirmed that single-walled carbon nanotubes modified electrodes can be exploited for better amplification signal as compared to multi-walled carbon nanotubes. Copyright © 2018. Published by Elsevier B.V.
A sweet new role for LCP enzymes in protein glycosylation
Amer, Brendan R.; Clubb, Robert T.
2014-11-21
The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less
Zhu, Xiali; Xie, Yingxia; Zhang, Yingjie; Huang, Heqing; Huang, Shengnan; Hou, Lin; Zhang, Huijuan; Li, Zhi; Shi, Jinjin; Zhang, Zhenzhong
2014-11-01
This report focuses on the thermo-sensitive liposomes loaded with doxorubicin and lysine-modified single-walled carbon nanotube drug delivery system, which was designed to enhance the anti-tumor effect and reduce the side effects of doxorubicin. Doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes was prepared by reverse-phase evaporation method, the mean particle size was 232.0 ± 5.6 nm, and drug entrapment efficiency was 86.5 ± 3.7%. The drug release test showed that doxorubicin released more quickly at 42℃ than at 37℃. Compared with free doxorubicin, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes could efficiently cross the cell membranes and afford higher anti-tumor efficacy on the human hepatic carcinoma cell line (SMMC-7721) cells in vitro. For in vivo experiments, the relative tumor volumes of the sarcomaia 180-bearing mice in thermo-sensitive liposomes group and doxorubicin group were significantly smaller than those of N.S. group. Meanwhile, the combination of near-infrared laser irradiation at 808 nm significantly enhanced the tumor growth inhibition both on SMMC-7721 cells and the sarcomaia 180-bearing mice. The quality of life such as body weight, mental state, food and water intake of sarcomaia 180 tumor-bearing mice treated with doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes were much higher than those treated with doxorubicin. In conclusion, doxorubicin-lysine/single-walled carbon nanotube-thermo-sensitive liposomes combined with near-infrared laser irradiation at 808 nm may potentially provide viable clinical strategies for targeting delivery of anti-cancer drugs. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.
Reem, Nathan T; Chen, Han-Yi; Hur, Manhoi; Zhao, Xuefeng; Wurtele, Eve Syrkin; Li, Xu; Li, Ling; Zabotina, Olga
2018-03-01
This research provides new insights into plant response to cell wall perturbations through correlation of transcriptome and metabolome datasets obtained from transgenic plants expressing cell wall-modifying enzymes. Plants respond to changes in their cell walls in order to protect themselves from pathogens and other stresses. Cell wall modifications in Arabidopsis thaliana have profound effects on gene expression and defense response, but the cell signaling mechanisms underlying these responses are not well understood. Three transgenic Arabidopsis lines, two with reduced cell wall acetylation (AnAXE and AnRAE) and one with reduced feruloylation (AnFAE), were used in this study to investigate the plant responses to cell wall modifications. RNA-Seq in combination with untargeted metabolome was employed to assess differential gene expression and metabolite abundance. RNA-Seq results were correlated with metabolite abundances to determine the pathways involved in response to cell wall modifications introduced in each line. The resulting pathway enrichments revealed the deacetylation events in AnAXE and AnRAE plants induced similar responses, notably, upregulation of aromatic amino acid biosynthesis and changes in regulation of primary metabolic pathways that supply substrates to specialized metabolism, particularly those related to defense responses. In contrast, genes and metabolites of lipid biosynthetic pathways and peroxidases involved in lignin polymerization were downregulated in AnFAE plants. These results elucidate how primary metabolism responds to extracellular stimuli. Combining the transcriptomics and metabolomics datasets increased the power of pathway prediction, and demonstrated the complexity of pathways involved in cell wall-mediated signaling.
Mu, Xiaoyu; Qiao, Juan; Qi, Li; Liu, Ying; Ma, Huimin
2014-08-13
Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.
A microplate assay for quantitative evaluation of plant cell wall degrading enzymes
USDA-ARS?s Scientific Manuscript database
Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Plant pathogenic fungi are a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass convers...
DOE Office of Scientific and Technical Information (OSTI.GOV)
Walker, Johnnie A.; Pattathil, Sivakumar; Bergeman, Lai F.
Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss ofmore » specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides. The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.« less
Walker, Johnnie A.; Pattathil, Sivakumar; Bergeman, Lai F.; ...
2017-02-02
Glycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions. Single, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss ofmore » specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides. The identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass.« less
Gan, Ning; Yang, Xin; Xie, Donghua; Wu, Yuanzhao; Wen, Weigang
2010-01-01
A disposable organophosphorus pesticides (OPs) enzyme biosensor based on magnetic composite nanoparticle-modified screen printed carbon electrodes (SPCE) has been developed. Firstly, an acetylcholinesterase (AChE)-coated Fe3O4/Au (GMP) magnetic nanoparticulate (GMP-AChE) was synthesized. Then, GMP-AChE was absorbed on the surface of a SPCE modified by carbon nanotubes (CNTs)/nano-ZrO2/prussian blue (PB)/Nafion (Nf) composite membrane by an external magnetic field. Thus, the biosensor (SPCE│CNTs/ZrO2/PB/Nf│GMP-AChE) for OPs was fabricated. The surface of the biosensor was characterized by scanning electron micrography (SEM) and X-ray fluorescence spectrometery (XRFS) and its electrochemical properties were studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The degree of inhibition (A%) of the AChE by OPs was determined by measuring the reduction current of the PB generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh). In pH = 7.5 KNO3 solution, the A was related linearly to the concentration of dimethoate in the range from 1.0 × 10−3–10 ng·mL−1 with a detection limit of 5.6 × 10−4 ng·mL−1. The recovery rates in Chinese cabbage exhibited a range of 88%–105%. The results were consistent with the standard gas chromatography (GC) method. Compared with other enzyme biosensors the proposed biosensor exhibited high sensitivity, good selectivity with disposable, low consumption of sample. In particular its surface can be easily renewed by removal of the magnet. The convenient, fast and sensitive voltammetric measurement opens new opportunities for OPs analysis. PMID:22315558
Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency.
Montalbano, Antonino; Juergensen, Lonny; Roeth, Ralph; Weiss, Birgit; Fukami, Maki; Fricke-Otto, Susanne; Binder, Gerhard; Ogata, Tsutomu; Decker, Eva; Nuernberg, Gudrun; Hassel, David; Rappold, Gudrun A
2016-12-01
Mutations in the homeobox gene SHOX cause SHOX deficiency, a condition with clinical manifestations ranging from short stature without dysmorphic signs to severe mesomelic skeletal dysplasia. In rare cases, individuals with SHOX deficiency are asymptomatic. To elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with SHOX deficiency. The variant p.Phe508Cys of the retinoic acid catabolizing enzyme CYP26C1 co-segregated with the SHOX variant p.Val161Ala in the affected individuals, while the SHOX mutant alone was present in asymptomatic individuals. Two further cases with SHOX deficiency and damaging CYP26C1 variants were identified in a cohort of 68 individuals with LWD The identified CYP26C1 variants affected its catabolic activity, leading to an increased level of retinoic acid. High levels of retinoic acid significantly decrease SHOX expression in human primary chondrocytes and zebrafish embryos. Individual morpholino knockdown of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. Together, our findings describe CYP26C1 as the first genetic modifier for SHOX deficiency. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.
Role of Melatonin in Cell-Wall Disassembly and Chilling Tolerance in Cold-Stored Peach Fruit.
Cao, Shifeng; Bian, Kun; Shi, Liyu; Chung, Hsiao-Hang; Chen, Wei; Yang, Zhenfeng
2018-06-06
Melatonin reportedly increases chilling tolerance in postharvest peach fruit during cold storage, but information on its effects on cell-wall disassembly in chilling-injured peaches is limited. In this study, we investigated the role of cell-wall depolymerization in chilling-tolerance induction in melatonin-treated peaches. Treatment with 100 μM melatonin alleviated chilling symptoms (mealiness) characterized by a decrease in fruit firmness and increase in juice extractability in treated peaches during storage. The loss of neutral sugars, such as arabinose and galactose, in both the 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA)- and Na 2 CO 3 -soluble fractions was observed at 7 days in treated peaches, but the contents increased after 28 days of storage. Atomic-force-microscopy (AFM) analysis revealed that the polysaccharide widths in the CDTA- and Na 2 CO 3 -soluble fractions in the treated fruit were mainly distributed in a shorter range, as compared with those in the control fruit. In addition, the expression profiles of a series of cell-wall-related genes showed that melatonin treatment maintained the balance between transcripts of PpPME and PpPG, which accompany the up-regulation of several other genes involved in cell-wall disassembly. Taken together, our results suggested that the reduced mealiness by melatonin was probably associated with its positive regulation of numerous cell-wall-modifying enzymes and proteins; thus, the depolymerization of the cell-wall polysaccharides in the peaches treated with melatonin was maintained, and the treated fruit could soften gradually during cold storage.
Stabilization of enzymes in ionic liquids via modification of enzyme charge.
Nordwald, Erik M; Kaar, Joel L
2013-09-01
Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.
Ao, Jie; Free, Stephen J
2017-04-01
The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. Copyright © 2017 Elsevier Inc. All rights reserved.
Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki; Kotake, Toshihisa; Yamazaki, Takashi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Fukui, Keiji; Osada, Ikuko; Kasahara, Haruo; Kamada, Motoshi
2015-01-01
Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions. PMID:26378793
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tittmann, B. R.; Xi, X.
This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which weremore » sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical
Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bartley, Laura; Wu, Y.; Zhu, L.
. These markers might be used to select switchgrass genotypes with improved composition in breeding programs for biofuel and forage production. Because the SSAC continues to be characterized by collaborators in the bioenergy community, the data generated will be used to identify additional markers in higher resolution genotyping data to approach identifying the genes and alleles that cause natural variation in switchgrass cell wall quality. For example, these markers can be surveyed in the 2100-member Oklahoma Southern and Northern Lowland switchgrass collections that this project also characterized. An orthogonal approach to biodiversity studies, using comparative functional genomics permits systematic querying of how much regulatory information is likely to be transferable from dicots to grasses and use of accumulated functional genomics resources for better-characterized grass species, such as rice, itself a biomass source in global agriculture and in certain regions. The project generated and tested a number of specific hypotheses regarding cell wall transcription factors and enzymes of grasses. To aid identification of cell wall regulators, the project assembled a novel, highdepth and -quality gene association network using a general linearized model scoring system to combine rice gene network data. Using known or putative orthologs of Arabidopsis cell wall biosynthesis genes and regulators, the project pulled from this network a cell wall sub-network that includes 96 transcription factors. Reverse genetics of a co-ortholog of the Arabidopsis MYB61 transcription factor in rice revealed that this regulatory node has evolved the ability to regulate grass-specific cell wall synthesis enzymes. A transcription factor with such activity has not been previously characterized to our knowledge, representing a major conclusion of this work. Changes in gene expression in a protoplast-based assay demonstrated positive or negative roles in cell wall regulation for eleven other
Phage lytic enzymes: a history.
Trudil, David
2015-02-01
There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of 'bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.
Zhang, J.; Bruton, B. D.; Biles, C. L.
2014-01-01
Didymella bryoniae is an important pathogen of cucurbits worldwide. Virulence factors of D. bryoniae were investigated in regard to fungal growth and the production of cell wall-degrading enzymes, polygalacturonase (PG), pectate lyase (PL), pectin lyase (PNL), β-galactosidase (β-Gal) and cellulase (Cx). Virulence levels of five D. bryoniae isolates were determined by the severity of inoculated cantaloupe fruit decay. The highly virulent isolates had more mycelial growth than the moderately virulent isolates in different media. PG activities produced by the highly virulent isolates in shake cultures and in decayed fruit were greater than those of the moderately virulent isolates. PNL, but not PL, in decayed fruit was higher with the highly virulent isolates compared to the moderately virulent ones. The highly virulent isolates showed higher Cx activity than the moderately virulent ones in decayed fruit and in fruit tissue shake culture. β-Gal activities of the highly virulent isolates in pectin shake culture and in decayed fruit were greater than those of the two moderately virulent isolates although fruit also produced β-Gal. Protein analysis showed two fungal β-Gal isozymes in decayed fruit compared to those of healthy fruit. Correlation analysis indicated that the activities of PG, PNL, β-Gal and Cx in cultures and in decayed fruit positively correlated with fungal growth and fruit decay severity. The results of this study suggest that PG, PNL, β-Gal, and Cx appear to be virulence factors of D. bryoniae in cantaloupe decay with PG and β-Gal as the most predominant fruit decay enzymes. PMID:25364138
Evolutionary transitions in enzyme activity of ant fungus gardens.
De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J
2010-07-01
Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.
Li, Junhua; Zhang, Fuxing; Wang, Jianqiu; Xu, Zhifeng; Zeng, Rongying
2009-05-01
Due to the strong cation-exchange ability of Nafion and the excellent properties of multi-walled carbon nanotubes (MWCNTs), a highly sensitive and mercury-free method of determining trace levels of In(3+) has been established based on the bifunctionality of a MWCNTs/Nafion modified glassy carbon electrode (GCE). The MWCNTs/Nafion modified GCE detects In(3+) in a 0.01 M HAc-NaAc buffer solution at pH 5.0 using anodic stripping voltammetry (ASV). The experimental results suggest that a sensitive anodic stripping peak appears at -0.58 V on anodic stripping voltammograms, which can be used as an analytical signal for the determination of In(3+). A good linear relationship between the stripping peak currents and the In(3+) concentration is obtained, covering the concentration range from 5.0 x 10(-10) to 2.0 x 10(-7) M, with a correlation coefficient of 0.999; the detection limit is 1.0 x 10(-11) M. This proposed method has been applied to detect In(3+) as a new way.
Niu, Meng; Hou, Gary G; Kindelspire, Julie; Krishnan, Padmanaban; Zhao, Siming
2017-05-15
With the utilization of enzymes including endoxylanase, glucose oxidase (GOX) and transglutaminase (TG), and emulsifiers comprising sodium stearoyl lactate (SSL) and soy lecithin, the microstructural, textural, and sensory properties of whole-wheat noodle (WWN) were modified. The development time and stability of whole-wheat dough (WWD) were enhanced by TG due to the formation of a more compact gluten network, and by SSL resulting from the enhanced gluten strength. Microstructure graphs by scanning electron microscopy (SEM) verified that TG and SSL promoted the connectivity of gluten network and the coverage of starch granules in WWN. TG increased the hardness and elasticity of cooked WWN, while two emulsifiers increased the noodle cohesiveness. Additionally, TG and SSL improved the sensory properties of noodle such as bite, springiness, and mouth-feel. The results suggest that TG and SSL are effective ingredients in enhancing the gluten strength of WWD and improving the qualities of WWN. Copyright © 2016 Elsevier Ltd. All rights reserved.
Moola, Z B; Scawen, M D; Atkinson, T; Nicholls, D J
1994-01-01
This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme. PMID:7945221
[Two-wall decompression without resection of the medial wall. Effect on squint angle].
Bertelmann, E; Rüther, K
2011-11-01
Postoperative new onset diplopia can be a disadvantage for surgical orbital decompression in patients with exophthalmos in thyroid eye disease. The various modifications of decompression (number and combination of walls) differ in their influence on the postoperative squint angle. We report on postoperative diplopia in a modified 2 wall decompression strategy (lateral wall and floor). This study was a retrospective analysis of 36 consecutive 2-wall decompressions performed between 2006-2010 in 24 patients with 6 months of stable exophthalmos in thyroid eye disease after medical therapy and radiotherapy. The preoperative and postoperative squint angle in prism cover test (PCT), motility, induction of diplopia, reduction of exophthalmos, visual acuity and complications were evaluated. In all 36 decompressions the postoperative squint angle was equal to or less than before surgery. In 8 eyes additional squint surgery was performed. The mean reduction in exopthalmos was 4.3 mm. An adverse effect of decompression on the postoperative squint angle was not evident in this study. New induction of diplopia was not observed at all. One possible explanation is the preservation of the medial wall.
Mold Pectinase Modified with Dialdehyde Derivatives of Dextran and Cellulose.
Kobayashi, M; Chiba, Y; Funane, K; Ohya, S; Kato, Y
1996-01-01
Chemical modification of mold pectinase with dextran- and cellulose-dialdehydes was examined to improve the enzyme characteristics. The modified pectinase with dextran-dialdehyde retained about 50% of the original activity, and more than 80% of the total amino groups were modified. HPLC gel filtration analysis showed an increase in molecular weight of the reaction product. Reaction with cellulose-dialdehyde provided an immobilized form of pectinase. The immobilized pectinase was resistant to both acidic and alkaline pHs, and also acquired heat stability at 60°C. The optimum pH of the modified enzyme shifted from pH 4.5 to 5.0-5.5, and this enzyme had higher activity at neutral pH regions than the native enzyme. A rather low recovery of immobilized enzyme (14.5%) should be improved by the combination with various methods hitherto established.
Ziyatdinova, Guzel; Kozlova, Ekaterina; Budnikov, Herman
2016-04-01
Phenolic antioxidants of wine were electrochemically oxidized on multi-walled carbon nanotubes modified glassy carbon electrode (MWNT/GCE) in phosphate buffer solution. Three oxidation peaks were observed at 0.39, 0.61 and 0.83V for red dry wine and 0.39, 0.80 and 1.18 V for white dry wine, respectively, using differential pulse voltammetry at pH 4.0. The oxidation potentials for individual phenolic antioxidants confirmed the integral nature of the analytical signals for the wines examined. A one-step chronocoulometric method at 0.83 and 1.18 V for red and white wines, respectively, has been developed for the evaluation of wine antioxidant capacity (AOC). The AOC is expressed in gallic acid equivalents per 1L of wine. The AOC of white wine was significantly less than red wine (386 ± 112 vs. 1224 ± 184, p<0.0001), as might be expected. Positive correlations were observed between gallic acid equivalent AOC of wine and total antioxidant capacity, based on coulometric titration with electrogenerated bromine (r=0.8957 at n=5 and r=0.8986 at n=4 for red and white wines, respectively). Copyright © 2015 Elsevier Ltd. All rights reserved.
Synthesis and Characterization of Magnetic Carriers Based on Immobilized Enzyme
NASA Astrophysics Data System (ADS)
Li, F. H.; Tang, N.; Wang, Y. Q.; Zhang, L.; Du, W.; Xiang, J.; Cheng, P. G.
2018-05-01
Several new types of carriers and technologies have been implemented to improve traditional enzyme immobilization in industrial biotechnology. The magnetic immobilized enzyme is a kind of new method of enzyme immobilization developed in recent years. An external magnetic field can be used to control the motion mode and direction of immobilized enzyme, and to improve the catalytic efficiency of immobilized enzyme. In this paper, Fe3O4-CaCO3-PDA complex and CaCO3/Fe3O4 composite modified by PEI were prepared. The results show that the morphology of Fe3O4-CaCO3-PDA complex formation is irregular, while the morphology of CaCO3/Fe3O4 composite modified by PEI is regular and has a porous structure.
Enzyme-Less Growth in Chara and Terrestrial Plants
Boyer, John S.
2016-06-21
Enzyme-less chemistry appears to control the growth rate of the green alga Chara corallina. The chemistry occurs in the wall where a calcium pectate cycle determines both the rate of wall enlargement and the rate of pectate deposition into the wall. The process is the first to indicate that a wall polymer can control how a plant cell enlarges after exocytosis releases the polymer to the wall. This raises the question of whether other species use a similar mechanism. Chara is one of the closest relatives of the progenitors of terrestrial plants and during the course of evolution, new wallmore » features evolved while pectate remained one of the most conserved components. In addition, charophytes contain auxin which affects Chara in ways resembling its action in terrestrial plants. Furthermore, this review considers whether more recently acquired wall features require different mechanisms to explain cell expansion.« less
Enzyme-Less Growth in Chara and Terrestrial Plants
DOE Office of Scientific and Technical Information (OSTI.GOV)
Boyer, John S.
Enzyme-less chemistry appears to control the growth rate of the green alga Chara corallina. The chemistry occurs in the wall where a calcium pectate cycle determines both the rate of wall enlargement and the rate of pectate deposition into the wall. The process is the first to indicate that a wall polymer can control how a plant cell enlarges after exocytosis releases the polymer to the wall. This raises the question of whether other species use a similar mechanism. Chara is one of the closest relatives of the progenitors of terrestrial plants and during the course of evolution, new wallmore » features evolved while pectate remained one of the most conserved components. In addition, charophytes contain auxin which affects Chara in ways resembling its action in terrestrial plants. Furthermore, this review considers whether more recently acquired wall features require different mechanisms to explain cell expansion.« less
Modification of enzymes by use of high-pressure homogenization.
Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi
2018-07-01
High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.
Ripening-induced changes in grape skin proanthocyanidins modify their interaction with cell walls.
Bindon, Keren A; Kennedy, James A
2011-03-23
Proanthocyanidins were isolated from the skins of Cabernet Sauvignon grapes at different stages of grape development in order to study the effect of proanthocyanidin modification on the interaction with grape cell wall material. After veraison, the degree of proanthocyanidin polymerization increased, and thereafter was variable between 24 and 33 subunits as ripening progressed. Affinity of skin cell wall material for proanthocyanidin decreased with proanthocyanidin ripeness following veraison. A significant negative relationship (R2=0.93) was found for average proanthocyanidin molecular mass and the proportion of high molecular mass proanthocyanidin adsorbed by skin cell wall material. This indicated that as proanthocyanidin polymerization increased, the affinity of a component of high molecular mass proanthocyanidins for skin cell wall material declined. This phenomenon was only associated with skin proanthocyanidins from colored grapes, as high molecular mass proanthocyanidins of equivalent subunit composition from colorless mutant Cabernet Sauvignon grapes had a higher affinity for skin cell wall material.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Byrne, Robert T.; Whelan, Fiona; Aller, Pierre
2013-06-01
The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group. Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo{sup 5}U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo{sup 5}U and was annotated as an S-adenosyl-l-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichiamore » coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-l-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry http://scripts.iucr.org/cgi-bin/cr.cgi?rm, suggests that the active site contains SCM-SAH and not SAM.« less
Plant cell wall engineering: applications in biofuel production and improved human health.
Burton, Rachel A; Fincher, Geoffrey B
2014-04-01
Plant cell walls consist largely of cellulose, non-cellulosic polysaccharides and lignin. Concerted attempts are underway to convert wall polysaccharides from crop plant residues into renewable transport fuels and other valuable products, and to exploit the dietary benefits of cereal grain wall polysaccharides in human health. Attempts to improve plant performance for these applications have involved the manipulation of the levels and structures of wall components. Some successes in altering non-cellulosic polysaccharides has been achieved, but it would appear that drastic changes in cellulose are more difficult to engineer. Nevertheless, future prospects for both genetically modified (GM) and non-GM technologies to modify plant cell wall composition and structure remain bright, and will undoubtedly find applications beyond the current focus on human health and biofuel production. Copyright © 2013. Published by Elsevier Ltd.
Intein-modified enzymes, their production and industrial applications
Apgar, James; Lessard, Philip; Raab, Michael R.; Shen, Binzhang; Lazar, Gabor; de la Vega, Humberto
2016-10-11
A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/ sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-.beta.-sheet junction or a loop-.alpha.-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.
Scott, Alison J; Oyler, Benjamin L; Goodlett, David R; Ernst, Robert K
2017-11-01
Strategies utilizing Toll-like receptor 4 (TLR4) agonists for treatment of cancer, infectious diseases, and other targets report promising results. Potent TLR4 antagonists are also gaining attention as therapeutic leads. Though some principles for TLR4 modulation by lipid A have been described, a thorough understanding of the structure-activity relationship (SAR) is lacking. Only through a complete definition of lipid A-TLR4 SAR is it possible to predict TLR4 signaling effects of discrete lipid A structures, rendering them more pharmacologically relevant. A limited 'toolbox' of lipid A-modifying enzymes has been defined and is largely composed of enzymes from mesophile human and zoonotic pathogens. Expansion of this 'toolbox' will result from extending the search into lipid A biosynthesis and modification by bacteria living at the extremes. Here, we review the fundamentals of lipid A structure, advances in lipid A uses in TLR4 modulation, and the search for novel lipid A-modifying systems in extremophile bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2017 Elsevier B.V. All rights reserved.
Enzyme-assisted extraction of bioactives from plants.
Puri, Munish; Sharma, Deepika; Barrow, Colin J
2012-01-01
Demand for new and novel natural compounds has intensified the development of plant-derived compounds known as bioactives that either promote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimized using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Here, we discuss the use of environmentally friendly enzyme-assisted extraction of bioactive compounds from plant sources, particularly for food and nutraceutical purposes. In particular, we discuss an enzyme-assisted extraction of stevioside from Stevia rebaudiana, as an example of a process of potential value to the food industry. Copyright © 2011 Elsevier Ltd. All rights reserved.
ZnO-Based Amperometric Enzyme Biosensors
Zhao, Zhiwei; Lei, Wei; Zhang, Xiaobing; Wang, Baoping; Jiang, Helong
2010-01-01
Nanostructured ZnO with its unique properties could provide a suitable microenvironment for immobilization of enzymes while retaining their biological activity, and thus lead to an expanded use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance. ZnO-based enzyme electrochemical biosensors are summarized in several tables for an easy overview according to the target biosensing analyte (glucose, hydrogen peroxide, phenol and cholesterol), respectively. Moreover, recent developments in enzyme electrochemical biosensors based on ZnO nanomaterials are reviewed with an emphasis on the fabrications and features of ZnO, approaches for biosensor construction (e.g., modified electrodes and enzyme immobilization) and biosensor performances. PMID:22205864
Considerations on the Use of Exogenous Fibrolytic Enzymes to Improve Forage Utilization
Mendoza, Germán D.; Plata-Pérez, Fernando X.
2014-01-01
Digestion of cell wall fractions of forage in the rumen is incomplete due to the complex links which limit their degradation. It is therefore necessary to find options to optimize the use of forages in ruminant production systems. One alternative is to use exogenous enzymes. Exogenous fibrolytic enzymes are of fungal or bacterial origin and increase nutrient availability from the cell wall, which consists of three fractions in different proportions depending on the species of forage: digestible, potentially digestible, and indigestible. The response to addition of exogenous enzymes varies with the type of forage; many researchers infer that there are enzyme-forage interactions but fail to explain the biological mechanism. We hypothesize that the response is related to the proportion of the potentially digestible fraction. The exogenous enzyme activity depends on several factors but if the general conditions for enzyme action are available, the potentially digestible fraction may determine the magnitude of the response. Results of experiments with exogenous fibrolytic enzymes in domestic ruminants are inconsistent. This, coupled with their high cost, has made their use unattractive to farmers. Development of cheaper products exploring other microorganisms with fibrolytic activity, such as Fomes fomentarius or Cellulomonas flavigena, is required. PMID:25379525
Troian, Rogério Fraga; Steindorff, Andrei Stecca; Ramada, Marcelo Henrique Soller; Arruda, Walquiria; Ulhoa, Cirano José
2014-10-01
Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.
Xia, Xue; Zhang, Hui-Ming; Offler, Christina E.; Patrick, John W.
2017-01-01
Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated. PMID:29259611
Xia, Xue; Zhang, Hui-Ming; Offler, Christina E; Patrick, John W
2017-01-01
Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans -differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta . Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.
Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.
Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard
2014-10-01
Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.
Enzyme-coated mesoporous silica nanoparticles as efficient antibacterial agents in vivo.
Li, Li-Li; Wang, Hao
2013-10-01
Despite the fact that pathogenic infections are widely treated by antibiotics in the clinic nowadays, the increasing risk of multidrug-resistance associated with abuse of antibiotics is becoming a major concern in global public health. The increased death toll caused by pathogenic bacterial infection calls for effective antibiotic alternatives. Lysozyme-coated mesoporous silica nanoparticles (MSNs⊂Lys) are reported as antibacterial agents that exhibit efficient antibacterial activity both in vitro and in vivo with low cytotoxicity and negligible hemolytic side effect. The Lys corona provides multivalent interaction between MSNs⊂Lys and bacterial walls and consequently raises the local concentration of Lys on the surface of cell walls, which promotes hydrolysis of peptidoglycans and increases membrane-perturbation abilities. The minimal inhibition concentration (MIC) of MSNs⊂Lys is fivefold lower than that of free Lys in vitro. The antibacterial efficacy of MSNs⊂Lys is evaluated in vivo by using an intestine-infected mouse model. Experimental results indicate that the number of bacteria surviving in the colon is three orders of magnitude lower than in the untreated group. These natural antibacterial enzyme-modified nanoparticles open up a new avenue for design and synthesis of next-generation antibacterial agents as alternatives to antibiotics. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Two endogenous proteins that induce cell wall extension in plants
NASA Technical Reports Server (NTRS)
McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.
1992-01-01
Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.
NASA Astrophysics Data System (ADS)
Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder
2016-02-01
Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.
A transonic wind tunnel wall interference prediction code
NASA Technical Reports Server (NTRS)
Phillips, Pamela S.; Waggoner, Edgar G.
1988-01-01
A small disturbance transonic wall interference prediction code has been developed that is capable of modeling solid, open, perforated, and slotted walls as well as slotted and solid walls with viscous effects. This code was developed by modifying the outer boundary conditions of an existing aerodynamic wing-body-pod-pylon-winglet analysis code. The boundary conditions are presented in the form of equations which simulate the flow at the wall, as well as finite difference approximations to the equations. Comparisons are presented at transonic flow conditions between computational results and experimental data for a wing alone in a solid wall wind tunnel and wing-body configurations in both slotted and solid wind tunnels.
Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum
DOE Office of Scientific and Technical Information (OSTI.GOV)
D /Souza, T.M.; Merritt, C.S.; Reddy, C.A.
1999-12-01
Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen shaken cultures were much greater than those seen in low-nitrogen, malt extract, or wool-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units aremore » structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HM cultures showed two laccase activity bands, where as isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, and 5.1. Low levels of MnP activity were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum.« less
Injuries on British climbing walls.
Limb, D
1995-01-01
A postal survey was carried out of the 90 most accessible climbing walls in England, Scotland and Wales to determine the incidence and nature of injuries requiring emergency treatment associated with their use. Over a two year period, representing 1.021 million visits to the 56 walls used by more than 30 climbers per week, 55 significant injuries were recorded. The rate of injury was not related to any identified design or safety feature of the walls, although upper limb injuries were proportionally more common in walls which provided thinner fixed landing mats rather than thicker, moveable crash mats. The overall rate of injury was very low and climbers seem to modify risk taking behaviour and thus compensate for the level of safety equipment available. It may be possible to reduce the injury rate further by providing seamless ground cover with matting of adequate energy absorbency. Images Figure 1 PMID:8800849
Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko
2015-11-15
The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.
How Do Enzymes 'Meet' Nanoparticles and Nanomaterials?
Chen, Ming; Zeng, Guangming; Xu, Piao; Lai, Cui; Tang, Lin
2017-11-01
Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial-enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.
Navarre, William Wiley; Schneewind, Olaf
1999-01-01
The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836
Microbial Enzymes: Tools for Biotechnological Processes
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
Modified Single-Wall Carbon Nanotubes for Reinforce Thermoplastic Polyimide
NASA Technical Reports Server (NTRS)
Lebron-COlon, Marisabel; Meador, Michael A.
2006-01-01
A significant improvement in the mechanical properties of the thermoplastic polyimide film was obtained by the addition of noncovalently functionalized single-wall carbon nanotubes (SWNTs). Polyimide films were reinforced using pristine SWNTs and functionalized SWNTs (F-SWNTs). The tensile strengths of the polyimide films containing F-SWNTs were found to be approximately 1.4 times higher than those prepared from pristine SWNTs.
NASA Astrophysics Data System (ADS)
Yang, Xiang I. A.; Park, George Ilhwan; Moin, Parviz
2017-10-01
Log-layer mismatch refers to a chronic problem found in wall-modeled large-eddy simulation (WMLES) or detached-eddy simulation, where the modeled wall-shear stress deviates from the true one by approximately 15 % . Many efforts have been made to resolve this mismatch. The often-used fixes, which are generally ad hoc, include modifying subgrid-scale stress models, adding a stochastic forcing, and moving the LES-wall-model matching location away from the wall. An analysis motivated by the integral wall-model formalism suggests that log-layer mismatch is resolved by the built-in physics-based temporal filtering. In this work we investigate in detail the effects of local filtering on log-layer mismatch. We show that both local temporal filtering and local wall-parallel filtering resolve log-layer mismatch without moving the LES-wall-model matching location away from the wall. Additionally, we look into the momentum balance in the near-wall region to provide an alternative explanation of how LLM occurs, which does not necessarily rely on the numerical-error argument. While filtering resolves log-layer mismatch, the quality of the wall-shear stress fluctuations predicted by WMLES does not improve with our remedy. The wall-shear stress fluctuations are highly underpredicted due to the implied use of LES filtering. However, good agreement can be found when the WMLES data are compared to the direct numerical simulation data filtered at the corresponding WMLES resolutions.
Leurs, Melanie; Tiller, Joerg C
2017-01-01
The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.
Qiu, Bin; Lin, Zhenyu; Wang, Jian; Chen, Zhihuang; Chen, Jinhua; Chen, Guonan
2009-04-15
A poly(nickel(II) tetrasulfophthalocyanine)/multi-walled carbon nanotubes composite modified electrode (polyNiTSPc/MWNTs) was fabricated by electropolymerization of NiTSPc on MWNTs-modified glassy carbon electrode (GCE). The modified electrode was found to be able to greatly improve the emission of luminol electrochemiluminescence (ECL) in a solution containing hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the surface of polyNiTSPc/MWNTs modified GC electrode by Nafion to establish an ECL glucose sensor. Under the optimum conditions, the linear response range of glucose was 1.0x10(-6) to 1.0x10(-4) mol L(-1) with a detection limit of 8.0x10(-8) mol L(-1) (defined as the concentration that could be detected at the signal-to-noise ratio of 3). The ECL sensor showed an outstanding well reproducibility and long-term stability. The established method has been applied to determine the glucose concentrations in real serum samples with satisfactory results.
Recent advances in material science for developing enzyme electrodes.
Sarma, Anil Kumar; Vatsyayan, Preety; Goswami, Pranab; Minteer, Shelley D
2009-04-15
The enzyme-modified electrode is the fundamental component of amperometric biosensors and biofuel cells. The selection of appropriate combinations of materials, such as: enzyme, electron transport mediator, binding and encapsulation materials, conductive support matrix and solid support, for construction of enzyme-modified electrodes governs the efficiency of the electrodes in terms of electron transfer kinetics, mass transport, stability, and reproducibility. This review investigates the varieties of materials that can be used for these purposes. Recent innovation in conductive electro-active polymers, functionalized polymers, biocompatible composite materials, composites of transition metal-based complexes and organometallic compounds, sol-gel and hydro-gel materials, nanomaterials, other nano-metal composites, and nano-metal oxides are reviewed and discussed here. In addition, the critical issues related to the construction of enzyme electrodes and their application for biosensor and biofuel cell applications are also highlighted in this article. Effort has been made to cover the recent literature on the advancement of materials sciences to develop enzyme electrodes and their potential applications for the construction of biosensors and biofuel cells.
Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates
NASA Astrophysics Data System (ADS)
Fuhrmann, Gregor; Grotzky, Andrea; Lukić, Ružica; Matoori, Simon; Luciani, Paola; Yu, Hao; Zhang, Baozhong; Walde, Peter; Schlüter, A. Dieter; Gauthier, Marc A.; Leroux, Jean-Christophe
2013-07-01
Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.
Subpolar addition of new cell wall is directed by DivIVA in mycobacteria
Meniche, Xavier; Otten, Renee; Siegrist, M. Sloan; Baer, Christina E.; Murphy, Kenan C.; Bertozzi, Carolyn R.; Sassetti, Christopher M.
2014-01-01
Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension. PMID:25049412
Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder
2016-02-05
Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.
Reconstitution of a secondary cell wall in a secondary cell wall-deficient Arabidopsis mutant.
Sakamoto, Shingo; Mitsuda, Nobutaka
2015-02-01
The secondary cell wall constitutes a rigid frame of cells in plant tissues where rigidity is required. Deposition of the secondary cell wall in fiber cells contributes to the production of wood in woody plants. The secondary cell wall is assembled through co-operative activities of many enzymes, and their gene expression is precisely regulated by a pyramidal cascade of transcription factors. Deposition of a transmuted secondary cell wall in empty fiber cells by expressing selected gene(s) in this cascade has not been attempted previously. In this proof-of-concept study, we expressed chimeric activators of 24 transcription factors that are preferentially expressed in the stem, in empty fiber cells of the Arabidopsis nst1-1 nst3-1 double mutant, which lacks a secondary cell wall in fiber cells, under the control of the NST3 promoter. The chimeric activators of MYB46, SND2 and ANAC075, as well as NST3, reconstituted a secondary cell wall with different characteristics from those of the wild type in terms of its composition. The transgenic lines expressing the SND2 or ANAC075 chimeric activator showed increased glucose and xylose, and lower lignin content, whereas the transgenic line expressing the MYB46 chimeric activator showed increased mannose content. The expression profile of downstream genes in each transgenic line was also different from that of the wild type. This study proposed a new screening strategy to identify factors of secondary wall formation and also suggested the potential of the artificially reconstituted secondary cell walls as a novel raw material for production of bioethanol and other chemicals. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
Pauchet, Y; Saski, C A; Feltus, F A; Luyten, I; Quesneville, H; Heckel, D G
2014-06-01
The ability of herbivorous beetles from the superfamilies Chrysomeloidea and Curculionoidea to degrade plant cell wall polysaccharides has only recently begun to be appreciated. The presence of plant cell wall degrading enzymes (PCWDEs) in the beetle's digestive tract makes this degradation possible. Sequences encoding these beetle-derived PCWDEs were originally identified from transcriptomes and strikingly resemble those of saprophytic and phytopathogenic microorganisms, raising questions about their origin; e.g. are they insect- or microorganism-derived? To demonstrate unambiguously that the genes encoding PCWDEs found in beetle transcriptomes are indeed of insect origin, we generated a bacterial artificial chromosome library from the genome of the leaf beetle Chrysomela tremula, containing 18 432 clones with an average size of 143 kb. After hybridizing this library with probes derived from 12 C. tremula PCWDE-encoding genes and sequencing the positive clones, we demonstrated that the latter genes are encoded by the insect's genome and are surrounded by genes possessing orthologues in the genome of Tribolium castaneum as well as in three other beetle genomes. Our analyses showed that although the level of overall synteny between C. tremula and T. castaneum seems high, the degree of microsynteny between both species is relatively low, in contrast to the more closely related Colorado potato beetle. © 2014 The Royal Entomological Society.
Ng, Jovyn K T; Schröder, Roswitha; Brummell, David A; Sutherland, Paul W; Hallett, Ian C; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W
2015-03-15
Substantial differences in softening behaviour can exist between fruit even within the same species. Apple cultivars 'Royal Gala' and 'Scifresh' soften at different rates despite having a similar genetic background and producing similar amounts of ethylene during ripening. An examination of cell wall metabolism from the fruitlet to the ripe stages showed that in both cultivars pectin solubilisation increased during cell expansion, declined at the mature stage and then increased again during ripening. This process was much less pronounced in the slower softening 'Scifresh' than in 'Royal Gala' at every developmental stage examined, consistent with less cell separation and softening in this cultivar. Both cultivars also exhibited a progressive loss of pectic galactan and arabinan side chains during development. The cell wall content of arabinose residues was similar in both cultivars, but the galactose residue content in 'Scifresh' remained higher than that of 'Royal Gala' at every developmental stage. The higher content of cell wall galactose residue in 'Scifresh' cell walls correlated with a lower β-galactosidase activity and more intense immunolabelling of RG-I galactan side chains in both microscopy sections and glycan microarrays. A high cell wall galactan content has been associated with reduced cell wall porosity, which may restrict access of cell wall-modifying enzymes and thus maintain better structural integrity later in development. The data suggest that the composition and structure of the cell wall at very early development stages may influence subsequent cell wall loosening, and may even predispose the wall's ensuing properties. Copyright © 2014 Elsevier GmbH. All rights reserved.
Bi, Xiaodong; Liu, Zhen
2014-12-16
Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.
Mitu, Shahida Akter; Bose, Utpal; Suwansa-Ard, Saowaros; Turner, Luke H; Zhao, Min; Elizur, Abigail; Ogbourne, Steven M; Shaw, Paul Nicholas; Cummins, Scott F
2017-11-07
The sea cucumber (phylum Echinodermata) body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra ; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.
Assembly and enlargement of the primary cell wall in plants
NASA Technical Reports Server (NTRS)
Cosgrove, D. J.
1997-01-01
Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.
Assembly and enlargement of the primary cell wall in plants.
Cosgrove, D J
1997-01-01
Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.
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. © 2015 Wiley Periodicals, Inc.
Second-order near-wall turbulence closures - A review
NASA Technical Reports Server (NTRS)
So, R. M. C.; Lai, Y. G.; Zhang, H. S.; Hwang, B. C.
1991-01-01
Advances in second-order near-wall turbulence closures are summarized. All closures under consideration are based on high-Reynolds-number models. Most near-wall closures proposed to date attempt to modify the high-Reynolds-number models for the dissipation function and the pressure redistribution term so that the resultant models are applicable all the way to the wall. The asymptotic behavior of the near-wall closures is examined and compared with the proper near-wall behavior of the exact Reynolds-stress equations. It is found that three second-order near-wall closures give the best correlations with simulated turbulence statistics. However, their predictions of near-wall Reynolds-stress budgets are considered to be incorrect. A proposed modification to the dissipitation-rate equation remedies part of those predictions. It is concluded that further improvements are required if a complete replication of all the turbulence properties and Reynolds-stress budgets by a statistical model of turbulence is desirable.
Chang, Hao-Xun; Yendrek, Craig R; Caetano-Anolles, Gustavo; Hartman, Glen L
2016-07-12
Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2
A review of near-wall Reynolds-stress
NASA Technical Reports Server (NTRS)
So, R. M. C.; Lai, Y. G.; Zhang, H. S.; Hwang, B. C.
1991-01-01
The advances made in second-order near-wall turbulence closures are summarized. All closures examined are based on some form of high Reynolds number models for the Reynolds stress and the turbulent kinetic energy dissipation rate equations. Consequently, most near-wall closures proposed to data attempt to modify the high Reynolds number models for the dissipation rate equation so that the resultant models are applicable all the way to the wall. The near-wall closures are examined for their asymptotic behavior so that they can be compared with the proper near-wall behavior of the exact equations. A comparison of the closure's performance in the calculation of a low Reynolds number plane channel flow is carried out. In addition, the closures are evaluated for their ability to predict the turbulence statistics and the limiting behavior of the structure parameters compared to direct simulation data.
2012-01-01
Background Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization), were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth. Results Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran. Conclusion This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided resulted in dissimilar enzyme
Ma, Liang; Salas, Omar; Bowler, Kyle
2017-01-01
ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. PMID:29162710
Dalkıran, Berna; Erden, Pınar Esra; Kılıç, Esma
2016-06-01
In this study, two enzyme electrodes based on graphene (GR), Co3O4 nanoparticles and chitosan (CS) or multi-walled carbon nanotubes (MWCNTs), Co3O4 nanoparticles, and CS, were fabricated as novel biosensing platforms for galactose determination, and their performances were compared. Galactose oxidase (GaOx) was immobilized onto the electrode surfaces by crosslinking with glutaraldehyde. Optimum working conditions of the biosensors were investigated and the analytical performance of the biosensors was compared with respect to detection limit, linearity, repeatability, and stability. The MWCNTs-based galactose biosensor provided about 1.6-fold higher sensitivity than its graphene counterpart. Moreover, the linear working range and detection limit of the MWCNTs-based galactose biosensor was superior to the graphene-modified biosensor. The successful application of the purposed biosensors for galactose biosensing in human serum samples was also investigated.
Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M
2016-05-01
Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.
Synthesis and Testing of Polymers Susceptible to Degradation by Proteolytic Enzymes
1975-05-01
diisocyanatohexane, was biodegraded by the enzymes urease and rennin and also by two fungi. The tensile strength was greater than 10,000 psi, with high...Copolymer Degradation by Urease Enzyme Copolymer Degradation by Rennin Enzyme Degradation of Modified Gelatins: Undrawn Bulk Material Degradation of...bacteria. Results with urease enzyme did indicate significant degradation, as shown by the following tables: Table 1. Copolymer Degradation by
Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose
Payne, Christina M.; Resch, Michael G.; Chen, Liqun; Crowley, Michael F.; Himmel, Michael E.; Taylor, Larry E.; Sandgren, Mats; Ståhlberg, Jerry; Stals, Ingeborg; Tan, Zhongping; Beckham, Gregg T.
2013-01-01
Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. PMID:23959893
Air film cooling in a nonadiabatic wall conical nozzle.
NASA Technical Reports Server (NTRS)
Boldman, D. R.; Papell, S. S.; Ehlers, R. C.
1972-01-01
Experimental data for an air-film cooled conical nozzle operating with a heated-air main stream and a water-cooled wall confirm the validity of Lieu's (1964) method for correlating film cooling data in the accelerated flow of a nonadiabatic-wall nozzle. The film cooling effectiveness modified for nonadiabatic walls by Lieu can be used to correlate film cooling under the condition that the main-stream to coolant velocity ratio at the slot is about 1. Such a ratio provides the optimum cooling effectiveness.
Effects of Wall-Normal and Angular Momentum Injections in Airfoil Separation Control
NASA Astrophysics Data System (ADS)
Munday, Phillip M.; Taira, Kunihiko
2018-05-01
The objective of this computational study is to quantify the influence of wall-normal and angular momentum injections in suppressing laminar flow separation over a canonical airfoil. Open-loop control of fully separated, incompressible flow over a NACA 0012 airfoil at $\\alpha = 9^\\circ$ and $Re = 23,000$ is examined with large-eddy simulations. This study independently introduces wall-normal momentum and angular momentum into the separated flow using swirling jets through model boundary conditions. The response of the flow field and the surface vorticity fluxes to various combinations of actuation inputs are examined in detail. It is observed that the addition of angular momentum input to wall-normal momentum injection enhances the suppression of flow separation. Lift enhancement and suppression of separation with the wall-normal and angular momentum inputs are characterized by modifying the standard definition of the coefficient of momentum. The effect of angular momentum is incorporated into the modified coefficient of momentum by introducing a characteristic swirling jet velocity based on the non-dimensional swirl number. With this single modified coefficient of momentum, we are able to categorize each controlled flow into separated, transitional, and attached flows.
Cerdeira Ferreira, Luís Marcos; da Costa, Eric Tavares; do Lago, Claudimir Lucio; Angnes, Lúcio
2013-09-15
This paper describes the development of a microfluidic system having as main component an enzymatic reactor constituted by a microchannel assembled in poly(methyl methacrylate) (PMMA) substrate connected to an amperometric detector. A CO2 laser engraving machine was used to make the channels, which in sequence were thermally sealed. The internal surfaces of the microchannels were chemically modified with polyethyleneimine (PEI), which showed good effectiveness for the immobilization of the glucose oxidase enzyme using glutaraldehyde as crosslinking agent, producing a very effective microreactor for the detection of glucose. The hydrogen peroxide generated by the enzymatic reaction was detected in an electrochemical flow cell localized outside of the reactor using a platinum disk as the working electrode. The proposed system was applied to the differential amperometric determination of glucose content in soft drinks showing good repeatability (DPR=1.72%, n=50), low detection limit (1.40×10(-6)molL(-1)), high sampling frequency (calculated as 345 samples h(-1)), and relatively good stability for long-term use. The results were in close agreement with those obtained by the classical spectrophotometric method utilized to quantify glucose in biological fluids. Copyright © 2013 Elsevier B.V. All rights reserved.
Characterization of the Sclerotinia sclerotiorum cell wall proteome.
Liu, Longzhou; Free, Stephen J
2016-08-01
We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. © 2015 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.
Near-wall k-epsilon turbulence modeling
NASA Technical Reports Server (NTRS)
Mansour, N. N.; Kim, J.; Moin, P.
1987-01-01
The flow fields from a turbulent channel simulation are used to compute the budgets for the turbulent kinetic energy (k) and its dissipation rate (epsilon). Data from boundary layer simulations are used to analyze the dependence of the eddy-viscosity damping-function on the Reynolds number and the distance from the wall. The computed budgets are used to test existing near-wall turbulence models of the k-epsilon type. It was found that the turbulent transport models should be modified in the vicinity of the wall. It was also found that existing models for the different terms in the epsilon-budget are adequate in the region from the wall, but need modification near the wall. The channel flow is computed using a k-epsilon model with an eddy-viscosity damping function from the data and no damping functions in the epsilon-equation. These computations show that the k-profile can be adequately predicted, but to correctly predict the epsilon-profile, damping functions in the epsilon-equation are needed.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gardiner, M.; Chrispeels, M.J.
1975-01-01
Pulse labeling of carrot root phloem parenchyma (Daucus carota L. ev. Nantes) tissue with /sup 14/C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identiy of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as /sup 14/C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major sitemore » of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma memebrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants. (auth)« less
Brunner, Patrick C; Torriani, Stefano F F; Croll, Daniel; Stukenbrock, Eva H; McDonald, Bruce A
2013-06-01
Zymoseptoria tritici is an important fungal pathogen on wheat that originated in the Fertile Crescent. Its closely related sister species Z. pseudotritici and Z. ardabiliae infect wild grasses in the same region. This recently emerged host-pathogen system provides a rare opportunity to investigate the evolutionary processes shaping the genome of an emerging pathogen. Here, we investigate genetic signatures in plant cell wall degrading enzymes (PCWDEs) that are likely affected by or driving coevolution in plant-pathogen systems. We hypothesize four main evolutionary scenarios and combine comparative genomics, transcriptomics, and selection analyses to assign the majority of PCWDEs in Z. tritici to one of these scenarios. We found widespread differential transcription among different members of the same gene family, challenging the idea of functional redundancy and suggesting instead that specialized enzymatic activity occurs during different stages of the pathogen life cycle. We also find that natural selection has significantly affected at least 19 of the 48 identified PCWDEs. The majority of genes showed signatures of purifying selection, typical for the scenario of conserved substrate optimization. However, six genes showed diversifying selection that could be attributed to either host adaptation or host evasion. This study provides a powerful framework to better understand the roles played by different members of multigene families and to determine which genes are the most appropriate targets for wet laboratory experimentation, for example, to elucidate enzymatic function during relevant phases of a pathogen's life cycle.
Truong, Thi Ngoc Lien; Tran, Dai Lam; Vu, Thi Hong An; Tran, Vinh Hoang; Duong, Tuan Quang; Dinh, Quang Khieu; Tsukahara, Toshifumi; Lee, Young Hoon; Kim, Jong Seung
2010-01-15
In this paper, we describe DNA electrochemical detection for genetically modified organism (GMO) based on multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole (PPy). DNA hybridization is studied by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). An increase in DNA complementary target concentration results in a decrease in the faradic charge transfer resistance (R(ct)) and signifying "signal-on" behavior of MWCNTs-PPy-DNA system. QCM and EIS data indicated that the electroanalytical MWCNTs-PPy films were highly sensitive (as low as 4pM of target can be detected with QCM technique). In principle, this system can be suitable not only for DNA but also for protein biosensor construction.
Wu, Xia; Paskaleva, Elena E.; Mehta, Krunal K.; Dordick, Jonathan S.; Kane, Ravi S.
2016-01-01
Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract. PMID:27759081
Pandey, Jitendra Kumar; Dash, Sidhartha Kumar; Biswal, Basanti
2017-07-01
The precise nature of the developmental modulation of the activity of cell wall hydrolases that breakdown the wall polysaccharides to maintain cellular sugar homeostasis under sugar starvation environment still remains unclear. In this work, the activity of β-galactosidase (EC 3.2.1.23), a cell-wall-bound enzyme known to degrade the wall polysaccharides, has been demonstrated to remarkably enhance during senescence-induced loss in photosynthesis in Arabidopsis thaliana. The enhancement in the enzyme activity reaches a peak at the terminal phase of senescence when the rate of photosynthesis is at its minimum. Although the precise nature of chemistry of the interface between the decline in photosynthesis and enhancement in the activity of the enzyme could not be fully resolved, the enhancement in its activity in dark and its suppression in light or with exogenous sugars may indicate the involvement of loss of photosynthetic production of sugars as a key factor that initiates and stimulates the activity of the enzyme. The hydrolase possibly participates in the catabolic network of cell wall polysaccharides to produce sugars for execution of energy-dependant senescence program in the background of loss of photosynthesis. Drought stress experienced by the senescing leaves accelerates the decline in photosynthesis with further stimulation in the activity of the enzyme. The stress recovery of photosynthesis and suppression of the enzyme activity on withdrawal of stress support the proposition of photosynthetic modulation of the cell-wall-bound enzyme activity.
Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G
2017-07-01
Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes
Artificial Enzymes with Thiazolium and Imidazolium Coenzyme Mimics
Zhao, Huanyu; Foss, Frank W.; Breslow, Ronald
2009-01-01
Hydrophobic thiazolium and imidazolium coenzyme mimics in the presence of modified-polyethylenimine enzyme mimics catalyze the benzoin condensation 2300–3300 times faster than the coenzyme mimics alone. Polycationic enzyme mimics provide not only a hydrophobic binding domain for coenzyme and substrate, but also electrostatic stabilization of anionic species that arise along the reaction pathway of the benzoin condensation. PMID:18763766
Building a plant cell wall at a glance.
Lampugnani, Edwin R; Khan, Ghazanfar Abbas; Somssich, Marc; Persson, Staffan
2018-01-29
Plant cells are surrounded by a strong polysaccharide-rich cell wall that aids in determining the overall form, growth and development of the plant body. Indeed, the unique shapes of the 40-odd cell types in plants are determined by their walls, as removal of the cell wall results in spherical protoplasts that are amorphic. Hence, assembly and remodeling of the wall is essential in plant development. Most plant cell walls are composed of a framework of cellulose microfibrils that are cross-linked to each other by heteropolysaccharides. The cell walls are highly dynamic and adapt to the changing requirements of the plant during growth. However, despite the importance of plant cell walls for plant growth and for applications that we use in our daily life such as food, feed and fuel, comparatively little is known about how they are synthesized and modified. In this Cell Science at a Glance article and accompanying poster, we aim to illustrate the underpinning cell biology of the synthesis of wall carbohydrates, and their incorporation into the wall, in the model plant Arabidopsis . © 2018. Published by The Company of Biologists Ltd.
Near-wall turbulence alteration through thin streamwise riblets
NASA Technical Reports Server (NTRS)
Wilkinson, Stephen P.; Lazos, Barry S.
1987-01-01
The possibility of improving the level of drag reduction associated with near-wall riblets is considered. The methodology involves the use of a hot-wire anemometer to study various surface geometries on small, easily constructed models. These models consist of small, adjacent rectangular channels on the wall aligned in the streamwise direction. The VITA technique is modified and applied to thin-element-array and smooth flat-plate data and the results are indicated schematically.
A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis
Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.
2014-01-01
Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210
Norman-Setterblad, C; Vidal, S; Palva, E T
2000-04-01
We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.
Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang
2016-01-15
A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. Copyright © 2015 Elsevier B.V. All rights reserved.
Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan
2014-02-01
The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
Bhattacharya, Kunal; Sacchetti, Cristiano; Costa, Pedro M; Sommertune, Jens; Brandner, Birgit D; Magrini, Andrea; Rosato, Nicola; Bottini, Nunzio; Bottini, Massimo; Fadeel, Bengt
2018-03-01
Polyethylene glycol (PEG)-modified carbon nanotubes have been successfully employed for intra-articular delivery in mice without systemic or local toxicity. However, the fate of the delivery system itself remains to be understood. In this study 2 kDa PEG-modified single-walled carbon nanotubes (PNTs) are synthesized, and trafficking and degradation following intra-articular injection into the knee-joint of healthy mice are studied. Using confocal Raman microspectroscopy, PNTs can be imaged in the knee-joint and are found to either egress from the synovial cavity or undergo biodegradation over a period of 3 weeks. Raman analysis discloses that PNTs are oxidatively degraded mainly in the chondrocyte-rich cartilage and meniscus regions while PNTs can also be detected in the synovial membrane regions, where macrophages can be found. Furthermore, using murine chondrocyte (ATDC-5) and macrophage (RAW264.7) cell lines, biodegradation of PNTs in activated, nitric oxide (NO)-producing chondrocytes, which is blocked upon pharmacological inhibition of inducible nitric oxide synthase (iNOS), can be shown. Biodegradation of PNTs in macrophages is also noted, but after a longer period of incubation. Finally, cell-free degradation of PNTs upon incubation with the peroxynitrite-generating compound, SIN-1 is demonstrated. The present study paves the way for the use of PNTs as delivery systems in the treatment of diseases of the joint. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Research and application of microbial enzymes--India's contribution.
Chand, Subhash; Mishra, Prashant
2003-01-01
Enzymes have attracted the attention of scientists world over due to their wide range of physiological, analytical and industrial applications. Although enzymes have been isolated, purified and studied from microbial, animal and plant sources, microorganisms represent the most common source of enzymes due to their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation. With the advent of molecular biology techniques, a number of genes of industrially important enzymes has been cloned and expressed in order to improve the production of enzymes, substrate utilization and other commercially useful properties. Special attention has been focused on enzymes isolated from thermophiles due to their inherent stability and industrial applications. In addition, a variety of methods have been employed to modify enzymes for their industrial usage including strain improvement, chemical modifications, modification of reaction environment, immobilization and protein engineering. A wide range of applications of enzymes in different bioprocess industries is discussed.
Goornavar, Virupaxi; Jeffers, Robert; Biradar, Santoshkumar; Ramesh, Govindarajan T
2014-07-01
In this work we report the improved performance an electrochemical glucose sensor based on a glassy carbon electrode (GCE) that has been modified with highly purified single wall carbon nanotubes (SWCNTs) dispersed in polyethyleneimine (PEI), polyethylene glycol (PEG) and polypyrrole (PPy). The single wall carbon nanotubes were purified by both thermal and chemical oxidation to achieve maximum purity of ~98% with no damage to the tubes. The SWCNTs were then dispersed by sonication in three different organic polymers (1.0mg/ml SWCNT in 1.0mg/ml of organic polymer). The stable suspension was coated onto the GCE and electrochemical characterization was performed by Cyclic Voltammetry (CV) and Amperometry. The electroactive enzyme glucose oxidase (GOx) was immobilized on the surface of the GCE/(organic polymer-SWCNT) electrode. The amperometric detection of glucose was carried out at 0.7 V versus Ag/AgCl. The GCE/(SWCNT-PEI, PEG, PPY) gave a detection limit of 0.2,633 μM, 0.434 μM, and 0.9,617 μM, and sensitivities of 0.2411 ± 0.0033 μA mM(-1), r(2)=0.9984, 0.08164 ± 0.001129 μA mM(-1), r(2)=0.9975, 0.04189 ± 0.00087 μA mM(-1), and r(2)=0.9944 respectively and a response time of less than 5s. The use of purified SWCNTs has several advantages, including fast electron transfer rate and stability in the immobilized enzyme. The significant enhancement of the SWCNT modified electrode as a glucose sensor can be attributed to the superior conductivity and large surface area of the well dispersed purified SWCNTs. Copyright © 2014 Elsevier B.V. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yuki, Tobimatsu; Sasikumar, Elumalai; Grabber, John H.
2012-04-01
The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers, such as rosmarinic acid (RA) and analogous catechol derivatives, into cell-wall lignins that are consequently less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that RA readily underwent peroxidase-catalyzed copolymerization with monolignols and lignin oligomers to form polymers with new benzodioxane inter-unit linkages. Incorporation of RA permitted extensive depolymerization of synthetic lignins by mild alkaline hydrolysis, presumably by cleavage of ester intra-unit linkages within RA. Copolymerization of RA with monolignols into maize cell walls by in situ peroxidases significantly enhanced alkaline lignin extractability andmore » promoted subsequent cell wall saccharification by fungal enzymes. Incorporating RA also improved cell wall saccharification by fungal enzymes and by rumen microflora even without alkaline pretreatments, possibly by modulating lignin hydrophobicity and/or limiting cell wall cross-linking. Consequently, we anticipate that bioengineering approaches for partial monolignol substitution with RA and analogous plant hydroxycinnamates would permit more efficient utilization of plant fiber for biofuels or livestock production.« less
Bacterial enzymes involved in lignin degradation.
de Gonzalo, Gonzalo; Colpa, Dana I; Habib, Mohamed H M; Fraaije, Marco W
2016-10-20
Lignin forms a large part of plant biomass. It is a highly heterogeneous polymer of 4-hydroxyphenylpropanoid units and is embedded within polysaccharide polymers forming lignocellulose. Lignin provides strength and rigidity to plants and is rather resilient towards degradation. To improve the (bio)processing of lignocellulosic feedstocks, more effective degradation methods of lignin are in demand. Nature has found ways to fully degrade lignin through the production of dedicated ligninolytic enzyme systems. While such enzymes have been well thoroughly studied for ligninolytic fungi, only in recent years biochemical studies on bacterial enzymes capable of lignin modification have intensified. This has revealed several types of enzymes available to bacteria that enable them to act on lignin. Two major classes of bacterial lignin-modifying enzymes are DyP-type peroxidases and laccases. Yet, recently also several other bacterial enzymes have been discovered that seem to play a role in lignin modifications. In the present review, we provide an overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignin-derived products. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.
Vertical regolith shield wall construction for lunar base applications
NASA Technical Reports Server (NTRS)
Kaplicky, Jan; Nixon, David; Wernick, Jane
1992-01-01
Lunar bases located on the lunar surface will require permanent protection from radiation and launch ejecta. This paper outlines a method of providing physical protection using lunar regolith that is constructed in situ as a modular vertical wall using specially devised methods of containment and construction. Deployable compartments, reinforced with corner struts, are elevated and filled by a moving gantry. The compartments interlock to form a stable wall. Different wall heights, thicknesses, and plan configurations are achieved by varying the geometry of the individual compartments, which are made from woven carbon fibers. Conventional terrestrial structural engineering techniques can be modified and used to establish the structural integrity and performance of the wall assembly.
Pauchet, Yannick; Kirsch, Roy; Giraud, Sandra; Vogel, Heiko; Heckel, David G
2014-06-01
Xylophagous insects have evolved to thrive in a highly challenging environment. For example, wood-boring beetles from the family Cerambycidae feed exclusively on woody tissues, and to efficiently access the nutrients present in this sub-optimal environment, they have to cope with the lignocellulose barrier. Whereas microbes of the insect's gut flora were hypothesized to be responsible for the degradation of lignin, the beetle itself depends heavily on the secretion of a range of enzymes, known as plant cell wall degrading enzymes (PCWDEs), to efficiently digest both hemicellulose and cellulose networks. Here we sequenced the larval gut transcriptome of the Mulberry longhorn beetle, Apriona japonica (Cerambycidae, Lamiinae), in order to investigate the arsenal of putative PCWDEs secreted by this species. We combined our transcriptome with all available sequencing data derived from other cerambycid beetles in order to analyze and get insight into the evolutionary history of the corresponding gene families. Finally, we heterologously expressed and functionally characterized the A. japonica PCWDEs we identified from the transcriptome. Together with a range of endo-β-1,4-glucanases, we describe here for the first time the presence in a species of Cerambycidae of (i) a xylanase member of the subfamily 2 of glycoside hydrolase family 5 (GH5 subfamily 2), as well as (ii) an exopolygalacturonase from family GH28. Our analyses greatly contribute to a better understanding of the digestion physiology of this important group of insects, many of which are major pests of forestry worldwide. Copyright © 2014 Elsevier Ltd. All rights reserved.
Molecular engineering of industrial enzymes: recent advances and future prospects.
Yang, Haiquan; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian
2014-01-01
Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.
Ferroelectric domain wall motion induced by polarized light
Rubio-Marcos, Fernando; Del Campo, Adolfo; Marchet, Pascal; Fernández, Jose F.
2015-01-01
Ferroelectric materials exhibit spontaneous and stable polarization, which can usually be reoriented by an applied external electric field. The electrically switchable nature of this polarization is at the core of various ferroelectric devices. The motion of the associated domain walls provides the basis for ferroelectric memory, in which the storage of data bits is achieved by driving domain walls that separate regions with different polarization directions. Here we show the surprising ability to move ferroelectric domain walls of a BaTiO3 single crystal by varying the polarization angle of a coherent light source. This unexpected coupling between polarized light and ferroelectric polarization modifies the stress induced in the BaTiO3 at the domain wall, which is observed using in situ confocal Raman spectroscopy. This effect potentially leads to the non-contact remote control of ferroelectric domain walls by light. PMID:25779918
Catalytic activity of enzymes immobilized on AlGaN /GaN solution gate field-effect transistors
NASA Astrophysics Data System (ADS)
Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M.
2006-10-01
Enzyme-modified field-effect transistors (EnFETs) were prepared by immobilization of penicillinase on AlGaN /GaN solution gate field-effect transistors. The influence of the immobilization process on enzyme functionality was analyzed by comparing covalent immobilization and physisorption. Covalent immobilization by Schiff base formation on GaN surfaces modified with an aminopropyltriethoxysilane monolayer exhibits high reproducibility with respect to the enzyme/substrate affinity. Reductive amination of the Schiff base bonds to secondary amines significantly increases the stability of the enzyme layer. Electronic characterization of the EnFET response to penicillin G indicates that covalent immobilization leads to the formation of an enzyme (sub)monolayer.
Micellar Polymer Encapsulation of Enzymes.
Besic, Sabina; Minteer, Shelley D
2017-01-01
Although enzymes are highly efficient and selective catalysts, there have been problems incorporating them into fuel cells. Early enzyme-based fuel cells contained enzymes in solution rather than immobilized on the electrode surface. One problem utilizing an enzyme in solution is an issue of transport associated with long diffusion lengths between the site of bioelectrocatalysis and the electrode. This issue drastically decreases the theoretical overall power output due to the poor electron conductivity. On the other hand, enzymes immobilized at the electrode surface have eliminated the issue of poor electron conduction due to close proximity of electron transfer between electrode and the biocatalyst. Another problem is inefficient and short term stability of catalytic activity within the enzyme that is suspended in free flowing solution. Enzymes in solutions are only stable for hours to days, whereas immobilized enzymes can be stable for weeks to months and now even years. Over the last decade, there has been substantial research on immobilizing enzymes at electrode surfaces for biofuel cell and sensor applications. The most commonly used techniques are sandwich or wired. Sandwich techniques are powerful and successful for enzyme immobilization; however, the enzymes optimal activity is not retained due to the physical distress applied by the polymer limiting its applications as well as the non-uniform distribution of the enzyme and the diffusion of analyte through the polymer is slowed significantly. Wired techniques have shown to extend the lifetime of an enzyme at the electrode surface; however, this technique is very hard to master due to specific covalent bonding of enzyme and polymer which changes the three-dimensional configuration of enzyme and with that decreases the optimal catalytic activity. This chapter details encapsulation techniques where an enzyme will be immobilized within the pores/pockets of the hydrophobically modified micellar polymers such as
Daniel J. Yelle; John Ralph; Charles R. Frihart
2008-01-01
A recently described plant cell wall dissolution system has been modified to use perdeuterated solvents to allow direct in-NMR-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent system containing dimethylsulfoxide-d6 and 1-methylimidazole-d6 in a ratio of 4:1 (v/...
Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S
2017-07-25
Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.
Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A
2017-10-01
Chardonnay grape pomace was treated with pressurized heat followed by enzymatic hydrolysis, with commercial or pure enzymes, in buffered conditions. The pomace was unfermented as commonly found for white winemaking wastes and treatments aimed to simulate biovalorization processing. Cell wall profiling techniques showed that the pretreatment led to depectination of the outer layers thereby exposing xylan polymers and increasing the extractability of arabinans, galactans, arabinogalactan proteins and mannans. This higher extractability is believed to be linked with partial degradation and opening-up of cell wall networks. Pectinase-rich enzyme preparations were presumably able to access the inner rhamnogalacturonan I dominant coating layers due to the hydrothermal pretreatment. Patterns of epitope abundance and the sequential release of cell wall polymers with specific combinations of enzymes led to a working model of the hitherto, poorly understood innermost xyloglucan-rich hemicellulose layers of unfermented grape pomace. Copyright © 2017 Elsevier Ltd. All rights reserved.
Safari, Masoumeh; Ghanati, Faezeh; Safarnejad, Mohammad Reza; Chashmi, Najmeh Ahmadian
2018-02-01
Treatment with aluminum triggers a unique response in tea seedlings resulting in biochemical modification of the cell wall, regulation of the activity of the loosening agents, and elongation of root. Unlike most terrestrial plants, tea (Camellia sinensis L.) responds to aluminum (Al) through the promotion of its root elongation; but the real mechanism(s) behind this phenomenon is not well understood. A plausible relationship between the modifications of the cell wall and the promotion of root elongation was examined in tea seedlings treated for 8 days with 400 µM Al. The mechanical properties of the cell wall, the composition of its polysaccharides and their capacity to absorb Al, the expression of genes, and the activities of the wall-modifying proteins were studied. With 6 h of the treatment, about 40% of the absorbed Al was bound to the cell wall; however, the amount did not increase thereafter. Meanwhile, the activity of pectin methylesterase, the level of pectin demethylation, the amounts and the average molecular mass of xyloglucan in the root apices significantly decreased upon exposure to Al, resulting in the reduction of Al binding sites. On the other hand, the activity and the gene expression of peroxidase decreased, whereas the activity and gene expression of xyloglucan-degrading enzymes, the expression of expansin A and the H + -ATPase4 genes increased in the Al-treated plants. Interestingly, it was accompanied by the increase of elastic and viscous extensibility of the root apices. From the results, it can be suggested that the biochemical modification of the cell walls reduces sites of Al binding to roots and triggers the activity of the loosening agents, thereby increasing the length of tea roots.
Dynamic moisture sorption characteristics of enzyme-resistant recrystallized cassava starch.
Mutungi, Christopher; Schuldt, Stefan; Onyango, Calvin; Schneider, Yvonne; Jaros, Doris; Rohm, Harald
2011-03-14
The interaction of moisture with enzyme-resistant recrystallized starch, prepared by heat-moisture treatment of debranched acid-modified or debranched non-acid-modified cassava starch, was investigated in comparison with the native granules. Crystallinities of the powdered products were estimated by X-ray diffraction. Moisture sorption was determined using dynamic vapor sorption analyzer and data fitted to various models. Percent crystallinities of native starch (NS), non-acid-modified recrystallized starch (NAMRS), and acid-modified recrystallized starch (AMRS) were 39.7, 51.9, and 56.1%, respectively. In a(w) below 0.8, sorption decreased in the order NS > NAMRS > AMRS in line with increasing sample crystallinities but did not follow this crystallinity dependence at higher a(w) because of condensation and polymer dissolution effects. Adsorbed moisture became internally absorbed in NS but not in NAMRS and AMRS, which might explain the high resistance of the recrystallized starches to digestion because enzyme and starch cannot approach each other over fairly sufficient surface at the molecular level.
Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph
2015-06-24
Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.
Sun, Wei; Cao, Lili; Deng, Ying; Gong, Shixing; Shi, Fan; Li, Gaonan; Sun, Zhenfan
2013-06-05
A graphene (GR) and multi-walled carbon nanotubes (MWCNT) hybrid was prepared and modified on a 1-hexylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE). Hemoglobin (Hb) was immobilized on GR-MWCNT/CILE surface with Nafion as the film forming material and the modified electrode was denoted as Nafion/Hb-GR-MWCNT/CILE. Spectroscopic results revealed that Hb molecules retained its native structure in the GR-MWCNT hybird. Electrochemical behaviors of Hb were carefully investigated by cyclic voltammetry with a pair of well-defined redox peaks obtained, which indicated that direct electron transfer of Hb was realized in the hybrid modified electrode. The result could be attributed to the synergistic effects of GR-MWCNT hybrid with enlarged surface area and improved conductivity through the formation of a three-dimensional network. Electrochemical parameters of the immobilized Hb on the electrode surface were further calculated with the results of the electron transfer number (n) as 1.03, the charge transfer coefficient (a) as 0.58 and the electron-transfer rate constant (ks) as 0.97 s(-1). The Hb modified electrode showed good electrocatalytic ability toward the reduction of different substrates such as trichloroacetic acid in the concentration range from 0.05 to 38.0 mmol L(-1) with a detection limit of 0.0153 mmol L(-1) (3σ), H2O2 in the concentration range from 0.1 to 516.0 mmol L(-1) with a detection limit of 34.9 nmol/L (3σ) and NaNO2 in the concentration range from 0.5 to 650.0 mmol L(-1) with a detection limit of 0.282 μmol L(-1) (3σ). So the proposed electrode had the potential application in the third-generation electrochemical biosensors without mediator. Copyright © 2013 Elsevier B.V. All rights reserved.
Polarization-induced local pore-wall functionalization for biosensing: from micropore to nanopore.
Liu, Jie; Pham, Pascale; Haguet, Vincent; Sauter-Starace, Fabien; Leroy, Loïc; Roget, André; Descamps, Emeline; Bouchet, Aurélie; Buhot, Arnaud; Mailley, Pascal; Livache, Thierry
2012-04-03
The use of biological-probe-modified solid-state pores in biosensing is currently hindered by difficulties in pore-wall functionalization. The surface to be functionalized is small and difficult to target and is usually chemically similar to the bulk membrane. Herein, we demonstrate the contactless electrofunctionalization (CLEF) approach and its mechanism. This technique enables the one-step local functionalization of the single pore wall fabricated in a silica-covered silicon membrane. CLEF is induced by polarization of the pore membrane in an electric field and requires a sandwich-like composition and a conducting or semiconducting core for the pore membrane. The defects in the silica layer of the micropore wall enable the creation of an electric pathway through the silica layer, which allows electrochemical reactions to take place locally on the pore wall. The pore diameter is not a limiting factor for local wall modification using CLEF. Nanopores with a diameter of 200 nm fabricated in a silicon membrane and covered with native silica layer have been successfully functionalized with this method, and localized pore-wall modification was obtained. Furthermore, through proof-of-concept experiments using ODN-modified nanopores, we show that functionalized nanopores are suitable for translocation-based biosensing.
Genovesi, Valeria; Fornalé, Silvia; Fry, Stephen C; Ruel, Katia; Ferrer, Pau; Encina, Antonio; Sonbol, Fathi-Mohamed; Bosch, Josep; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David
2008-01-01
Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.
Ballistic Limit Equation for Single Wall Titanium
NASA Technical Reports Server (NTRS)
Ratliff, J. M.; Christiansen, Eric L.; Bryant, C.
2009-01-01
Hypervelocity impact tests and hydrocode simulations were used to determine the ballistic limit equation (BLE) for perforation of a titanium wall, as a function of wall thickness. Two titanium alloys were considered, and separate BLEs were derived for each. Tested wall thicknesses ranged from 0.5mm to 2.0mm. The single-wall damage equation of Cour-Palais [ref. 1] was used to analyze the Ti wall's shielding effectiveness. It was concluded that the Cour-Palais single-wall equation produced a non-conservative prediction of the ballistic limit for the Ti shield. The inaccurate prediction was not a particularly surprising result; the Cour-Palais single-wall BLE contains shield material properties as parameters, but it was formulated only from tests of different aluminum alloys. Single-wall Ti shield tests were run (thicknesses of 2.0 mm, 1.5 mm, 1.0 mm, and 0.5 mm) on Ti 15-3-3-3 material custom cut from rod stock. Hypervelocity impact (HVI) tests were used to establish the failure threshold empirically, using the additional constraint that the damage scales with impact energy, as was indicated by hydrocode simulations. The criterion for shield failure was defined as no detached spall from the shield back surface during HVI. Based on the test results, which confirmed an approximately energy-dependent shield effectiveness, the Cour-Palais equation was modified.
USDA-ARS?s Scientific Manuscript database
Nucleotide-activated sugars are essential substrates for plant cell wall carbohydrate-polymer biosynthetic glycosyltransferase enzymes. The most prevalent sugars in grass cell walls include glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the uridine di...
Domozych, David; Lietz, Anna; Patten, Molly; Singer, Emily; Tinaz, Berke; Raimundo, Sandra C
2017-01-01
The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.
Plant cell wall proteomics: the leadership of Arabidopsis thaliana
Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth
2013-01-01
Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247
Scandurra, Graziella; Antonella, Arena; Ciofi, Carmine; Saitta, Gaetano; Lanza, Maurizio
2014-01-01
A conducting composite prepared by dispersing multi-walled carbon nanotubes (MWCNTs) into a host matrix consisting of Nafion, electrochemically doped with copper, has been prepared, characterized and used to modify one of the gold electrodes of simply designed electrochemical cells having copier grade transparency sheets as substrates. Electrical measurements performed in deionized water show that the Au/Nafion/Au-MWCNTs–Nafion:Cu cells can be successfully used in order to detect the presence of p-aminophenol (PAP) in water, without the need for any supporting electrolyte. The intensity of the redox peaks arising when PAP is added to deionized water is found to be linearly related to the analyte in the range from 0.2 to 1.6 μM, with a detection limit of 90 nM and a sensitivity of 7 μA·(μM−1)·cm−2. PMID:24854357
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine
DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 ( MWL-2), the most closely related gene to MWL-1 in themore » protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/ mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/ mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Lastly, our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.« less
Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine; ...
2016-03-01
DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 ( MWL-2), the most closely related gene to MWL-1 in themore » protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/ mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/ mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Lastly, our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.« less
Gunawan, Christa; Xue, Saisi; Pattathil, Sivakumar; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh
2017-01-01
Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans. In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4- O -methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme
Huang, Danni; Wang, Xianying; Deng, Chunhui; Song, Guoxin; Cheng, Hefa; Zhang, Xiangmin
2014-01-17
In this study, novel raisin-bread sandwich-structured magnetic graphene/mesoporous silica composites with C18-modified interior pore-walls (mag-graphene@mSiO2-C18) were synthesized by coating mesoporous silica layers onto each side of magnetic graphene through a surfactant-mediated co-condensation sol-gel process. The prepared functionalized nanocomposites possessed marvelous properties of extended plate-like morphology, fine water dispersibility, high magnetic response, large surface area (315.4cm(2)g(-1)), uniform pore size (3.3nm) and C18-modified interior pore-walls. Several kinds of phthalates were selected as model analytes to systematically evaluate the performance of adsorbents in extracting hydrophobic molecules followed by gas chromatography-mass spectrometry analyses. Various extraction parameters, including pH value of sample solution, amounts of adsorbents, adsorption time, species and volume of eluting solvent, and desorption time were optimized. The anti-interference ability to macromolecular proteins was also investigated. Method validations such as linearity, recovery, reproducibility, and limit of detection were also studied. Finally, mag-graphene@mSiO2-C18 composites were successfully applied to analyzing phthalates in environmental water samples. The results indicated that this novel approach offered an attractive alternative for rapid, convenient, efficient and selective magnetic solid-phase extraction for targeted hydrophobic compounds. Copyright © 2013 Elsevier B.V. All rights reserved.
A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.
Esipov, R S; Abramchik, Yu A; Fateev, I V; Konstantinova, I D; Kostromina, M A; Muravyova, T I; Artemova, K G; Miroshnikov, A I
2016-01-01
We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D -pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp . 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D -pentoses into 5-phosphates that were further converted into 5-phospho-α- D -pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D -ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.
Perini, M A; Sin, I N; Villarreal, N M; Marina, M; Powell, A L T; Martínez, G A; Civello, P M
2017-04-01
Firmness, one of the major determinants of postharvest quality and shelf life of fruits is determined by the mechanical resistance imposed by the plant cell wall. Expansins (EXP) are involved in the non-hydrolytic metabolic disassembly of plant cell walls, particularly in processes where relaxation of the wall is necessary, such as fruit development and ripening. As many carbohydrate-associated proteins, expansins have a putative catalytic domain and a carbohydrate-binding module (CBM). Several strategies have been pursued to control the loss of fruit firmness during storage. Most of the approaches have been to suppress the expression of key enzymes involved in the cell wall metabolism, but this is the first time that a CBM was overexpressed in a fruit aimed to control cell wall degradation and fruit softening. We report the constitutive overexpression of the CBM of Solanum lycopersicum expansin 1 (CBM-SlExp1) in the cell wall of tomato plants, and its effects on plant and fruit phenotype. Overexpression of CBM-SlExp1 increased the mechanical resistance of leaves, whereas it did not modify plant growth and general phenotype. However, transgenic plants showed delayed softening and firmer fruits. In addition, fruits were less susceptible to Botrytis cinerea infection, and the "in vitro" growth of the fungus on media containing AIR from the pericarp of transgenic fruits was lower than controls. The possibility of overexpressing a CBM of a fruit-specific expansin to control cell wall degradation and fruit softening is discussed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Activity, Stability, and Structure of Native and Modified by Woodward Reagent K Mushroom Tyrosinase
NASA Astrophysics Data System (ADS)
Emami, S.; Piri, H.; Gheibi, N.
2018-01-01
Mushroom tyrosinase (MT) was considered a good model for studying the inhibition, activation, and mutation of tyrosinase as the key enzyme of melanogenesis. In the present study, the activity, structure, reduction, and stability of native and modified enzymes were investigated after the modification of MT carboxylic residues by the Woodward reagent K (WRK). The relative activity of the sole enzyme was reduced from 100 to 77.9, 53.8, 39.4, and 26.4% after its modification by 2.5, 5, 25, and 50 ratios of [WRK]/[MT], respectively. The Tm values were calculated from thermal denaturation curves at 61.2, 60.1, 58.3, 53.9, and 45.5oC for the sole and modified enzymes. The reduction of the Δ {G}_{{H}_2O} values for the modified enzyme in chemical denaturation indicated instability. A structural study by CD and intrinsic fluorescence technique revealed the fluctuation of the secondary and tertiary structures of MT.
Cang-Rong, Jason Teng; Pastorin, Giorgia
2009-06-24
In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they
NASA Astrophysics Data System (ADS)
Cang-Rong, Jason Teng; Pastorin, Giorgia
2009-06-01
In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they
Samalova, Marketa; Mélida, Hugo; Vilaplana, Francisco; Bulone, Vincent; Soanes, Darren M.; Talbot, Nicholas J.
2016-01-01
Abstract The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β‐1,3‐glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate‐binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72 + GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3‐glucans have a higher degree of polymerization and are less branched than the wild‐type strain. The mutant showed significant differences in global patterns of gene expression, a hyper‐branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium‐mediated plant infection. PMID:27568483
Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall
Scholz, Matthew J.; Weiss, Taylor L.; Jinkerson, Robert E.; Jing, Jia; Roth, Robyn; Goodenough, Ursula; Posewitz, Matthew C.
2014-01-01
Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes. PMID:25239976
Inhibitors targeting on cell wall biosynthesis pathway of MRSA.
Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui
2012-11-01
Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.
A versatile strategy for grafting polymers to wood cell walls.
Keplinger, T; Cabane, E; Chanana, M; Hass, P; Merk, V; Gierlinger, N; Burgert, I
2015-01-01
The hierarchical structure of wood is composed of a cellulose skeleton of high structural order at various length scales. At the nanoscale and microscale the specific structural features of the cells and cell walls result in a lightweight structure with an anisotropic material profile of excellent mechanical performance. By being able to specifically functionalize wood at the level of cell and cell walls one can insert new properties and inevitably upscale them along the intrinsic hierarchical structure, to a level of large-scale engineering materials applications. For this purpose, however, precise control of the spatial distribution of the modifying substances in the complex wood structure is needed. Here we demonstrate a method to insert methacryl groups into wood cell walls using two different chemistry routes. By using these methacryl groups as the anchor points for grafting, various polymers can be inserted into the wood structure. Strikingly, depending on the methacryl precursor, the spatial distribution of the polymer differs strongly. As a proof of concept we grafted polystyrene as a model compound in the second modification step. In the case of methacryloyl chloride the polymer was located mainly at the interface between the cell lumina and the cell wall covering the inner surface of the cells and being traceable up to 2-3 μm in the cell wall, whereas in the case of methacrylic anhydride the polymer was located inside the whole cell wall. Scanning electron microscopy, Fourier transform infrared spectroscopy and especially Raman spectroscopy were used for an in-depth analysis of the modified wood at the cell wall level. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Pumera, Martin; Smíd, Bretislav
2007-10-01
Double wall carbon nanotubes are noncovalently functionalized with redox protein and such assembly is used for construction of electrochemical binder-less glucose biosensor. Redox protein glucose oxidase performs as biorecognition element and double wall carbon nanotubes act both as immobilization platform for redox enzyme and as signal transducer. The double carbon nanotubes are characterized by cyclic voltammetry and specific surface area measurements; the redox protein noncovalently functionalized double wall carbon nanotubes are characterized in detail by X-ray photoelectron spectroscopy, cyclic voltammetry, amperometry, and transmission electron microscopy.
Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff
2013-09-01
Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
Dissecting enzyme function with microfluidic-based deep mutational scanning.
Romero, Philip A; Tran, Tuan M; Abate, Adam R
2015-06-09
Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.
Extending enzyme molecular recognition with an expanded amino acid alphabet
Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam
2017-01-01
Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894
Patterns of functional enzyme activity in fungus farming ambrosia beetles.
De Fine Licht, Henrik H; Biedermann, Peter H W
2012-06-06
In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray
Mechanisms, biology and inhibitors of deubiquitinating enzymes.
Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L
2007-11-01
The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.
Xiao, Fei; Zhao, Faqiong; Li, Jiangwen; Yan, Rui; Yu, Jingjing; Zeng, Baizhao
2007-07-16
A novel composite film modified glassy carbon electrode has been fabricated and characterized by scanning electron microscope (SEM) and voltammetry. The composite film comprises of single-wall carbon nanotube (SWNT), gold nanoparticle (GNP) and ionic liquid (i.e. 1-octyl-3-methylimidazolium hexafluorophosphate), thus has the characteristics of them. The resulting electrode shows good stability, high accumulation efficiency and strong promotion to electron transfer. On it, chloramphenicol can produce a sensitive cathodic peak at -0.66 V (versus SCE) in pH 7.0 phosphate buffer solutions. Parameters influencing the voltammetric response of chloramphenicol are optimized, which include the composition of the film and the operation conditions. Under the optimized conditions, the peak current is linear to chloramphenicol concentration in the range of 1.0x10(-8)-6.0x10(-6) M, and the detection limit is estimated to be 5.0x10(-9) M after an accumulation for 150 s on open circuit. The electrode is applied to the determination of chloramphenicol in milk samples, and the recoveries for the standards added are 97.0% and 100.3%. In addition, the electrochemical reaction of chloramphenicol and the effect of single-wall carbon nanotube, gold nanoparticle and ionic liquid are discussed.
Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele; Schlag, Martin; Götz, Friedrich; Tran, Dat Q.; Schaal, Justin B.; Ouellette, André J.; Selsted, Michael E.; Sahl, Hans-Georg
2014-01-01
θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin. PMID:25632351
Ma, Xiaoyan; Yang, Hongqiao; Xiong, Huabin; Li, Xiaofen; Gao, Jinting; Gao, Yuntao
2016-01-01
In this paper, the multi-walled carbon nanotubes modified screen-printed electrode (MWCNTs/SPE) was prepared and the MWCNTs/SPE was employed for the electrochemical determination of the antioxidant substance chlorogenic acids (CGAs). A pair of well-defined redox peaks of CGA was observed at the MWCNTs/SPE in 0.10 mol/L acetic acid-sodium acetate buffer (pH 6.2) and the electrode process was adsorption-controlled. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods for the determination of CGA were proposed based on the MWCNTs/SPE. Under the optimal conditions, the proposed method exhibited linear ranges from 0.17 to 15.8 µg/mL, and the linear regression equation was Ipa (µA) = 4.1993 C (×10−5 mol/L) + 1.1039 (r = 0.9976) and the detection limit for CGA could reach 0.12 µg/mL. The recovery of matrine was 94.74%–106.65% (RSD = 2.92%) in coffee beans. The proposed method is quick, sensitive, reliable, and can be used for the determination of CGA. PMID:27801797
Lugo-Morales, Leyda Z; Loziuk, Philip L; Corder, Amanda K; Toups, J Vincent; Roberts, James G; McCaffrey, Katherine A; Sombers, Leslie A
2013-09-17
Neurotransmission occurs on a millisecond time scale, but conventional methods for monitoring nonelectroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, nonelectroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically generated H2O2 without the need for additional strategies to eliminate interfering agents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM(-1) and 13.1 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many nonelectroactive enzyme substrates.
Zhi, Huanhuan; Liu, Qiqi; Xu, Juan; Dong, Yu; Liu, Mengpei; Zong, Wei
2017-12-01
Ultrasound has been applied in fruit pre-washing processes. However, it is not sufficient to protect fruit from pathogenic infection throughout the entire storage period, and sometimes ultrasound causes tissue damage. The goal of this study was to investigate the effects of calcium chloride (CaCl 2 , 10 g L -1 ) and ultrasound (350 W at 40 kHz), separately and in combination, on jujube fruit quality, antioxidant status, tissue Ca 2+ content and distribution along with cell wall metabolism at 20 °C for 6 days. All three treatments significantly maintained fruit firmness and peel color, reduced respiration rate, decay incidence, superoxide anion, hydrogen peroxide and malondialdehyde and preserved higher enzymatic (superoxide dismutase, catalase and peroxidase) and non-enzymatic (ascorbic acid and glutathione) antioxidants compared with the control. Moreover, the combined treatment was more effective in increasing tissue Ca 2+ content and distribution, inhibiting the generation of water-soluble and CDTA-soluble pectin fractions, delaying the solubilization of Na 2 CO 3 -soluble pectin and having lower activities of cell wall-modifying enzymes (polygalacturonase and pectate lyase) during storage. These results demonstrated that the combination of CaCl 2 and ultrasound has potential commercial application to extend the shelf life of jujube fruit by facilitating Ca 2+ absorption and stabilizing the cell wall structure. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
Mechanistic insights into the regulation of metabolic enzymes by acetylation
2012-01-01
The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120
Modification of cell wall polysaccharides during retting of cassava roots.
Ngolong Ngea, Guillaume Legrand; Guillon, Fabienne; Essia Ngang, Jean Justin; Bonnin, Estelle; Bouchet, Brigitte; Saulnier, Luc
2016-12-15
Retting is an important step in traditional cassava processing that involves tissue softening of the roots to transform the cassava into flour and various food products. The tissue softening that occurs during retting was attributed to the degradation of cell wall pectins through the action of pectin-methylesterase and pectate-lyase that possibly originated from a microbial source or the cassava plant itself. Changes in cell wall composition were investigated during retting using chemical analysis, specific glycanase degradation and immuno-labelling of cell wall polysaccharides. Pectic 1,4-β-d-galactan was the main cell wall polysaccharide affected during the retting of cassava roots. This result suggested that better control of pectic galactan degradation and a better understanding of the degradation mechanism by endogenous endo-galactanase and/or exogenous microbial enzymes might contribute to improve the texture properties of cassava products. Copyright © 2016 Elsevier Ltd. All rights reserved.
NASA Technical Reports Server (NTRS)
Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.
1988-01-01
A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.
Yeroslavsky, Gil; Girshevitz, Olga; Foster-Frey, Juli; Donovan, David M; Rahimipour, Shai
2015-01-27
Antibiotic resistance and the colonization of bacteria on surfaces, often as biofilms, prolong hospitalization periods, increase mortality, and are thus major concerns for health care providers. There is an urgent need for antimicrobial and antibiofilm surface treatments that are permanent, can eradicate both biofilms and planktonic pathogens over long periods of time, and do not select for resistant strains. In this study, we have demonstrated a simple, robust, and biocompatible method that utilizes the adhesive property of polydopamine (PDA) to covalently attach the antimicrobial enzyme lysostaphin (Lst) to a variety of surfaces to generate antibacterial and antibiofilm interfaces. The immobilization of the recombinant Lst onto PDA-coated surfaces was carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of the enzyme, minimizing the losses of bioagent activity. The modified surfaces were extensively characterized by X-ray photoelectron spectroscopy and peak force quantitative nanomechanical mapping (PeakForce QNM) AFM-based method, and the presence of Lst on the surfaces was further confirmed immunochemically using anti-Lst antibody. We also found that, in contrast to the physically adsorbed Lst, the covalently attached Lst does not leach from the surfaces and maintains its endopeptidase activity to degrade the staphylococcal cell wall, avoiding most intracellular bacterial resistance mechanisms. Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureus in less than 15 min and prevent biofilm formation. This immobilization method should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment.
Kinetics of leather dyeing pretreated with enzymes: role of acid protease.
Kanth, Swarna Vinodh; Venba, Rajangam; Jayakumar, Gladstone Christopher; Chandrababu, Narasimhan Kannan
2009-04-01
In the present investigation, kinetics of dyeing involving pretreatment with acid protease has been presented. Application of acid protease in dyeing process resulted in increased absorption and diffusion of dye into the leather matrix. Enzyme treatment at 1% concentration, 60 min duration and 50 degrees C resulted in maximum of 98% dye exhaustion and increased absorption rate constants. The final exhaustion (C(infinity)) for the best fit of CI Acid Black 194 dye has been 98.5% with K and r2 values from the modified Cegarra-Puente isotherm as 0.1033 and 0.0631. CI Acid Black 194 being a 2:1 metal complex acid dye exhibited higher absorption rate than the acid dye CI Acid Black 210. A reduction in 50% activation energy calculated from Arrhenius equation has been observed in enzyme assisted dyeing process of both the dyes that substantiates enhanced dye absorption. The absorption rate constant calculated with modified Cegarra-Puente equation confirm higher rate constants and faster kinetics for enzyme assisted dyeing process. Enzyme treated leather exhibited richness of color and shade when compared with control. The present study substantiates the essential role of enzyme pretreatment as an eco-friendly leather dyeing process.
Vermerris, Wilfred; Abril, Alejandra
2015-04-01
Cellulose from plant biomass can serve as a sustainable feedstock for fuels, chemicals and polymers that are currently produced from petroleum. In order to enhance economic feasibility, the efficiency of cell wall deconstruction needs to be enhanced. With the use of genetic and biotechnological approaches cell wall composition can be modified in such a way that interactions between the major cell wall polymers—cellulose, hemicellulosic polysaccharides and lignin—are altered. Some of the resulting plants are compromised in their growth and development, but this may be caused in part by the plant's overcompensation for metabolic perturbances. In other cases novel structures have been introduced in the cell wall without negative effects. The first field studies with engineered bioenergy crops look promising, while detailed structural analyses of cellulose synthase offer new opportunities to modify cellulose itself. Copyright © 2014 Elsevier Ltd. All rights reserved.
Apetrei, Irina Mirela; Apetrei, Constantin
2013-01-01
A biosensor comprising tyrosinase immobilized on a single-walled carbon nanotube-modified glassy carbon electrode has been developed. The sensitive element, ie, tyrosinase, was immobilized using a drop-and-dry method followed by cross-linking. Tyrosinase maintained high bioactivity on this nanomaterial, catalyzing the oxidation of epinephrine to epinephrine-quinone, which was electrochemically reduced (−0.07 V versus Ag/AgCl) on the biosensor surface. Under optimum conditions, the biosensor showed a linear response in the range of 10–110 μM. The limit of detection was calculated to be 2.54 μM with a correlation coefficient of 0.977. The repeatability, expressed as the relative standard deviation for five consecutive determinations of 10−5 M epinephrine solution was 3.4%. A good correlation was obtained between results obtained by the biosensor and those obtained by ultraviolet spectrophotometric methods. PMID:24348034
Noncanonical Radical SAM Enzyme Chemistry Learned from Diphthamide Biosynthesis.
Dong, Min; Zhang, Yugang; Lin, Hening
2018-05-10
Radical S-adenosylmethionine (SAM) enzymes are a superfamily of enzymes that use SAM and reduced [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical to catalyze numerous challenging reactions. We have reported a type of noncanonical radical SAM enzymes in the diphthamide biosynthesis pathway. These enzymes also use SAM and reduced [4Fe-4S] clusters, but generate a 3-amino-3-carboxypropyl (ACP) radical to modify the substrate protein, translation elongation factor 2. The regioselective cleavage of a different C-S bond of the sulfonium center of SAM in these enzymes comparing to canonical radical SAM enzymes is intriguing. Here, we highlight some recent findings in the mechanism of these types of enzymes, showing that the diphthamide biosynthetic radial SAM enzymes bound SAM with a distinct geometry. In this way, the unique iron of the [4Fe-4S] cluster in the enzyme can only attack the carbon on the ACP group to form an organometallic intermediate. The homolysis of the organometallic intermediate releases the ACP radical and generates the EF2 radial.
Characterizing visible and invisible cell wall mutant phenotypes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Carpita, Nicholas C.; McCann, Maureen C.
2015-04-06
About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basismore » of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.« less
Enzymatic degradation of cell wall and related plant polysaccharides.
Ward, O P; Moo-Young, M
1989-01-01
Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.
Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research.
Dedieu, L; Serveau-Avesque, C; Kremer, L; Canaan, S
2013-01-01
Tuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB. Copyright © 2012 Elsevier Masson SAS. All rights reserved.
2013-01-01
Background The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis. Results We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain. Conclusions The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1
Yue, Chao-Yan; Ying, Chun-Mei
2017-01-01
To explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method. Serum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation. There was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions. Pre-mixing protocol did not influence the results of fresh serum or frozen serum incubation <3days at 4°C and -80°C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy. Copyright © 2016 Elsevier B.V. All rights reserved.
Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques.
King, S. P.; Lunn, J. E.; Furbank, R. T.
1997-01-01
Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning. PMID:12223695
Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques.
King, S. P.; Lunn, J. E.; Furbank, R. T.
1997-05-01
Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.
Action of multi-enzyme complex on protein extraction to obtain a protein concentrate from okara.
de Figueiredo, Vitória Ribeiro Garcia; Yamashita, Fábio; Vanzela, André Luis Laforga; Ida, Elza Iouko; Kurozawa, Louise Emy
2018-04-01
The objective of this study was to optimize the extraction of protein by applying a multi-enzymatic pretreatment to okara, a byproduct from soymilk processing. The multi-enzyme complex Viscozyme, containing a variety of carbohydrases, was used to hydrolyze the okara cell walls and facilitate extraction of proteins. Enzyme-assisted extraction was carried out under different temperatures (37-53 °C), enzyme concentrations (1.5-4%) and pH values (5.5-6.5) according to a central composite rotatable design. After extraction, the protein was concentrated by isoelectric precipitation. The optimal conditions for maximum protein content and recovery in protein concentrate were 53 °C, pH 6.2 and 4% of enzyme concentration. Under these conditions, protein content of 56% (dry weight basis) and a recovery of 28% were obtained, representing an increase of 17 and 86%, respectively, compared to the sample with no enzymatic pretreatment. The multi-enzyme complex Viscozyme hydrolyzed the structural cell wall polysaccharides, improving extraction and obtaining protein concentrate from the okara. An electrophoretic profile of the protein concentrate showed two distinct bands, corresponding to the acidic and basic subunits of the protein glycinin. There were no limiting amino acids in the protein concentrate, which had a greater content of arginine.
Effects of multivalent cations on cell wall-associated acid phosphatase activity
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tu, S.I.; Brouillette, J.N.; Nagahashi, G.
1988-09-01
Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu/sup 2 +/, Mg/sup 2 +/, Za/sup 2 +/, and Mn/sup 2 +/; unaffected by Ba/sup 2 +/, Cd/sup 2 +/, and Pb/sup 2 +/; and inhibited by Al/sup 3 +/. The bound acid phosphatase of PCW was stimulatedmore » by a low concentration but inhibited by a higher concentration of Hg/sup 2 +/. On the other hand, in the case of corn root cells walls (CCW), only inhibition of the bound acid phosphatase by Al/sup 3 +/ and Hg/sup 2 +/ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg/sup 2 +/. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca/sup 2 +/ significantly reduced the effects of Hg/sup 2 +/ or Al/sup 3 +/, but not Mg/sup 2 +/, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg/sup 2 +/ or Al/sup 3 +/ which caused a Ca/sup 2 +/-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.« less
Palmero, Paola; Colle, Ines; Lemmens, Lien; Panozzo, Agnese; Nguyen, Tuyen Thi My; Hendrickx, Marc; Van Loey, Ann
2016-01-15
High-pressure homogenization disrupts cell structures, assisting carotenoid release from the matrix and subsequent micellarization. However, lycopene bioaccessibility of tomato puree upon high-pressure homogenization is limited by the formation of a process-induced barrier. In this context, cell wall-degrading enzymes were applied to hydrolyze the formed barrier and enhance lycopene bioaccessibility. The effectiveness of the enzymes in degrading their corresponding substrates was evaluated (consistency, amount of reducing sugars, molar mass distribution and immunolabeling). An in vitro digestion procedure was applied to evaluate the effect of the enzymatic treatments on lycopene bioaccessibility. Enzymatic treatments with pectinases and cellulase were proved to effectively degrade their corresponding cell wall polymers; however, no further significant increase in lycopene bioaccessibility was obtained. A process-induced barrier consisting of cell wall material is not the only factor governing lycopene bioaccessibility upon high-pressure homogenization. © 2015 Society of Chemical Industry.
Liu, Xiuhui; Bu, Caihong; Nan, Zhihan; Zheng, Lichun; Qiu, Yu; Lu, Xiaoquan
2013-02-15
We report on a new approach for the electrochemical detection of hydrogen peroxide (H2O2) based on Cytochrome C (Cyt c) immobilized ionic liquid (IL)-functionalized multi-walled carbon nanotubes (MWCNTs) modified glass carbon electrode (GCE). Functionalization of multi-walled carbon nanotube with amine-terminated ionic liquid materials was characterized using fourier transform infrared spectroscopy (FTIR), UV-vis spectra, and electrochemical impedance spectroscopy (EIS), and the results showed that the covalent modification of MWCNTs with ILs exhibited a high surface area for enzyme immobilization and provided a good microenvironment for Cyt c to retain its bioelectrocatalytic activity toward H2O2. Amperometry was used to evaluate the catalytic activity of the cyt c towards H2O2. The proposed biosensor exhibited a wide linear response range nearly 4 orders of magnitude of H2O2 (4.0 × 10(-8)M-1.0 × 10(-4)M) with a good linearity (0.9980) and a low detection limit of 1.3 × 10(-8)M (based on S/N=3). Furthermore, the biosensor also displays some other excellent characteristics such as high selectivity, good reproducibility and long-term stability. Thus, the biosensor constructed in this study has great potential for detecting H2O2 in the complex biosystems. Copyright © 2012 Elsevier B.V. All rights reserved.
USDA-ARS?s Scientific Manuscript database
Octenylsuccinic anhydride (OSA)-modified starches with degree of substitution of 0.018 (OS-S-L) and 0.092 (OS-S-H) were prepared from granular native waxy maize starch in an aqueous slurry system. The substitution distribution of OS groups was investigated by enzyme hydrolysis followed by chromatogr...
NASA Technical Reports Server (NTRS)
Everhart, J. L.
1983-01-01
The theoretical development of a simple and consistent method for removing the interference in adaptive-wall wind tunnels is reported. A Cauchy integral formulation of the velocities in an imaginary infinite extension of the real wind-tunnel flow is obtained and evaluated on a closed contour dividing the real and imaginary flow. The contour consists of the upper and lower effective wind-tunnel walls (wall plus boundary-layer displacement thickness) and upstream and downstream boundaries perpendicular to the axial tunnel flow. The resulting integral expressions for the streamwise and normal perturbation velocities on the contour are integrated by assuming a linear variation of the velocities between data-measurement stations along the contour. In an iterative process, the velocity components calculated on the upper and lower boundaries are then used to correct the shape of the wall to remove the interference. Convergence of the technique is shown numerically for the cases of a circular cylinder and a lifting and nonlifting NACA 0012 airfoil in incompressible flow. Experimental convergence at a transonic Mach number is demonstrated by using an NACA 0012 airfoil at zero lift.
Enzyme and Chemical Encapsulation in Polymeric Microcapsules,
1995-01-01
Polypyrrole microcapsules (prepared via the template method) were used for immobilization of both enzymatic and chemical catalytic systems. Enzymes...immobilized Pd nanoparticles for catalysis of hydrogen peroxide decomposition. Microcapsules loaded with glucose oxidase (GOD) were found to have...from the capsules; no leakage was observed. Subtilisin was used to show that these microcapsules can be used in non-aqueous solvents. The effect of capsule wall thickness on the rate of enzymatic reaction was also explored.
NASA Technical Reports Server (NTRS)
Xu, W.; Purugganan, M. M.; Polisensky, D. H.; Antosiewicz, D. M.; Fry, S. C.; Braam, J.
1995-01-01
Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.
Peptide inhibitor modified magnetic particles for pepsin separation.
Filuszová, Michaela; Kucerová, Zdenka; Tichá, Marie
2009-06-01
Synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) was coupled to glyoxal-activated magnetic agarose particles via the free peptide amino group. The peptide-modified magnetic particles were used for the separation of pepsins. Porcine pepsin A and human pepsin A were adsorbed to the magnetic peptide-modified affinity carrier, while the rat pepsin C and human pepsin C did not interact with the immobilized ligand. Conditions of pepsin adsorption to peptide-modified magnetic particles, as well as elution buffers were optimized. Porcine pepsin A did not interact with the immobilized peptide in the presence of pepsin inhibitor pepstatin A, indicating that the enzyme binding site is involved in the studied interaction. The elaborated method represents a rapid and simple technique not only for the separation of pepsins but also, in combination with MS, for the enzyme detection and determination.
Venditti, Tullio; D'hallewin, Guy; Ladu, Gianfranca; Petretto, Giacomo L; Pintore, Giorgio; Labavitch, John M
2018-03-25
The present study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with NaHCO 3 , at two different pH (5 and 8.3), on cell wall-degrading enzymes activity, over a period of 72 h. The treatment with NaHCO 3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, if compared to the control or the same treatment at pH 5. On the contrary, the pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5 treated plugs up to 72 h. The rapid increase in pectin methyl esterase activity, under alkaline conditions, is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. This article is protected by copyright. All rights reserved.
Role of conformational dynamics in the evolution of novel enzyme function.
Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia
2018-05-21
The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.
Synthetic metabolism: metabolic engineering meets enzyme design.
Erb, Tobias J; Jones, Patrik R; Bar-Even, Arren
2017-04-01
Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Asgharzadeh, Mohammad; Mohammadzadeh Asl, Yalda
2017-10-01
Enzymatic inactivation is one of the most important mechanisms of resistance to aminoglycosides. The aim of this study was to investigate the prevalence of armA and diversity of the genes encoding aminoglycoside-modifying enzymes (AMEs) and their associations with resistance phenotypes in Enterobacteriaceae isolates. Three hundred and seven Enterobacteriaceae isolates were collected from five hospitals in northwest Iran. The disk diffusion method for amikacin, gentamicin, tobramycin, kanamycin, and streptomycin, as well as the minimum inhibitory concentration for amikacin, gentamicin, tobramycin, and kanamycin were done for susceptibility testing. Thirteen AME genes and armA methylase were screened using the PCR and sequencing assays. Two hundred and twenty (71.7%) of isolates were resistant to aminoglycosides and 155 (70.5%) of them were positive for aminoglycoside resistance genes. The most prevalent AME genes were ant(3″)-Ia and aph(3″)-Ib with the frequency 35.9% and 30.5%, respectively. Also, 21 (9.5%) of resistant isolates were positive for armA methylase gene. The prevalence of resistance to aminoglycoside is high and AME genes frequently are disseminated in Enterobacteriaceae isolates. There is an association between phenotypic resistance and the presence of some aminoglycoside genes.
A small cellulose binding domain protein in Phytophtora is cell wall localized
USDA-ARS?s Scientific Manuscript database
Cellulose binding domains (CBD) are structurally conserved regions linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are not generally present in plant pathogenic fungi. A genome wide survey of CBDs w...
Froestl, Wolfgang; Pfeifer, Andrea; Muhs, Andreas
2013-01-01
Cognitive enhancers (nootropics) are drugs to treat cognition deficits in patients suffering from Alzheimer's disease, schizophrenia, stroke, attention deficit hyperactivity disorder, or aging. Cognition refers to a capacity for information processing, applying knowledge, and changing preferences. It involves memory, attention, executive functions, perception, language, and psychomotor functions. The term nootropics was coined in 1972 when memory enhancing properties of piracetam were observed in clinical trials. In the meantime, hundreds of drugs have been evaluated in clinical trials or in preclinical experiments. To classify the compounds, a concept is proposed assigning drugs to 19 categories according to their mechanism(s) of action, in particular drugs interacting with receptors, enzymes, ion channels, nerve growth factors, re-uptake transporters, antioxidants, metal chelators, and disease modifying drugs, meaning small molecules, vaccines, and monoclonal antibodies interacting with amyloid-β and tau. For drugs, whose mechanism of action is not known, they are either classified according to structure, e.g., peptides, or their origin, e.g., natural products. The review covers the evolution of research in this field over the last 25 years.
Uraji, Misugi; Kimura, Masayo; Inoue, Yosikazu; Kawakami, Kayoko; Kumagai, Yuya; Harazono, Koichi; Hatanaka, Tadashi
2013-11-01
Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.
Nasseau, M; Boublik, Y; Meier, W; Winterhalter, M; Fournier, D
2001-12-05
How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. Copyright 2001 John Wiley & Sons, Inc.
SigrafW: An Easy-to-Use Program for Fitting Enzyme Kinetic Data
ERIC Educational Resources Information Center
Leone, Francisco Assis; Baranauskas, Jose Augusto; Furriel, Rosa Prazeres Melo; Borin, Ivana Aparecida
2005-01-01
SigrafW is Windows-compatible software developed using the Microsoft[R] Visual Basic Studio program that uses the simplified Hill equation for fitting kinetic data from allosteric and Michaelian enzymes. SigrafW uses a modified Fibonacci search to calculate maximal velocity (V), the Hill coefficient (n), and the enzyme-substrate apparent…
Abeta-degrading enzymes in Alzheimer's disease.
Miners, James Scott; Baig, Shabnam; Palmer, Jennifer; Palmer, Laura E; Kehoe, Patrick G; Love, Seth
2008-04-01
In Alzheimer's disease (AD) Abeta accumulates because of imbalance between the production of Abeta and its removal from the brain. There is increasing evidence that in most sporadic forms of AD, the accumulation of Abeta is partly, if not in some cases solely, because of defects in its removal--mediated through a combination of diffusion along perivascular extracellular matrix, transport across vessel walls into the blood stream and enzymatic degradation. Multiple enzymes within the central nervous system (CNS) are capable of degrading Abeta. Most are produced by neurons or glia, but some are expressed in the cerebral vasculature, where reduced Abeta-degrading activity may contribute to the development of cerebral amyloid angiopathy (CAA). Neprilysin and insulin-degrading enzyme (IDE), which have been most extensively studied, are expressed both neuronally and within the vasculature. The levels of both of these enzymes are reduced in AD although the correlation with enzyme activity is still not entirely clear. Other enzymes shown capable of degrading Abetain vitro or in animal studies include plasmin; endothelin-converting enzymes ECE-1 and -2; matrix metalloproteinases MMP-2, -3 and -9; and angiotensin-converting enzyme (ACE). The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 are reported to be reduced in AD. Reductions in neprilysin, IDE and plasmin in AD have been associated with possession of APOEepsilon4. We found no change in the level or activity of MMP-2, -3 or -9 in AD. The level and activity of ACE are increased, the level being directly related to Abeta plaque load. Up-regulation of some Abeta-degrading enzymes may initially compensate for declining activity of others, but as age, genetic factors and diseases such as hypertension and diabetes diminish the effectiveness of other Abeta-clearance pathways, reductions in the activity of particular Abeta-degrading enzymes may become critical, leading to the development of AD and CAA.
Near-wall modelling of compressible turbulent flows
NASA Technical Reports Server (NTRS)
So, Ronald M. C.
1990-01-01
Work was carried out to extend the near-wall models formulated for the incompressible Reynolds stress equations to compressible flows. The idea of splitting the compressible dissipation function into a solenoidal part that is not sensitive to changes of compressibility indicators and a compressible part that is directly affected by these changes is adopted. This means that all models involving the dissipation rate could be expressed in terms of the solenoidal dissipation rate and an equation governing its transport could be formulated to close the set of compressible Reynolds stress equations. The near-wall modelling of the dissipation rate equation is investigated and its behavior near a wall is studied in detail using k-epsilon closure. It is found that all existing modelled equations give the wrong behavior for the dissipation rate near a wall. Improvements are suggested and the resultant behavior is found to be in good agreement with near-wall data. Furthermore, the present modified k-epsilon closure is used too calculate a flat plate boundary layer and the results are compared with four existing k-epsilon closures. These comparisons show that all closures tested give essentially the same flow properties, except in a region very close to the wall. In this region, the present k-epsilon closure calculations are in better agreement with measurements and direct simulation data; in particular, the behavior of the dissipation rate.
Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.
Yeats, Trevor H; Bacic, Antony; Johnson, Kim L
2018-04-18
Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.
Modifying lignin to improve bioenergy feedstocks: strengthening the barrier against pathogens?†
Sattler, Scott E.; Funnell-Harris, Deanna L.
2013-01-01
Lignin is a ubiquitous polymer present in cell walls of all vascular plants, where it rigidifies and strengthens the cell wall structure through covalent cross-linkages to cell wall polysaccharides. The presence of lignin makes the cell wall recalcitrant to conversion into fermentable sugars for bioenergy uses. Therefore, reducing lignin content and modifying its linkages have become major targets for bioenergy feedstock development through either biotechnology or traditional plant breeding. In addition, lignin synthesis has long been implicated as an important plant defense mechanism against pathogens, because lignin synthesis is often induced at the site of pathogen attack. This article explores the impact of lignin modifications on the susceptibility of a range of plant species to their associated pathogens, and the implications for development of feedstocks for the second-generation biofuels industry. Surprisingly, there are some instances where plants modified in lignin synthesis may display increased resistance to associated pathogens, which is explored in this article. PMID:23577013
Cao, Xu-Ni; Lin, Li; Zhou, Yu-Yan; Shi, Guo-Yue; Zhang, Wen; Yamamoto, Katsunobu; Jin, Li-Tong
2003-07-27
In this paper, multi-wall carbon nanotubes functionalized with carboxylic groups modified electrode (MWNT-COOH CME) was fabricated. This chemically modified electrode (CME) can be used as the working electrode in the liquid chromatography for the determination of 6-mercaptopurine (6-MP). The results indicate that the CME exhibits efficiently electrocatalytic oxidation for 6-MP with relatively high sensitivity, stability and long-life. The peak currents of 6-MP are linear to its concentrations ranging from 4.0 x 10(-7) to 1.0 x 10(-4) mol l(-1) with the calculated detection limit (S/N=3) of 2.0 x 10(-7) mol l(-1). Coupled with microdialysis, the method has been successfully applied to the pharmacokinetic study of 6-MP in rabbit blood. This method provides a fast, sensible and simple technique for the pharmacokinetic study of 6-MP in vivo.
NASA Astrophysics Data System (ADS)
Yang, Rui; Wang, Siqun; Zhou, Dingguo; Zhang, Jie; Lan, Ping; Jia, Chong
2018-01-01
Dimethyldichlorosilane was used to improve the hydrophobicity of wood surface. The water contact angle of the treated wood surface increased from 85° to 143°, which indicated increased hydrophobicity. The nanomechanical properties of the wood cell wall were evaluated using a nanoindentation test to analyse the hydrophobic mechanism on the nano scale. The elastic modulus of the cell wall was significantly affected by the concentration but the influence of treatment time is insignificant. The hardness of the cell wall for treated samples was significantly affected by both treatment time and concentration. The interaction between treatment time and concentration was extremely significant for the elastic modulus of the wood cell wall.
Mehta, Krunal K; Paskaleva, Elena E; Wu, Xia; Grover, Navdeep; Mundra, Ruchir V; Chen, Kevin; Zhang, Yongrong; Yang, Zhiyong; Feng, Hanping; Dordick, Jonathan S; Kane, Ravi S
2016-12-01
Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Carere, Jason; Colgrave, Michelle L; Stiller, Jiri; Liu, Chunji; Manners, John M; Kazan, Kemal; Gardiner, Donald M
2016-11-01
Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes. Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed. Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted. The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies. © 2016 CSIRO. New Phytologist © 2016 New Phytologist Trust.
Enzymes- An Existing and Promising Tool of Food Processing Industry.
Ray, Lalitagauri; Pramanik, Sunita; Bera, Debabrata
2016-01-01
The enzyme catalyzed process technology has enormous potential in the food sectors as indicated by the recent patents studies. It is very well realized that the adaptation of the enzyme catalyzed process depends on the availability of enzyme in affordable prices. Enzymes may be used in different food sectors like dairy, fruits & vegetable processing, meat tenderization, fish processing, brewery and wine making, starch processing and many other. Commercially only a small number of enzymes are used because of several factors including instability of enzymes during processing and high cost. More and more enzymes for food technology are now derived from specially selected or genetically modified microorganisms grown in industrial scale fermenters. Enzymes with microbial source have commercial advantages of using microbial fermentation rather than animal and plant extraction to produce food enzymes. At present only a relatively small number of enzymes are used commercially in food processing. But the number is increasing day by day and field of application will be expanded more and more in near future. The purpose of this review is to describe the practical applications of enzymes in the field of food processing.
A potentiometric enzyme electrode for the direct measurement of organophosphate (OP)
nerve agents was developed. The basic element of this enzyme electrode was a pH electrode
modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking
OPH ...
Sun, Yuan; Zhang, Zhizhou
2016-09-01
Polydimethylsiloxane (PDMS) with exceptional fouling-release properties is extremely susceptible to the microfouling resulted from the colonization of the pioneer microorganisms in the marine environment. In this study, six carboxyl-modified multi-walled carbon nanotubes (cMWNTs) nanoparticles were incorporated into the PDMS matrix, respectively, in order to produce the cMWNTs-filled PDMS nanocomposites (CPs) with improved antifouling (AF) properties. The AF properties of the six CPs were examined via the field assays conducted in Weihai, China. The effects of the anti-biofouling potential of the CPs (i.e. the P3 surface) on the colonization of the pioneer prokaryotic and eukaryotic microbes were investigated using the single-stranded conformation polymorphism technique via the comparison of the diversity indices. Different CPs have displayed differential and better AF properties as compared to that of the unfilled PDMS (P0). The P3 surface has exhibited exceptional anti-biofouling capacity compared with the other CPs surfaces, which can effectively prevent biofouling for more than 14 weeks in the field. The SSCP analysis revealed that the P3 surface may have significant modulating effect on the pioneer microbial communities. The pioneer eukaryotic microbes seemed more susceptible than the pioneer prokaryotic microbes to be subjected to the major perturbations exerted by the P3 surface. The dramatically reduced eukaryotic-microbial diversity may contribute to the impeding and weakening of the development and growth of the biofilm. The P3 surface has the potential to be used for future maritime applications.
Mbéguié-A-Mbéguié, D.; Hubert, O.; Baurens, F. C.; Matsumoto, T.; Chillet, M.; Fils-Lycaon, B.; Sidibé-Bocs, S.
2009-01-01
Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1–4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates. PMID:19357434
Regional cardiac wall motion from gated myocardial perfusion SPECT studies
NASA Astrophysics Data System (ADS)
Smith, M. F.; Brigger, P.; Ferrand, S. K.; Dilsizian, V.; Bacharach, S. L.
1999-06-01
A method for estimating regional epicardial and endocardial wall motion from gated myocardial perfusion SPECT studies has been developed. The method uses epicardial and endocardial boundaries determined from four long-axis slices at each gate of the cardiac cycle. The epicardial and endocardial wall position at each time gate is computed with respect to stationary reference ellipsoids, and wall motion is measured along lines normal to these ellipsoids. An initial quantitative evaluation of the method was made using the beating heart from the dynamic mathematical cardiac torso (MCAT) phantom, with and without a 1.5-cm FWHM Gaussian blurring filter. Epicardial wall motion was generally well-estimated within a fraction of a 3.56-mm voxel, although apical motion was overestimated with the Gaussian filter. Endocardial wall motion was underestimated by about two voxels with and without the Gaussian filter. The MCAT heart phantom was modified to model hypokinetic and dyskinetic wall motion. The wall motion analysis method enabled this abnormal motion to be differentiated from normal motion. Regional cardiac wall motion also was analyzed for /sup 201/Tl patient studies. Estimated wall motion was consistent with a nuclear medicine physician's visual assessment of motion from gated long-axis slices for male and female study examples. Additional research is required for a comprehensive evaluation of the applicability of the method to patient studies with normal and abnormal wall motion.
A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification.
Sojitra, Uttam V; Nadar, Shamraja S; Rathod, Virendra K
2016-12-15
The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.
Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L.; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.; Mengin-Lecreulx, Dominique; Wilson, Ian A.
2011-01-01
Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships. PMID:21445265
Das, Debanu; Hervé, Mireille; Feuerhelm, Julie; Farr, Carol L; Chiu, Hsiu-Ju; Elsliger, Marc-André; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A
2011-03-18
Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In gram-negative bacteria, ∼30-60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.
Azzolina, B A; Yuan, X; Anderson, M S; El-Sherbeini, M
2001-04-01
We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes. Copyright 2001 Academic Press.
Kronish, Donald P.; Mohan, Raam R.; Schwartz, Benjamin S.
1964-01-01
Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581–587. 1964.—Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C14-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed. Images PMID:14127573
Trützschler, Julia; Sentosun, Kadir; Mozooni, Babak; Mattheis, Roland; McCord, Jeffrey
2016-08-04
High density magnetic domain wall gratings are imprinted in ferromagnetic-antiferromagnetic thin films by local ion irradiation by which alternating head-to-tail-to-head-to-tail and head-to-head-to-tail-to-tail spatially overlapping domain wall networks are formed. Unique magnetic domain processes result from the interaction of anchored domain walls. Non-linear magnetization response is introduced by the laterally distributed magnetic anisotropy phases. The locally varying magnetic charge distribution gives rise to localized and guided magnetization spin-wave modes directly constrained by the narrow domain wall cores. The exchange coupled multiphase material structure leads to unprecedented static and locally modified dynamic magnetic material properties.
NASA Astrophysics Data System (ADS)
Trützschler, Julia; Sentosun, Kadir; Mozooni, Babak; Mattheis, Roland; McCord, Jeffrey
2016-08-01
High density magnetic domain wall gratings are imprinted in ferromagnetic-antiferromagnetic thin films by local ion irradiation by which alternating head-to-tail-to-head-to-tail and head-to-head-to-tail-to-tail spatially overlapping domain wall networks are formed. Unique magnetic domain processes result from the interaction of anchored domain walls. Non-linear magnetization response is introduced by the laterally distributed magnetic anisotropy phases. The locally varying magnetic charge distribution gives rise to localized and guided magnetization spin-wave modes directly constrained by the narrow domain wall cores. The exchange coupled multiphase material structure leads to unprecedented static and locally modified dynamic magnetic material properties.
Jung, S K; Wilson, G S
1996-02-15
An oxidase-based glucose sensor has been developed that uses a mercaptosilane-modified platinum electrode to achieve selectivity of electrochemical interferants. A platinum-iridium (9:1) wire (0.178 mm o.d., sensing area of 1.12 mm2) is modified with (3-mercaptopropyl)trimethoxysilane. The modified sensors show excellent operational stability for more than 5 days. Glucose oxidase is immobilized on the modified surface (i) by using 3-maleimidopropionic acid as a linker or (ii) by cross-liking with bovine serum albumin using glutaraldehyde. Sensitivities in the range of 9.97 nA/mM glucose are observed when the enzyme is immobilized by method ii. Lower sensitivities (1.13 x 10(-1) nA/mM glucose) are observed when immobilization method i is employed. In terms of linear response range, the sensor enzyme-immobilized by method i is superior to that immobilized by method ii. The linearity is improved upon coating the enzyme layer with polyurethane. The sensor immobilized by method ii and coated with polyurethane exhibits a linear range to 15 mM glucose and excellent selectivity to glucose (0.47 nA/mM) against interferants such as ascorbic acid, uric acid, and acetaminophen.
Du, Fuying; Fung, Ying-Sing
2018-06-01
Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4-nitrophenol (4-NP) in the form of free phenol, glucuronide (4-NP-G) or as a sulfate ester (4-NP-S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC-CE device incorporating post-CE amperometric detection using multi-walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4-NP, 4-NP-G, and 4-NP-S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC-CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4-NP, 4-NP-G, and 4-NP-S in human urine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Wang, Qiong; Yang, Jichun; Dong, Yuanyuan; Zhang, Lei
2015-05-20
Benomyl, as one kind of agricultural pesticide, has adverse impact on human health and the environment. It is urgent to develop effective and rapid methods for quantitative determination of benomyl. A simple and sensitive electroanalytical method for determination of benomyl using a magnetic nickel ferrite (NiFe2O4)/multi-walled carbon nanotubes (MWCNTs) nanohybrid-modified glassy carbon electrode (GCE) was presented. The electrocatalytic properties and electroanalysis of benomyl on the modified electrode were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In the phosphate-buffered saline (PBS) of pH 6.0, this constructed biosensor exhibited two linear relationships with the benomyl concentration range from 1.00 × 10(-7) to 5.00 × 10(-7) mol/L and from 5.00 × 10(-7) to 1.00 × 10(-5) mol/L, respectively. The detection limit was 2.51 × 10(-8) mol/L (S/N = 3). Moreover, the proposed method was successfully applied to determine benomyl in real samples with satisfactory results. The NiFe2O4/MWCNTs/GCE showed good reproducibility and stability, excellent catalytic activity, and anti-interference.
An Assessment of Regional Variations in Martian Modified Impact Crater Morphology
NASA Astrophysics Data System (ADS)
Craddock, Robert A.; Bandeira, Lourenço.; Howard, Alan D.
2018-03-01
Impact craters on Mars have been extensively modified by ancient geologic processes that may have included rainfall and surface runoff, snow and ice, denudation by lava flows, burial by eolian material, or others. Many of these processes can leave distinct signatures on the morphometry of the modified impact crater as well as the surrounding landscape. To look for signs of potential regional differences in crater modification processes, we conducted an analysis of different morphometric parameters related to modified impact craters located in the Margaritifer Sinus, Sinus Sabaeus, Iapygia, Mare Tyrrhenum, Aeolis, and Eridania quadrangles, including depth, crater wall slope, crater floor slope, the curvature between the interior wall and the crater floor slope, and the curvature between the interior wall and surrounding landscape. A Welch's t test analysis comparing these parameters shows that fresh impact craters (Type 4) have consistent morphologies regardless of their geographic location examined in this study, which is not unexpected. Modified impact craters both in the initial (Type 3) and terminal stages (Type 1) of modification also have statistically consistent morphologies. This would suggest that the processes that operated in the late Noachian were globally ubiquitous, and that modified craters eventually reached a stable crater morphology. However, craters preserved in advanced (but not terminal) stages of modification (Type 2) have morphologies that vary across the quadrangles. It is possible that these variations reflect spatial differences in the types and intensity of geologic processes that operated during the Noachian, implying that the ancient climate also varied across regions.
NASA Astrophysics Data System (ADS)
Khaldi, Khadidja; Sam, Sabrina; Lounas, Amel; Yaddaden, Chafiaa; Gabouze, Noure-Eddine
2017-11-01
In this work, Acetylcholinesterase enzyme (AChE) was immobilized on porous silicon (PSi) surface using two strategies. In the first method, acid chains were covalently grafted on the hydrogenated PSi by hydrosilylation reaction. The obtained acid-terminated surface was activated by a reaction with N-hydroxysuccinimide (NHS) in the presence of a peptide-coupling agent N-ethyl-N‧-(3-dimethylaminopropyl)-carbodiimide (EDC), and then reacted with the amino linker of the lysine residues AChE to anchor the enzyme by a covalent amide bond. In the second procedure, the PSi surface was first hydroxylated in piranha solution, followed by a silanization reaction with 3-aminopropyltriethoxysilane (APTES) to form amine-terminated surface. Finally, AChE was attached to the terminal amine groups by an aminolysis reaction with carboxylic acid groups of AChE in the presence of NHS/EDC mixture. Fourier transform infrared spectroscopy (FTIR) confirmed the efficiency of the surface modifications. The enzymatic activity of immobilized AChE was determined by means of a colorimetric test and was discussed according to the enzyme orientation on the surface which was revealed by contact angle measurements.
Modified RF coaxial connector ends vacuum chamber wiring problem
NASA Technical Reports Server (NTRS)
Weiner, D.
1964-01-01
A standard radio frequency coaxial connector is modified so that a plastic insulating sleeve can be mounted in the wall of a vacuum chamber. This eliminates ground loops and interference from cable connections.
Dhar, Supurna; Kumari, Hansi; Balasubramanian, Deepak; Mathee, Kalai
2018-01-01
The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.
Scherbahn, V; Putze, M T; Dietzel, B; Heinlein, T; Schneider, J J; Lisdat, F
2014-11-15
Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved. Copyright © 2014 Elsevier B.V. All rights reserved.
Moro, Camila Fernandes; Gaspar, Marilia; da Silva, Felipe Rodrigues; Pattathil, Sivakumar; Hahn, Michael G; Salgado, Ione; Braga, Marcia Regina
2017-03-01
Nitric oxide (NO) exerts pleiotropic effects on plant development; however, its involvement in cell wall modification during root hair formation (RHF) has not yet been addressed. Here, mutants of Arabidopsis thaliana with altered root hair phenotypes were used to assess the involvement of S-nitrosoglutathione (GSNO), the primary NO source, in cell wall dynamics and gene expression in roots induced to form hairs. GSNO and auxin restored the root hair phenotype of the hairless root hair defective 6 (rhd6) mutant. A positive correlation was observed between increased NO production and RHF induced by auxin in rhd6 and transparent testa glabra (ttg) mutants. Deposition of an epitope within rhamnogalacturonan-I recognized by the CCRC-M2 antibody was delayed in root hair cells (trichoblasts) compared with nonhair cells (atrichoblasts). GSNO, but not auxin, restored the wild-type root glycome and transcriptome profiles in rhd6, modulating the expression of a large number of genes related to cell wall composition and metabolism, as well as those encoding ribosomal proteins, DNA and histone-modifying enzymes and proteins involved in post-translational modification. Our results demonstrate that NO plays a key role in cell wall remodelling in trichoblasts and suggest that it also participates in chromatin modification in root cells of A. thaliana. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David
2006-02-01
The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.
Dubald, M; Barakate, A; Mandaron, P; Mache, R
1993-11-01
Exopolygalacturonase (exoPG) is a pectin-degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.
Sequential Reactions of Surface-Tethered Glycolytic Enzymes
Mukai, Chinatsu; Bergkvist, Magnus; Nelson, Jacquelyn L.; Travis, Alexander J.
2014-01-01
SUMMARY The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices. PMID:19778729
Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique
2014-01-01
Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256
Characterizing visible and invisible cell wall mutant phenotypes.
Carpita, Nicholas C; McCann, Maureen C
2015-07-01
About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Brill, Elizabeth; van Thournout, Michel; White, Rosemary G.; Llewellyn, Danny; Campbell, Peter M.; Engelen, Steven; Ruan, Yong-Ling; Arioli, Tony; Furbank, Robert T.
2011-01-01
Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars. PMID:21757635
Pidgeon, Sean E; Pires, Marcos M
2017-07-21
Drug-resistant bacterial infections threaten to overburden our healthcare system and disrupt modern medicine. A large class of potent antibiotics, including vancomycin, operate by interfering with bacterial cell wall biosynthesis. Vancomycin-resistant enterococci (VRE) evade the blockage of cell wall biosynthesis by altering cell wall precursors, rendering them drug insensitive. Herein, we reveal the phenotypic plasticity and cell wall remodeling of VRE in response to vancomycin in live bacterial cells via a metabolic probe. A synthetic cell wall analog was designed and constructed to monitor cell wall structural alterations. Our results demonstrate that the biosynthetic pathway for vancomycin-resistant precursors can be hijacked by synthetic analogs to track the kinetics of phenotype induction. In addition, we leveraged this probe to interrogate the response of VRE cells to vancomycin analogs and a series of cell wall-targeted antibiotics. Finally, we describe a proof-of-principle strategy to visually inspect drug resistance induction. Based on our findings, we anticipate that our metabolic probe will play an important role in further elucidating the interplay among the enzymes involved in the VRE biosynthetic rewiring.
Koley, Tanmay Kumar; Walia, Shweta; Nath, Prerna; Awasthi, O P; Kaur, Charanjit
2011-05-01
Zizyphus (Indian ber) is an excellent source of several phenolic compounds. The effect of two cell wall degrading enzymes, namely pectinase and viscozyme, on the nutraceutical composition of Zizyphus juice was investigated in the present study. Enzyme assisted processing significantly (P < 0.05) improved the juice yield, total soluble solids, total phenolics and total antioxidant activity (AOX). There was significant increase in recovery of antioxidants, to the tune of 70.51%, 66%, and 45% respectively in ascorbic acid, total phenolics and total flavonoids through viscozyme. The in-vitro total AOX of juice extracted via enzyme-assisted processing was 20.9 and 15.59 μmol Trolox/ml in ferric-reducing antioxidant power and cupric-reducing antioxidant capacity assays, respectively. There was 41% increase in AOX of juice extracted with enzyme over straight pressed juice. Results indicate that enzyme-assisted processing can significantly improve the functional properties of the Zizyphus juice.
Grinyer, Jasmine; Hunt, Sybille; McKay, Matthew; Herbert, Ben R; Nevalainen, Helena
2005-06-01
Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea, therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of the R. solani cell walls. Twenty-four protein spots were identified using matrix-assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and N-terminal sequencing. Identified up-regulated proteins include known fungal cell wall-degrading enzymes such as N-acetyl-beta-D: -glucosaminidase and 42-kDa endochitinase. Three novel proteases of T. atroviride were identified, containing sequence similarity to vacuolar serine protease, vacuolar protease A and a trypsin-like protease from known fungal proteins. Eukaryotic initiation factor 4a, superoxide dismutase and a hypothetical protein from Neurospora crassa were also up-regulated as a response to R. solani cell walls. Several cell wall-degrading enzymes were identified from the T. atroviride culture supernatant, providing further evidence that a cellular response indicative of biological control had occurred.
Wang, Hongjuan; Qian, Duo; Xiao, Xilin; Deng, Chunyan; Liao, Lifu; Deng, Jian; Lin, Ying-Wu
2018-06-01
An innovative electrochemical sensor was fabricated for the sensitive and selective determination of tinidazole (TNZ), based on a carbon paste electrode (CPE) modified with multi-walled carbon nanotubes (MWCNTs) and boron-embedded molecularly imprinted composite membranes (B-MICMs). Density functional theory (DFT) calculations were carried out to investigate the utility of template-monomer interactions to screen appropriate monomers for the rational design of B-MICMs. The distinct synergic effect of MWCNTs and B-MICMs was evidenced by the positive shift of the reduction peak potential of TNZ at B-MICMs/MWCNTs modified CPE (B-MICMs/MWCNTs/CPE) by about 200 mV, and the 12-fold amplification of the peak current, compared with a bare carbon paste electrode (CPE). Moreover, the coordinate interactions between trisubstituted boron atoms embedded in B-MICMs matrix and nitrogen atoms of TNZ endow the sensor with advanced affinity and specific directionality. Thereafter, a highly sensitive electrochemical analytical method for TNZ was established by different pulse voltammetry (DPV) at B-MICMs/MWCNTs/CPE with a lower detection limit (1.25 × 10 -12 mol L -1 ) (S/N = 3). The practical application of the sensor was demonstrated by determining TNZ in pharmaceutical and biological samples with good precision (RSD 1.36% to 3.85%) and acceptable recoveries (82.40%-104.0%). Copyright © 2018 Elsevier B.V. All rights reserved.
Kistler, Erik B; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W
2012-08-01
In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress
Kistler, Erik B.; Alsaigh, Tom; Chang, Marisol; Schmid-Schönbein, Geert W.
2012-01-01
In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small bowel lumen. It is unresolved, however, whether ischemically-mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access, and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of non-ischemic rats was perfused for two hours with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase and lipase. Control (n=6) and experimental animals perfused with pancreatic enzymes only (n=6) or single enzymes (n=3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n=6) developed mild hypotension (p<0.001 compared to groups perfused with pancreatic enzymes only after 90 minutes) and increased intestinal permeability to intralumenally perfused FITC-dextrans-20kD (p<0.05) compared to control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n=6) developed hypotension and increased intestinal permeability (p<0.001 after 90 minutes). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These
Mechanism of cassava tuber cell wall weakening by dilute sodium hydroxide steeping.
Odoch, Martin; Buys, Elna M; Taylor, John R N
2017-08-01
Steeping of cassava root pieces in 0.75% NaOH in combination with wet milling was investigated to determine whether and how dilute NaOH modifies cassava cell walls. Gas chromatography data of cell wall constituent sugar composition and Fourier transform infrared (FTIR) data showed that NaOH steeping reduced the level of pectin in cassava cell walls. FTIR and wide-angle X-ray scattering spectroscopy also indicated that NaOH steeping combined with fine milling slightly reduced cellulose crystallinity. Scanning electron microscopy showed that NaOH steeping produced micropores in the cell walls and light microscopy revealed that NaOH steeping increased disaggregation of parenchyma cells. Steeping of ground cassava in NaOH resulted in a 12% decrease in large residue particles and approx. 4% greater starch yield with wet milling. Therefore dilute NaOH steeping can improve the effectiveness of wet milling in disintegrating cell walls through solubilisation of pectin, thereby reduced cell wall strength. Copyright © 2017 Elsevier Ltd. All rights reserved.
Determination of the Airborne Sound Insulation of a Straw Bale Partition Wall
NASA Astrophysics Data System (ADS)
Teslík, Jiří; Fabian, Radek; Hrubá, Barbora
2017-06-01
This paper describes the results of a scientific project focused on determining of the Airborne Sound Insulation of a peripheral non-load bearing wall made of straw bales expressed by Weighted Sound Reduction Index. Weighted Sound Reduction Index was determined by measuring in the certified acoustic laboratory at the Faculty of Mechanical Engineering at Brno University of Technology. The measured structure of the straw wall was modified in combinations with various materials, so the results include a wide range of possible compositions of the wall. The key modification was application of plaster on both sides of the straw bale wall. This construction as is frequently done in actual straw houses. The additional measurements were performed on the straw wall with several variants of additional wall of slab materials. The airborne sound insulation value has been also measured in separate stages of the construction. Thus it is possible to compare and determinate the effect of the single layers on the airborne sound insulation.
Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?
Jing, Xiaomin; Evangelista Falcon, Wilfredo; Baudry, Jerome; Serpersu, Engin H
2017-07-27
Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher T m . Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.
Immobilization, stabilization and patterning techniques for enzyme based sensor systems.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Flounders, A.W.; Carichner, S.C.; Singh, A.K.
1997-01-01
Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less
Domain walls in the extensions of the Standard Model
NASA Astrophysics Data System (ADS)
Krajewski, Tomasz; Lalak, Zygmunt; Lewicki, Marek; Olszewski, Paweł
2018-05-01
Our main interest is the evolution of domain walls of the Higgs field in the early Universe. The aim of this paper is to understand how dynamics of Higgs domain walls could be influenced by yet unknown interactions from beyond the Standard Model. We assume that the Standard Model is valid up to certain, high, energy scale Λ and use the framework of the effective field theory to describe physics below that scale. Performing numerical simulations with different values of the scale Λ we are able to extend our previous analysis [1]. Our recent numerical simulations show that evolution of Higgs domain walls is rather insensitive to interactions beyond the Standard Model as long as masses of new particles are grater than 1012 GeV. For lower values of Λ the RG improved effective potential is strongly modified at field strengths crucial to the evolution of domain walls. However, we find that even for low values of Λ, Higgs domain walls decayed shortly after their formation for generic initial conditions. On the other hand, in simulations with specifically chosen initial conditions Higgs domain walls can live longer and enter the scaling regime. We also determine the energy spectrum of gravitational waves produced by decaying domain walls of the Higgs field. For generic initial field configurations the amplitude of the signal is too small to be observed in planned detectors.
An improved k-epsilon model for near wall turbulence
NASA Technical Reports Server (NTRS)
Shih, T. H.; Hsu, Andrew T.
1991-01-01
An improved k-epsilon model for low Reynolds number turbulence near a wall is presented. In the first part of this work, the near-wall asymptotic behavior of the eddy viscosity and the pressure transport term in the turbulent kinetic energy equation are analyzed. Based on these analyses, a modified eddy viscosity model with the correct near-wall behavior is suggested, and a model for the pressure transport term in the k-equation is proposed. In addition, a modeled dissipation rate equation is reformulated, and a boundary condition for the dissipation rate is suggested. In the second part of the work, one of the deficiencies of the existing k-epsilon models, namely, the wall distance dependency of the equations and the damping functions, is examined. An improved model that does not depend on any wall distance is introduced. Fully developed turbulent channel flows and turbulent boundary layers over a flat plate are studied as validations for the proposed new models. Numerical results obtained from the present and other previous k-epsilon models are compared with data from direct numerical simulation. The results show that the present k-epsilon model, with added robustness, performs as well as or better than other existing models in predicting the behavior of near-wall turbulence.
Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.
Botyanszki, Zsofia; Tay, Pei Kun R; Nguyen, Peter Q; Nussbaumer, Martin G; Joshi, Neel S
2015-10-01
Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. © 2015 Wiley Periodicals, Inc.
NASA Astrophysics Data System (ADS)
Pavlov, Alexey; Pavlova, Larisa; Pavlova, Lyudmila
2018-03-01
In article results of research of versions of offered types of heaters on the basis of products from the modified fibers for designing energy efficient building enclosures residential high-rise buildings are presented. Traditional building materials (reinforced concrete, brick, wood) are not able to provide the required value of thermal resistance in areas with a temperate and harsh Russia climate in a single-layered enclosing structure. It can be achieved in a multi-layered enclosing structure, where the decisive role is played by new insulating materials with high thermal properties. In general, modern design solutions for external walls are based on the use of new effective thermal insulation materials with the use of the latest technology. The relevance of the proposed topic is to research thermoinsulation properties of new mineral heaters. Theoretical researches of offered heaters from mineral wool on slime-colloidal binder, bentocolloid and microdispersed binders are carried out. In addition, theoretical studies were carried out with several types of facade systems. Comprehensive studies were conducted on the resistance to heat transfer, resistance to vapor permeation and air permeability. According to the received data, recommendations on the use of insulation types depending on the number of storeys of buildings are proposed.
Fent, János; Bihari, Péter; Vippola, Minnamari; Sarlin, Essi; Lakatos, Susan
2015-08-01
Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood. Copyright © 2015 Elsevier Ltd. All rights reserved.
Lenarz, Christopher J; Moed, Berton R
2007-09-01
The purpose of this study was to describe a form of anterior wall acetabular fracture, which has been inadequately defined in the literature. Retrospective analysis of consecutive patients. Level 1 trauma center. A retrospective analysis of consecutive acetabulum patients treated by a single surgeon between 1999 and 2005 identified 6 patients with a form of anterior wall fracture without involvement of the pelvic brim. All fractures were treated operatively. Open reduction and internal fixation through an anterior surgical approach. Final radiographic appearance and modified Merle d'Aubigne score. All 6 cases demonstrated characteristics of an atypical fracture of the anterior wall, involving the anterior rim of the acetabulum similar in nature to those described for the posterior wall, rather than the standard anterior wall fracture type described by Letournel. Of the 6 cases that were identified, 5 had follow-up 1 year or greater with a mean modified Merle d'Aubigne Score of 17 (range: 17-18). A form of anterior wall acetabular fracture exists, which involves the anterior acetabular rim without involvement of the pelvic brim. It can occur in young patients with high-energy mechanisms of injury, as well as in the elderly with low-energy trauma. With appropriate surgical management, using a modified Smith-Peterson approach, good to excellent clinical outcomes should be expected.
Bacterial Enzymes and Antibiotic Resistance- Oral Presentation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Maltz, Lauren
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 ofmore » 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.« less
Sucrose Synthase Is Associated with the Cell Wall of Tobacco Pollen Tubes1[W
Persia, Diana; Cai, Giampiero; Del Casino, Cecilia; Faleri, Claudia; Willemse, Michiel T.M.; Cresti, Mauro
2008-01-01
Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes. PMID:18344420
Li, Ran; Ibeagha-Awemu, Eveline M
2017-05-01
Recently we showed that 5% linseed oil (LSO) and 5% safflower oil (SFO) supplementation of cow's diets reduced milk fat yield by 30·38 and 32·42% respectively, accompanied by differential expression of genes and regulation by microRNAs (miRNA). This research communication addresses the hypothesis that epigenetic regulation could be involved in the observed milk fat reduction. Thus, this study investigated the gene expression pattern of major epigenetic modifying enzymes in response to dietary supplementation with LSO or SFO. Twenty-six Canadian Holstein cows in mid lactation were randomly assigned to two groups (13/group) and fed a control diet for 28 d (day -28 to -1) (control period- CP) followed by a treatment period (TP) (control diet supplemented with 5% LSO (LSO treatment) or 5% SFO (SFO treatment) of 28 d (day +1 to +28). After treatment, cows in the two groups were returned to the control diet for another 28 d (day +29 to +56) (post treatment period-PTP). Milk samples were collected on day -1 (CP), +7, +28 (TP) and +56 (PTP) for RNA isolation and measurement of the expression of thirteen epigenetic modifying genes including two DNA methytrasferases (DNMT1, DNMT3A), four histone acetylases (HAT1, KAT2A, KAT5 and CREBBP), five histone deacetylases (HDAC1, HDAC2, HDAC3, SIRT1 and SIRT2) and two histone methytransferases (EHMT2 and PRMT1) by qPCR. Linseed oil supplementation significantly repressed the expression of EHMT2, HDAC2 and HDAC3 on day +7 (P < 0·05) and KAT2A and SIRT2 on day +28 (P < 0·05) as compared with the control period (day -1) while SFO had no effect. When LSO was withdrawn, the expression of some of the genes increased slightly but did not reach control (day -1) levels at the end of the PTP. Our study demonstrated a significant role of LSO in the epigenetic regulation of fatty acid synthesis as compared to SFO. The effect of LSO may be related to its higher degree of unsaturation and might represent a different regulatory mechanism which
Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.
Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto
2016-06-01
Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Peptide nanotube-modified electrodes for enzyme-biosensor applications.
Yemini, Miri; Reches, Meital; Gazit, Ehud; Rishpon, Judith
2005-08-15
The fabrication and notably improved performance of composite electrodes based on modified self-assembled diphenylalanine peptide nanotubes is described. Peptide nanotubes were attached to gold electrodes, and we studied the resulting electrochemical behavior using cyclic voltammetry and chronoamperometry. The peptide nanotube-based electrodes demonstrated a direct and unmediated response to hydrogen peroxide and NADH at a potential of +0.4 V (vs SCE). This biosensor enables a sensitive determination of glucose by monitoring the hydrogen peroxide produced by an enzymatic reaction between the glucose oxidase attached to the peptide nanotubes and glucose. In addition, the marked electrocatalytic activity toward NADH enabled a sensitive detection of ethanol using ethanol dehydrogenase and NAD+. The peptide nanotube-based amperometric biosensor provides a potential new tool for sensitive biosensors and biomolecular diagnostics.
Roberfroid, M; Poncelet, F; Lambotte-Vandepaer, M; Duverger-Van Bogaert, M; de Meester, C; Mercier, M
1978-01-01
Styrene is commonly used in western Europe for the manufacture of plastics suitable for packaging foodstuffs. This report demonstrates that, injected intraperitoneally at a dose as low as 10 mg/kg, styrene modifies the catalytic properties of aryl hydrocarbon hydroxylase by reducing its KM value. A similar effect is reported for two potent chemical carcinogens, 3-methylcholanthrene and benzo(a)pyrene. Ethylbenzene and benzo(e)pyrene and phenobarbital do not produce the same effect. Pretreatments of the rats with chemicals which modify aryl hydrocarbon hydroxylase also increase the capacity of the liver enzymes to activate benzopyrene to a mutagenic intermediate in vitro, as measured by the Ames test for mutagenicity. Exposure to both styrene and the other modifiers of the xenobiotic-metabolizing enzymes could thus influence the carcinogenic and toxic effects of chemicals which are activated by these enzymes. This hypothesis needs further investigation.
Carvajal, N; González, R; Morán, A; Oyarce, A M
1985-01-01
Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.
Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; de Sousa, Paulo Robson Monteiro; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G.; Maguire, Glenn E.M.; Lamichhane, Gyanu; Lameira, Jerônimo
2015-01-01
SUMMARY Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. PMID:25701501
Cotton fiber tips have diverse morphologies and show evidence of apical cell wall synthesis
Stiff , Michael R.; Haigler, Candace H.
2016-01-01
Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip. PMID:27301434
Haddad, R; Cosnier, S; Maaref, A; Holzinger, M
2009-12-01
Single-walled carbon nanotubes were functionalized with biotin using either electropolymerization or formation of pi-stacking interactions for the construction of biosensors. Thanks to the high affinity of the avidin-biotin interactions, a biotinylated glucose oxidase (B-GOX) as a biomolecule model was immobilized on the biotinylated nanotubes. The influence of the biosensor configuration on their amperometric performances was investigated by changing the amount of nanotubes and the numbers of avidin/B-GOX layers. By increasing the amount of nanotube and avidin/B-GOX layers, both sensor setups show a perfect linear increase of immobilized enzymes reflecting a high reproducibility of our systems. The highest sensitivities (up to 5.2 mA M(-1) cm(-2)) and maximum current densities (up to 55 microA cm(-2)) were obtained using nanotube deposits modified by electrochemical coatings. In contrast, non-covalently functionalized biotin-nanotubes show a better permeability for the enzymatically generated hydrogen peroxide.
Cell-Wall Recycling of the Gram-Negative Bacteria and the Nexus to Antibiotic Resistance.
Dik, David A; Fisher, Jed F; Mobashery, Shahriar
2018-05-30
The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC β-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the β-lactam antibiotics, is the exemplar of this challenge. This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma.
Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis.
Zou, Yi; Garcia-Borràs, Marc; Tang, Mancheng C; Hirayama, Yuichiro; Li, Dehai H; Li, Li; Watanabe, Kenji; Houk, K N; Tang, Yi
2017-03-01
Epoxides are highly useful synthons and biosynthons for the construction of complex natural products during total synthesis and biosynthesis, respectively. Among enzyme-catalyzed epoxide transformations, a reaction that is notably missing, in regard to the synthetic toolbox, is cationic rearrangement that takes place under strong acid. This is a challenging transformation for enzyme catalysis, as stabilization of the carbocation intermediate upon epoxide cleavage is required. Here, we discovered two Brønsted acid enzymes that can catalyze two unprecedented epoxide transformations in biology. PenF from the penigequinolone pathway catalyzes a cationic epoxide rearrangement under physiological conditions to generate a quaternary carbon center, while AsqO from the aspoquinolone pathway catalyzes a 3-exo-tet cyclization to forge a cyclopropane-tetrahydrofuran ring system. The discovery of these new epoxide-modifying enzymes further highlights the versatility of epoxides in complexity generation during natural product biosynthesis.
Analytical nanosphere sensors using quantum dot-enzyme conjugates for urea and creatinine.
Ruedas-Rama, Maria J; Hall, Elizabeth A H
2010-11-01
An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to coimmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pKa in the immobilized system > 8. QD-CaR absorption shows spectral overlap with QD530 emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FRET) efficiency, due to excited state proton transfer (λ(ex) = 540 nm; λ(em) = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and creatinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min.
A Near-Wall Reynolds-Stress Closure Without Wall Normals
NASA Technical Reports Server (NTRS)
Yuan, S. P.; So, R. M. C.
1997-01-01
Turbulent wall-bounded complex flows are commonly encountered in engineering practice and are of considerable interest in a variety of industrial applications. The presence of a wall significantly affects turbulence characteristics. In addition to the wall effects, turbulent wall-bounded flows become more complicated by the presence of additional body forces (e.g. centrifugal force and Coriolis force) and complex geometry. Most near-wall Reynolds stress models are developed from a high-Reynolds-number model which assumes turbulence is homogenous (or quasi-homogenous). Near-wall modifications are proposed to include wall effects in near-wall regions. In this process, wall normals are introduced. Good predictions could be obtained by Reynolds stress models with wall normals. However, ambiguity arises when the models are applied in flows with multiple walls. Many models have been proposed to model turbulent flows. Among them, Reynolds stress models, in which turbulent stresses are obtained by solving the Reynolds stress transport equations, have been proved to be the most successful ones. To apply the Reynolds stress models to wall-bounded flows, near-wall corrections accounting for the wall effects are needed, and the resulting models are called near-wall Reynolds stress models. In most of the existing near-wall models, the near-wall corrections invoke wall normals. These wall-dependent near-wall models are difficult to implement for turbulent flows with complex geometry and may give inaccurate predictions due to the ambiguity of wall normals at corners connecting multiple walls. The objective of this study is to develop a more general and flexible near-wall Reynolds stress model without using any wall-dependent variable for wall-bounded turbulent flows. With the aid of near-wall asymptotic analysis and results of direct numerical simulation, a new near-wall Reynolds stress model (NNWRS) is formulated based on Speziale et al.'s high-Reynolds-stress model with wall
Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification
NASA Astrophysics Data System (ADS)
Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo
An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.
Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong
2015-01-01
Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the
Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong
2015-01-01
Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the
Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.
1999-01-01
Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997
Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; Scheller, Henrik V.
2017-01-01
Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy. PMID:28900439
Zhang, Qi; Chen, Shuo; Wang, Hua; Yu, Hongtao
2018-03-14
Hydrogen peroxide (H 2 O 2 ) is a key reactant in the Fenton process. As a byproduct of enzymatic reaction, H 2 O 2 can be obtained via catalytical oxidation of glucose using glucose oxidase in the presence of O 2 . Another oxidation product (gluconic acid) can suitably adjust the microenvironmental pH contributing to the Fe 3+ /Fe 2+ cycle in the Fenton reaction. Enzymes are extremely efficient at catalyzing a variety of reactions with high catalytic activity, substrate specificity, and yields in living organisms. Inspired by the multiple functions of natural multienzyme systems, an exquisite nanozyme-modified α-FeOOH/porous carbon (PC) biomimetic catalyst constructed by in situ growth of glucose oxidase-mimicking Au nanoparticles and crystallization of adsorbed ferric ions within carboxyl into hierarchically PC is developed as an efficient enzyme-Fenton catalyst. The products (H 2 O 2 , ∼4.07 mmol·L -1 ) of the first enzymatic reaction are immediately used as substrates for the second Fenton-like reaction to generate the valuable • OH (∼96.84 μmol·L -1 ), thus mimicking an enzyme cascade pathway. α-FeOOH nanocrystals, attached by C-O-Fe bondings, are encapsulated into the mesoporous PC frameworks, facilitating the electron transfer between α-FeOOH and the PC support and greatly suppressing iron leaching. This study paves a new avenue for designing biomimetic enzyme-based Fenton catalysts mimicking a natural system for • OH production.
Error in Dasibi flight measurements of atmospheric ozone due to instrument wall-loss
NASA Technical Reports Server (NTRS)
Ainsworth, J. E.; Hagemeyer, J. R.; Reed, E. I.
1981-01-01
Theory suggests that in laminar flow the percent loss of a trace constituent to the walls of a measuring instrument varies as P to the -2/3, where P is the total gas pressure. Preliminary laboratory ozone wall-loss measurements confirm this P to the -2/3 dependence. Accurate assessment of wall-loss is thus of particular importance for those balloon-borne instruments utilizing laminar flow at ambient pressure, since the ambient pressure decreases by a factor of 350 during ascent to 40 km. Measurements and extrapolations made for a Dasibi ozone monitor modified for balloon flight indicate that the wall-loss error at 40 km was between 6 and 30 percent and that the wall-loss error in the derived total ozone column-content for the region from the surface to 40 km altitude was between 2 and 10 percent. At 1000 mb, turbulence caused an order of magnitude increase in the Dasibi wall-loss.
Biofunctionalization of carbon nanostructures through enzyme immobilization in colloidal silica
NASA Astrophysics Data System (ADS)
Goulet, Evan M.
Multi-walled carbon nanotubes (MWNT) and carbon nanopipettes (CNP) provide interesting high aspect ratio scaffolds on which to base functionally gradient materials. In this dissertation, we present a general method for the production of an enzymatically active composite material based on MWNTs. Polyethyleneimine (PEI) was applied to purified MWNTs, generating a positive electrostatic potential on the MWNTs. This positive potential was used to apply negatively charged colloidal silica particle in the presence of a high concentration of enzyme. The silica coating continued to grow via localized condensation of silica particles driven by the buffered saline conditions, immobilizing the enzyme within the coating. The mesoporous nanostructure was characterized via transmission electron microscopy. Optical spectroscopy experiments on the material employed as an active suspension showed that the immobilized enzymes horseradish peroxidase (HRP) and tyrosinase (TV) retained their activity upon incorporation into the material. Using HRP as a model enzyme, it was determined that the MWNT-HRP-Silica material showed similar pH and temperature dependencies in activity to those of free HRP in solution. An examination of the Michaelis-Menten kinetics showed that the material had a slightly higher value of KM than did free HRP. The MWNT-HRP-Silica material was also employed as an active filter membrane, which allowed us to explore the reusable nature of the material. We were able to show the denaturation of the filter due to the loss of Ca2+ cations at low pH and then restore the activity by soaking the filter membrane in 1 mM CaCl2. The MWNT-HRP-Silica material was used to modify a carbon microelectrode and produce a functioning electrochemical sensor for H2O2 . Utilizing cyclic voltammetry, the sensor was shown to have a linear response in limiting current versus concentration of H2O2 of 4.26 pA/microM. We also determined a lower detection limit of 0.67 microM H2O2. CNPs were
USDA-ARS?s Scientific Manuscript database
Plant cell wall polysaccharides, which consist of polymeric backbones with various types of substitution, were studied using the concept of combinatorial enzyme technology for conversion of agricultural fibers to functional products. Using citrus pectin as the starting substrate, an active oligo spe...
Parre, Elodie; Geitmann, Anja
2005-02-01
The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.
Latgé, Jean-Paul; Beauvais, Anne; Chamilos, Georgios
2017-09-08
More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.
Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium
NASA Astrophysics Data System (ADS)
Freeman, Michael F.; Helf, Maximilian J.; Bhushan, Agneya; Morinaka, Brandon I.; Piel, Jörn
2017-04-01
Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning β-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C-methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C-methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature.
Influence of strong perturbations on wall-bounded flows
NASA Astrophysics Data System (ADS)
Buxton, O. R. H.; Ewenz Rocher, M.; Rodríguez-López, E.
2018-01-01
found to modify the nature of the inner-wall peak of turbulent kinetic energy.
Activity of selected hydrolytic enzymes in Allium sativum L. anthers.
Winiarczyk, Krystyna; Gębura, Joanna
2016-05-01
The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
Molecular imprinting at walls of silica nanotubes for TNT recognition.
Xie, Chenggen; Liu, Bianhua; Wang, Zhenyang; Gao, Daming; Guan, Guijian; Zhang, Zhongping
2008-01-15
This paper reports the molecular imprinting at the walls of highly uniform silica nanotubes for the recognition of 2,4,6-trinitrotoluene (TNT). It has been demonstrated that TNT templates were efficiently imprinted into the matrix of silica through the strong acid-base pairing interaction between TNT and 3-aminopropyltriethoxysilane (APTS). TNT-imprinted silica nanotubes were synthesized by the gelation reaction between APTS and tetraethylorthosilicate (TEOS), selectively occurring at the porous walls of APTS-modified alumina membranes. The removal of the original TNT templates leaves the imprinted cavities with covalently anchored amine groups at the cavity walls. A high density of recognition sites with molecular selectivity to the TNT analyte was created at the wall of silica nanotubes. Furthermore, most of these recognition sites are situated at the inside and outside surfaces of tubular walls and in the proximity of the two surfaces due to the ultrathin wall thickness of only 15 nm, providing a better site accessibility and lower mass-transfer resistance. Therefore, greater capacity and faster kinetics of uptaking target species were achieved. The silica nanotube reported herein is an ideal form of material for imprinting various organic or biological molecules toward applications in chemical/biological sensors and bioassay.
Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB
Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne
2014-01-01
Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772
Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.
Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne
2015-01-01
The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.
Antony, B; Johny, J; Aldosari, S A; Abdelazim, M M
2017-08-01
Plant cell wall degrading enzymes (PCWDEs) from insects were recently identified as a multigene family of proteins that consist primarily of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) and play essential roles in the degradation of the cellulose/hemicellulose/pectin network in the invaded host plant. Here we applied transcriptomic and degenerate PCR approaches to identify the PCWDEs from a destructive pest of palm trees, Rhynchophorus ferrugineus, followed by a gut-specific and stage-specific differential expression analysis. We identified a total of 27 transcripts encoding GH family members and three transcripts of the CE family with cellulase, hemicellulase and pectinase activities. We also identified two GH9 candidates, which have not previously been reported from Curculionidae. The gut-specific quantitative expression analysis identified key cellulases, hemicellulases and pectinases from R. ferrugineus. The expression analysis revealed a pectin methylesterase, RferCE8u02, and a cellulase, GH45c34485, which showed the highest gut enriched expression. Comparison of PCWDE expression patterns revealed that cellulases and pectinases are significantly upregulated in the adult stages, and we observed specific high expression of the hemicellulase RferGH16c4170. Overall, our study revealed the potential of PCWDEs from R. ferrugineus, which may be useful in biotechnological applications and may represent new tools in R. ferrugineus pest management strategies. © 2017 The Royal Entomological Society.
Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.; Ding, Shi-You; Ciesielski, Peter N.; Yang, Shihui; Tucker, Melvin P.; Himmel, Michael E.
2015-01-01
Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed. PMID:26029221
Wei, Hui; Brunecky, Roman; Donohoe, Bryon S; Ding, Shi-You; Ciesielski, Peter N; Yang, Shihui; Tucker, Melvin P; Himmel, Michael E
2015-01-01
Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.
Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.; ...
2015-05-13
Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, for this study, we utilize a CaCl 2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This hasmore » led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. Finally, the implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.
Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, for this study, we utilize a CaCl 2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This hasmore » led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. Finally, the implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.« less
Rough-Wall Channel Analysis Using Suboptimal Control Theory
NASA Technical Reports Server (NTRS)
Flores, O.; Jimenez, J.; Tenpleton, J.
2003-01-01
The original aim of this work was to shed some light on the physics of turbulence over rough walls using large-eddy simulations and the suboptimal-control wall boundary conditions introduced by Nicoud et al. It was hoped that, if that algorithm was used to fit the mean velocity profile of the simulations to that of a rough-walled channel, instead of to a smooth one, the wall stresses introduced by the control algorithm would give some indication of what aspects of rough walls are most responsible for the modification of the flow in real turbulence. It was similarly expected that the structure of the resulting velocity fluctuations would share some of the characteristics of rough-walled flows, thus again suggesting what is intrinsic and what is accidental in the effect of geometric wall roughness. A secondary goal was to study the effect of 'unphysical' boundary conditions on the outside flow by observing how a relatively major change of the target velocity profile, and therefore presumably of the applied wall stresses, modifies properties such as the dominant length scales of the velocity fluctuations away from the wall. As will be seen below, this secondary goal grew more important during the course of the study, which was carried out during a short summer visit of the first two authors to the CTR. It became clear that there are open questions about the way in which the control algorithm models the boundary conditions, even for smooth walls, and that these questions make the physical interpretation of the results difficult. Considerable more work in that area seems to be needed before even relatively advanced large-eddy simulations, such as these, can be used to draw conclusions about the physics of wall-bounded turbulent flows. The numerical method is the same as in Nicoud et al. The modifications introduced in the original code are briefly described in section 2, but the original paper should be consulted for a full description of the algorithm. The results are
Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.
Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki
2014-02-01
A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.
Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D
2014-12-01
Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.
NASA Astrophysics Data System (ADS)
Kaya, Hatice; Bulut, Osman; Kamali, Ali Reza; Ege, Duygu
2018-06-01
Favorable implant-tissue interactions are crucial to achieve successful osseointegration of the implants. Poly(ether ether ketone) (PEEK) is an interesting alternative to titanium in orthopedics because of its low cost, high biocompatibility and comparable mechanical properties with cancellous bone. Despite these advantages; however, the untreated surface of PEEK fails to osseointegrate due to its bioinert and hydrophobic behavior. This paper deals with the surface modification of PEEK with a novel method. For this, PEEK was first treated with concentrated sulfuric acid to prepare sulfonated PEEK (SPEEK) films using a solvent casting method. Then, 1 and 2 wt% multi-walled carbon nanotube was incorporated into SPEEK to form nanocomposite films. The samples were characterized with Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy. After successful preparation of the nanocomposite films, L-arginine was covalently conjugated on the nanocomposite films to further improve their surface properties. Subsequently, the samples were characterized using X-ray Photoemission Spectroscopy (XPS), water contact angle measurements and Atomic Force Microscopy (AFM) and Dynamic Mechanical Thermal Analysis (DMTA). Finally, cell culture studies were carried out by using Alamar Blue assay to evaluate the biocompatibility of the films. The results obtained indicate the successful preparation of L-arginine-conjugated MWCNT/SPEEK nanocomposite films. The modified surface shows potential to improve implants' mechanical and biological performances.
Cell wall proteome analysis of Arabidopsis thaliana mature stems.
Duruflé, Harold; Clemente, Hélène San; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth
2017-04-01
Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid
NASA Astrophysics Data System (ADS)
Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.
2012-08-01
Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a
Morisaki, Keiko; Sawada, Yuji; Sano, Ryosuke; Yamamoto, Atsushi; Kurata, Tetsuya; Suzuki, Shiro; Matsuda, Mami; Hasunuma, Tomohisa; Hirai, Masami Yokota
2016-01-01
Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic
Yim, Grace; Kalan, Lindsay; Koteva, Kalinka; Thaker, Maulik N; Waglechner, Nicholas; Tang, Irene; Wright, Gerard D
2014-11-24
In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Evolution of plant parasitism in the phylum Nematoda.
Quist, Casper W; Smant, Geert; Helder, Johannes
2015-01-01
Within the species-rich and trophically diverse phylum Nematoda, at least four independent major lineages of plant parasites have evolved, and in at least one of these major lineages plant parasitism arose independently multiple times. Ribosomal DNA data, sequence information from nematode-produced, plant cell wall-modifying enzymes, and the morphology and origin of the style(t), a protrusible piercing device used to penetrate the plant cell wall, all suggest that facultative and obligate plant parasites originate from fungivorous ancestors. Data on the nature and diversification of plant cell wall-modifying enzymes point at multiple horizontal gene transfer events from soil bacteria to bacterivorous nematodes resulting in several distinct lineages of fungal or oomycete-feeding nematodes. Ribosomal DNA frameworks with sequence data from more than 2,700 nematode taxa combined with detailed morphological information allow for explicit hypotheses on the origin of agronomically important plant parasites, such as root-knot, cyst, and lesion nematodes.
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
Cells, walls, and endless forms.
Monniaux, Marie; Hay, Angela
2016-12-01
A key question in biology is how the endless diversity of forms found in nature evolved. Understanding the cellular basis of this diversity has been aided by advances in non-model experimental systems, quantitative image analysis tools, and modeling approaches. Recent work in plants highlights the importance of cell wall and cuticle modifications for the emergence of diverse forms and functions. For example, explosive seed dispersal in Cardamine hirsuta depends on the asymmetric localization of lignified cell wall thickenings in the fruit valve. Similarly, the iridescence of Hibiscus trionum petals relies on regular striations formed by cuticular folds. Moreover, NAC transcription factors regulate the differentiation of lignified xylem vessels but also the water-conducting cells of moss that lack a lignified secondary cell wall, pointing to the origin of vascular systems. Other novel forms are associated with modified cell growth patterns, including oriented cell expansion or division, found in the long petal spurs of Aquilegia flowers, and the Sarracenia purpurea pitcher leaf, respectively. Another good example is the regulation of dissected leaf shape in C. hirsuta via local growth repression, controlled by the REDUCED COMPLEXITY HD-ZIP class I transcription factor. These studies in non-model species often reveal as much about fundamental processes of development as they do about the evolution of form. Copyright © 2016 Elsevier Ltd. All rights reserved.
Manabe, Yuzuki; Nafisi, Majse; Verhertbruggen, Yves; Orfila, Caroline; Gille, Sascha; Rautengarten, Carsten; Cherk, Candice; Marcus, Susan E.; Somerville, Shauna; Pauly, Markus; Knox, J. Paul; Sakuragi, Yumiko; Scheller, Henrik Vibe
2011-01-01
Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea. PMID:21212300
Turner, Emma L; Malo, Mackenzie E; Pisclevich, Marnie G; Dash, Megan D; Davies, Gerald F; Arnason, Terra G; Harkness, Troy A A
2010-10-01
The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G(1). Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56(Ac)) levels were as reduced as those of total H3, indicating that loading histones with H3K56(Ac) is unaffected in APC mutants. However, under restrictive conditions, H3K9(Ac) and dimethylated H3K79 (H3K79(me2)) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5(CA) (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5(CA) temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5(CA) cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5(CA) defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G(1) and following G(1) arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G(1). To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.
Mäkelä, Miia R; Dilokpimol, Adiphol; Koskela, Salla M; Kuuskeri, Jaana; de Vries, Ronald P; Hildén, Kristiina
2018-04-26
Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; Monteiro de Sousa, Paulo Robson; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G; Maguire, Glenn E M; Lamichhane, Gyanu; Lameira, Jerônimo
2015-03-01
Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. Copyright © 2015. Published by Elsevier Ltd.
Runge-Kutta method for wall shear stress of blood flow in stenosed artery
NASA Astrophysics Data System (ADS)
Awaludin, Izyan Syazana; Ahmad, Rokiah@Rozita
2014-06-01
A mathematical model of blood flow through stenotic artery is considered. A stenosis is defined as the partial occlusion of the blood vessels due to the accumulation of cholesterols, fats and the abnormal growth of tissue on the artery walls. The development of stenosis in the artery is one of the factors that cause problem in blood circulation system. This study was conducted to determine the wall shear stress of blood flow in stenosed artery. Modified mathematical model is used to analyze the relationship of the wall shear stress versus the length and height of stenosis. The existing models that have been created by previous researchers are solved using fourth order Runge-Kutta method. Numerical results show that the wall shear stress is proportionate to the length and height of stenosis.
The role of endoxyloglucan transferase in the organization of plant cell walls.
Nishitani, K
1997-01-01
The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.
Mathew, Sindhu; Abraham, T Emilia
2004-01-01
Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.