Sample records for decomposing complex enzyme
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Microbial Decomposers Not Constrained by Climate History Along a Mediterranean Climate Gradient
NASA Astrophysics Data System (ADS)
Baker, N. R.; Khalili, B.; Martiny, J. B. H.; Allison, S. D.
2017-12-01
The return of organic carbon to the atmosphere through terrestrial decomposition is mediated through the breakdown of complex organic polymers by extracellular enzymes produced by microbial decomposer communities. Determining if and how these decomposer communities are constrained in their ability to degrade plant litter is necessary for predicting how carbon cycling will be affected by future climate change. To address this question, we deployed fine-pore nylon mesh "microbial cage" litterbags containing grassland litter with and without local inoculum across five sites in southern California, spanning a gradient of 10.3-22.8° C in mean annual temperature and 100-400+ mm mean annual precipitation. Litterbags were deployed in October 2014 and collected four times over the course of 14 months. Recovered litter was assayed for mass loss, litter chemistry, microbial biomass, extracellular enzymes (Vmax and Km), and enzyme temperature sensitivities. We hypothesized that grassland litter would decompose most rapidly in the grassland site, and that access to local microbial communities would enhance litter decomposition rates and microbial activity in the other sites along the gradient. We determined that temperature and precipitation likely interact to limit microbial decomposition in the extreme sites along our gradient. Despite their unique climate history, grassland microbes were not restricted in their ability to decompose litter under different climate conditions. Although we observed a strong correlation between bacterial biomass and mass loss across the gradient, litter that was inoculated with local microbial communities lost less mass despite having greater bacterial biomass and potentially accumulating more microbial residues. Our results suggest that microbial community composition may not constrain C-cycling rates under climate change in our system. However, there may be community constraints on decomposition if climate change alters litter chemistry, a mechanism only indirectly addressed by our design.
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Pedersen, Kristine S. K.; Aanen, Duur K.
2017-01-01
ABSTRACT Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished. PMID:29269491
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da Costa, Rafael R; Hu, Haofu; Pilgaard, Bo; Vreeburg, Sabine M E; Schückel, Julia; Pedersen, Kristine S K; Kračun, Stjepan K; Busk, Peter K; Harholt, Jesper; Sapountzis, Panagiotis; Lange, Lene; Aanen, Duur K; Poulsen, Michael
2018-03-01
Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites. IMPORTANCE Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished. Copyright © 2018 da Costa et al.
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Baker, Nameer R; Khalili, Banafshe; Martiny, Jennifer B H; Allison, Steven D
2018-06-01
Microbial decomposers mediate the return of CO 2 to the atmosphere by producing extracellular enzymes to degrade complex plant polymers, making plant carbon available for metabolism. Determining if and how these decomposer communities are constrained in their ability to degrade plant litter is necessary for predicting how carbon cycling will be affected by future climate change. We analyzed mass loss, litter chemistry, microbial biomass, extracellular enzyme activities, and enzyme temperature sensitivities in grassland litter transplanted along a Mediterranean climate gradient in southern California. Microbial community composition was manipulated by caging litter within bags made of nylon membrane that prevent microbial immigration. To test whether grassland microbes were constrained by climate history, half of the bags were inoculated with local microbial communities native to each gradient site. We determined that temperature and precipitation likely interact to limit microbial decomposition in the extreme sites along our gradient. Despite their unique climate history, grassland microbial communities were not restricted in their ability to decompose litter under different climate conditions across the gradient, although microbial communities across our gradient may be restricted in their ability to degrade different types of litter. We did find some evidence that local microbial communities were optimized based on climate, but local microbial taxa that proliferated after inoculation into litterbags did not enhance litter decomposition. Our results suggest that microbial community composition does not constrain C-cycling rates under climate change in our system, but optimization to particular resource environments may act as more general constraints on microbial communities. © 2018 by the Ecological Society of America.
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Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan
2015-08-01
Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.
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Kaiser, Christina; Franklin, Oskar; Richter, Andreas; Dieckmann, Ulf
2015-01-01
The chemical structure of organic matter has been shown to be only marginally important for its decomposability by microorganisms. The question of why organic matter does accumulate in the face of powerful microbial degraders is thus key for understanding terrestrial carbon and nitrogen cycling. Here we demonstrate, based on an individual-based microbial community model, that social dynamics among microbes producing extracellular enzymes (‘decomposers') and microbes exploiting the catalytic activities of others (‘cheaters') regulate organic matter turnover. We show that the presence of cheaters increases nitrogen retention and organic matter build-up by downregulating the ratio of extracellular enzymes to total microbial biomass, allowing nitrogen-rich microbial necromass to accumulate. Moreover, increasing catalytic efficiencies of enzymes are outbalanced by a strong negative feedback on enzyme producers, leading to less enzymes being produced at the community level. Our results thus reveal a possible control mechanism that may buffer soil CO2 emissions in a future climate. PMID:26621582
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Cheng, Minyi; Takenaka, Shinji; Aoki, Shunsuke; Murakami, Shuichiro; Aoki, Kenji
2009-04-01
A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.
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Integrating microbial physiology and enzyme traits in the quality model
NASA Astrophysics Data System (ADS)
Sainte-Marie, Julien; Barrandon, Matthieu; Martin, Francis; Saint-André, Laurent; Derrien, Delphine
2017-04-01
Microbe activity plays an undisputable role in soil carbon storage and there have been many calls to integrate microbial ecology in soil carbon (C) models. With regard to this challenge, a few trait-based microbial models of C dynamics have emerged during the past decade. They parameterize specific traits related to decomposer physiology (substrate use efficiency, growth and mortality rates...) and enzyme properties (enzyme production rate, catalytic properties of enzymes…). But these models are built on the premise that organic matter (OM) can be represented as one single entity or are divided into a few pools, while organic matter exists as a continuum of many different compounds spanning from intact plant molecules to highly oxidised microbial metabolites. In addition, a given molecule may also exist in different forms, depending on its stage of polymerization or on its interactions with other organic compounds or mineral phases of the soil. Here we develop a general theoretical model relating the evolution of soil organic matter, as a continuum of progressively decomposing compounds, with decomposer activity and enzyme traits. The model is based on the notion of quality developed by Agren and Bosatta (1998), which is a measure of molecule accessibility to degradation. The model integrates three major processes: OM depolymerisation by enzyme action, OM assimilation and OM biotransformation. For any enzyme, the model reports the quality range where this enzyme selectively operates and how the initial quality distribution of the OM subset evolves into another distribution of qualities under the enzyme action. The model also defines the quality range where the OM can be uptaken and assimilated by microbes. It finally describes how the quality of the assimilated molecules is transformed into another quality distribution, corresponding to the decomposer metabolites signature. Upon decomposer death, these metabolites return to the substrate. We explore here the how microbial physiology and enzyme traits can be incorporated in a model based on a continuous representation of the organic matter and evaluate how it can improve our ability to predict soil C cycling. To do so, we analyse the properties of the model by implementing different scenarii and test the sensitivity of its parameters. Agren, G. I., & Bosatta, E. (1998). Theoretical ecosystem ecology: understanding element cycles. Cambridge University Press.
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Steffen, K T; Hofrichter, M; Hatakka, A
2000-12-01
Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.
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In Vitro Assembly of Catalase*
Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars
2014-01-01
Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. PMID:25148685
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Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin.
Kowalczyk, Joanna E; Lubbers, Ronnie J M; Peng, Mao; Battaglia, Evy; Visser, Jaap; de Vries, Ronald P
2017-09-27
Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.
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Doi, Hidetaka; Tokura, Yuriko; Mori, Yukiko; Mori, Kenichi; Asakura, Yoko; Usuda, Yoshihiro; Fukuda, Hiroo; Chinen, Akito
2017-02-01
Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2 T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.
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Draft genome sequence of the white-rot fungus Obba rivulosa 3A-2
Otto Miettinen; Robert Riley; Kerrie Barry; Daniel Cullen; Ronald P. de Vries; Matthieu Hainaut; Annele Hatakka; Bernard Henrissat; Kristiina Hilden; Rita Kuo; Kurt LaButti; Anna Lipzen; Miia R. Makela; Laura Sandor; Joseph W. Spatafora; Igor V. Grigoriev; David S. Hibbett
2016-01-01
We report here the first genome sequence of the white-rot fungus Obba rivulsa (Polyporales, Basidiomycota), a polypore known for its lignin-decomposing ability. The genome is based on the homokaryon 3A-2 originating in Finland. The genome is typical in size and carbohydrate active enzyme (CAZy) content for wood-decomposing basidiomycetes.
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In vitro assembly of catalase.
Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars
2014-10-10
Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Colombo, Lívia Tavares; de Oliveira, Marcelo Nagem Valério; Carneiro, Deisy Guimarães; de Souza, Robson Assis; Alvim, Mariana Caroline Tocantins; Dos Santos, Josenilda Carlos; da Silva, Cynthia Canêdo; Vidigal, Pedro Marcus Pereira; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes
2016-09-01
Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.
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Ethylene biosynthesis by 1-aminocyclopropane-1-carboxylic acid oxidase: a DFT study.
Bassan, Arianna; Borowski, Tomasz; Schofield, Christopher J; Siegbahn, Per E M
2006-11-24
The reaction catalyzed by the plant enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) was investigated by using hybrid density functional theory. ACCO belongs to the non-heme iron(II) enzyme superfamily and carries out the bicarbonate-dependent two-electron oxidation of its substrate ACC (1-aminocyclopropane-1-carboxylic acid) concomitant with the reduction of dioxygen and oxidation of a reducing agent probably ascorbate. The reaction gives ethylene, CO(2), cyanide and two water molecules. A model including the mononuclear iron complex with ACC in the first coordination sphere was used to study the details of O-O bond cleavage and cyclopropane ring opening. Calculations imply that this unusual and complex reaction is triggered by a hydrogen atom abstraction step generating a radical on the amino nitrogen of ACC. Subsequently, cyclopropane ring opening followed by O-O bond heterolysis leads to a very reactive iron(IV)-oxo intermediate, which decomposes to ethylene and cyanoformate with very low energy barriers. The reaction is assisted by bicarbonate located in the second coordination sphere of the metal.
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The role of metals in carcinogenesis: biochemistry and metabolism.
Jennette, K W
1981-01-01
The oxyanions of vanadium, chromium, molybdenum, arsenic, and selenium are stable forms of these elements in high oxidation states which cross cell membranes using the normal phosphate and/or sulfate transport systems of the cell. Once inside the cell, these oxyanions may sulfuryl transfer reactions. Often the oxyanions serve as alternate enzyme substrates but form ester products which are hydrolytically unstable compared with the sulfate and phosphate esters and, therefore, decompose readily in aqueous solution. Arsenite and selenite are capable of reacting with sulfhydryl groups in proteins. Some cells are able to metabolize redox active oxyanions to forms of the elements in other stable oxidation states. Specific enzymes may be involved in the metabolic processes. The metabolites of these elements may form complexes with small molecules, proteins and nucleic acids which inhibit their ability to function properly. The divalent ions of beryllium, manganese, cobalt, nickel, cadmium, mercury, and lead are stable forms of these elements which may mimic essential divalent ions such as magnesium, calcium, iron, copper, or zinc. These ions may complex small molecules, enzymes, and nucleic acids in such a way that the normal activity of these species is altered. Free radicals may be produced in the presence of these metal ions which damage critical cellular molecules. PMID:7023933
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NASA Astrophysics Data System (ADS)
Baker, N. R.; Allison, S. D.
2013-12-01
Traditional decomposition models developed in mesic ecosystems often consistently underestimate rates of decomposition in more arid ecosystems such as deserts and Mediterranean grasslands. Photodegradation of plant litter by ultraviolet radiation (UV) is hypothesized to be one of the mechanisms accounting for the greater-than-expected rates of decomposition observed in these ecosystems. Putatively, photodegradation preferentially degrades complex aromatic compounds in litter such as lignin, whose decomposition is considered a rate-limiting step in the microbial decomposition of plant litter. This study tested the effects of attenuated ultraviolet radiation on the decomposition of two litter types over the course of a year in a Southern California Mediterranean grassland. The two types of litter differed primarily in lignin content to test for a differential effect of UV on high-lignin versus low-lignin litter. Rates of litter mass loss, changes in litter chemistry, and changes in microbial activity and microbial biomass were observed, and assays of extracellular enzymes were conducted at 5 points through the year, beginning during the dry season and continuing until the end of the following dry season. Litter exposed to attenuated ultraviolet radiation during the dry season had lower rates of mass loss than litter exposed to ambient radiation (6.1% vs. 8.6%, respectively, p < 0.04). Extracellular enzyme activities were significantly affected by UV attenuation, as low lignin samples exposed to attenuated UV displayed elevated cellulase enzyme activity potential during the wet season, while high lignin samples displayed decreased oxidative enzyme activity potential during the wet season. For example, potential activity of the cellulase cellobiohydrolase in low-lignin, ambient UV samples was 5286 μmol/hr*g during the wet season, compared to 7969 μmol/hr*g in attenuated UV samples (p < 0.003). Conversely, potential activity of the oxidative enzyme peroxidase in high-lignin, ambient UV samples was 85.9 μmol/hr*g during the wet season, compared to 44.1 μmol/hr*g in attenuated UV samples (p < 0.028). This increased potential cellulase activity under attenuated UV may indicate that dry season photodegradation primes low-lignin litter for wet season decomposition, reducing the selective pressure for microbial decomposers to invest in costly extracellular enzyme production. Similarly, the reduced potential oxidative enzyme activity in high-lignin samples exposed to attenuated UV may indicate that photodegradation is necessary to facilitate the breakdown of more complex compounds such as lignin by microbial decomposers. We conclude that while abiotic factors such as photodegradation can have a significant effect on the mechanisms of plant matter decomposition in semiarid ecosystems, these effects are not only restricted to the dry season and may also facilitate wet season decomposition.
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NASA Astrophysics Data System (ADS)
Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.
2011-09-01
Peatlands are carbon (C) storage ecosystems sustained by a high water table (WT). High WT creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WT drawdown caused by climate and/or land-use change. Aerobic decomposers are directly affected by WT drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WT drawdown on aerobic decomposer activity in plant litter at two stages of decomposition (incubated in the field for 1 or 2 years). We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen (N), phosphorus (P) and sulphur. Our study sites represented a three-stage chronosequence from pristine to short-term (years) and long-term (decades) WT drawdown conditions under two nutrient regimes (bog and fen). The litter types included reflected the prevalent vegetation: Sphagnum mosses, graminoids, shrubs and trees. Litter type was the main factor shaping microbial activity patterns and explained about 30 % of the variation in enzyme activities and activity allocation. Overall, enzyme activities were higher in vascular plant litters compared to Sphagnum litters, and the allocation of enzyme activities towards C or nutrient acquisition was related to the initial litter quality (chemical composition). Direct effects of WT regime, site nutrient regime and litter decomposition stage (length of incubation period) summed to only about 40 % of the litter type effect. WT regime alone explained about 5 % of the variation in enzyme activities and activity allocation. Generally, enzyme activity increased following the long-term WT drawdown and the activity allocation turned from P and N acquisition towards C acquisition. This caused an increase in the rate of litter decomposition. The effects of the short-term WT drawdown were minor compared to those of the long-term WT drawdown: e.g., the increase in the activity of C-acquiring enzymes was up to 120 % (bog) or 320 % (fen) higher after the long-term WT drawdown compared to the short-term WT drawdown. In general, the patterns of microbial activity as well as their responses to WT drawdown depended on peatland type: e.g., the shift in activity allocation to C-acquisition was up to 100 % stronger at the fen compared to the bog. Our results imply that changes in plant community composition in response to persistent WT drawdown will strongly affect the C dynamics of peatlands. The predictions of decomposer activity under changing climate and/or land-use thus cannot be based on the direct effects of the changed environment only, but need to consider the indirect effects of environmental changes: the changes in plant community composition, their dependence on peatland type, and their time scale.
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The complexity of divisibility.
Bausch, Johannes; Cubitt, Toby
2016-09-01
We address two sets of long-standing open questions in linear algebra and probability theory, from a computational complexity perspective: stochastic matrix divisibility, and divisibility and decomposability of probability distributions. We prove that finite divisibility of stochastic matrices is an NP-complete problem, and extend this result to nonnegative matrices, and completely-positive trace-preserving maps, i.e. the quantum analogue of stochastic matrices. We further prove a complexity hierarchy for the divisibility and decomposability of probability distributions, showing that finite distribution divisibility is in P, but decomposability is NP-hard. For the former, we give an explicit polynomial-time algorithm. All results on distributions extend to weak-membership formulations, proving that the complexity of these problems is robust to perturbations.
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Bödeker, Inga T M; Clemmensen, Karina E; de Boer, Wietse; Martin, Francis; Olson, Åke; Lindahl, Björn D
2014-07-01
In northern forests, belowground sequestration of nitrogen (N) in complex organic pools restricts nutrient availability to plants. Oxidative extracellular enzymes produced by ectomycorrhizal fungi may aid plant N acquisition by providing access to N in macromolecular complexes. We test the hypotheses that ectomycorrhizal Cortinarius species produce Mn-dependent peroxidases, and that the activity of these enzymes declines at elevated concentrations of inorganic N. In a boreal pine forest and a sub-arctic birch forest, Cortinarius DNA was assessed by 454-sequencing of ITS amplicons and related to Mn-peroxidase activity in humus samples with- and without previous N amendment. Transcription of Cortinarius Mn-peroxidase genes was investigated in field samples. Phylogenetic analyses of Cortinarius peroxidase amplicons and genome sequences were performed. We found a significant co-localization of high peroxidase activity and DNA from Cortinarius species. Peroxidase activity was reduced by high ammonium concentrations. Amplification of mRNA sequences indicated transcription of Cortinarius Mn-peroxidase genes under field conditions. The Cortinarius glaucopus genome encodes 11 peroxidases - a number comparable to many white-rot wood decomposers. These results support the hypothesis that some ectomycorrhizal fungi--Cortinarius species in particular--may play an important role in decomposition of complex organic matter, linked to their mobilization of organically bound N. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.
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NASA Astrophysics Data System (ADS)
Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.
2015-12-01
Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.
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Nguyen, Le Truc; Yang, Kun-Lin
2017-05-01
Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction. Copyright © 2017 Elsevier Inc. All rights reserved.
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Natural image statistics and low-complexity feature selection.
Vasconcelos, Manuela; Vasconcelos, Nuno
2009-02-01
Low-complexity feature selection is analyzed in the context of visual recognition. It is hypothesized that high-order dependences of bandpass features contain little information for discrimination of natural images. This hypothesis is characterized formally by the introduction of the concepts of conjunctive interference and decomposability order of a feature set. Necessary and sufficient conditions for the feasibility of low-complexity feature selection are then derived in terms of these concepts. It is shown that the intrinsic complexity of feature selection is determined by the decomposability order of the feature set and not its dimension. Feature selection algorithms are then derived for all levels of complexity and are shown to be approximated by existing information-theoretic methods, which they consistently outperform. The new algorithms are also used to objectively test the hypothesis of low decomposability order through comparison of classification performance. It is shown that, for image classification, the gain of modeling feature dependencies has strongly diminishing returns: best results are obtained under the assumption of decomposability order 1. This suggests a generic law for bandpass features extracted from natural images: that the effect, on the dependence of any two features, of observing any other feature is constant across image classes.
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A Tale of Three Classes: Case Studies in Course Complexity
ERIC Educational Resources Information Center
Gill, T. Grandon; Jones, Joni
2010-01-01
This paper examines the question of decomposability versus complexity of teaching situations by presenting three case studies of MIS courses. Because all three courses were highly successful in their observed outcomes, the paper hypothesizes that if the attributes of effective course design are decomposable, one would expect to see a large number…
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NASA Astrophysics Data System (ADS)
Berezin, L. V.; Khamova, O. F.; Paderina, E. V.; Gindemit, A. M.
2014-11-01
The abundance and activity of the soil microflora were studied in a field experiment with the use of green manure crops to assess the impact of reclamation measures on the biological activity of soils of the solonetz complex. The number of microorganisms in the plow soil horizon increased in the background of the green fallows as compared to the black ones. Coefficients of mineralization, immobilization, and transformation of organic compounds were calculated for different variants of the soil treatment. The value of the mineralization coefficient indicates the intense decomposition of the green manure that entered the soil. In the first year, peas were actively decomposed, while oats, in the second year (aftereffect). The activity of the soil enzymes (invertase, urease, and catalase) was determined. A close relationship between the catalase activity and the intensity of the microbiological processes in the soils was revealed.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu, Chaowei; Harrold, Duff R.; Claypool, Joshua T.
Microorganisms involved in biomass deconstruction are an important resource for organic waste recycling and enzymes for lignocellulose bioconversion. The goals of this paper were to examine the impact of nitrogen amendment on microbial community restructuring, secretion of xylanases and endoglucanases, and potential for biomass deconstruction. Communities were cultivated aerobically at 55 °C on green waste (GW) amended with varying levels of NH 4Cl. Bacterial and fungal communities were determined using 16S rRNA and ITS region gene sequencing and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to predict relative abundance of genes involved in lignocellulose hydrolysis.more » Nitrogen amendment significantly increased secretion of xylanases and endoglucanases, and microbial activity; enzyme activities and cumulative respiration were greatest when nitrogen level in GW was between 4.13–4.56 wt% (g/g), but decreased with higher nitrogen levels. The microbial community shifted to one with increasing potential to decompose complex polymers as nitrogen increased with peak potential occurring between 3.79–4.45 wt% (g/g) nitrogen amendment. Finally, the results will aid in informing the management of nitrogen level to foster microbial communities capable of secreting enzymes that hydrolyze recalcitrant polymers in lignocellulose and yield rapid decomposition of green waste.« less
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Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes.
Geeraerts, Zachary; Celis, Arianna I; Mayfield, Jeffery A; Lorenz, Megan; Rodgers, Kenton R; DuBois, Jennifer L; Lukat-Rodgers, Gudrun S
2018-03-06
O 2 -evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO 2 - ) to O 2 and Cl - . Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO 2 - , albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10 -9 M, the K D for the KpCld-CN - complex is 2 orders of magnitude smaller than that of DaCld-CN - and indicates an affinity for CN - that is greater than that of most heme proteins. The difference in CN - affinity between Kp- and DaClds is predominantly due to differences in k off . The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p K a of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 396 cm -1 ) and KpCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 356 cm -1 ) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.
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NASA Astrophysics Data System (ADS)
Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.
2011-02-01
Peatlands are carbon (C) storage ecosystems sustained by a high water level (WL). High WL creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WL drawdown caused by land-use and/or climate change. Aerobic decomposers are directly affected by WL drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WL drawdown on aerobic decomposer activity in plant litter. We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen, phosphorus and sulphur. Our study sites represented a three-stage chronosequence from pristine (undrained) to short-term (years) and long-term (decades) WL drawdown conditions under two nutrient regimes. The litter types included reflected the prevalent vegetation, i.e., Sphagnum mosses, graminoids, shrubs and trees. WL drawdown had a direct and positive effect on microbial activity. Enzyme allocation shifted towards C acquisition, which caused an increase in the rate of decomposition. However, litter type overruled the direct effects of WL drawdown and was the main factor shaping microbial activity patterns. Our results imply that changes in plant community composition in response to persistent WL drawdown will strongly affect the C dynamics of peatlands.
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Cusack, Daniela F; Lee, Joseph K; McCleery, Taylor L; LeCroy, Chase S
2015-12-01
Urban areas are expanding rapidly in tropical regions, with potential to alter ecosystem dynamics. In particular, exotic grasses and atmospheric nitrogen (N) deposition simultaneously affect tropical urbanized landscapes, with unknown effects on properties like soil carbon (C) storage. We hypothesized that (H1) soil nitrate (NO3 (-) ) is elevated nearer to the urban core, reflecting N deposition gradients. (H2) Exotic grasslands have elevated soil NO3 (-) and decreased soil C relative to secondary forests, with higher N promoting decomposer activity. (H3) Exotic grasslands have greater seasonality in soil NO3 (-) vs. secondary forests, due to higher sensitivity of grassland soil moisture to rainfall. We predicted that NO3 (-) would be positively related to dissolved organic C (DOC) production via changes in decomposer activity. We measured six paired grassland/secondary forest sites along a tropical urban-to-rural gradient during the three dominant seasons (hurricane, dry, and early wet). We found that (1) soil NO3 (-) was generally elevated nearer to the urban core, with particularly clear spatial trends for grasslands. (2) Exotic grasslands had lower soil C than secondary forests, which was related to elevated decomposer enzyme activities and soil respiration. Unexpectedly, soil NO3 (-) was negatively related to enzyme activities, and was lower in grasslands than forests. (3) Grasslands had greater soil NO3 (-) seasonality vs. forests, but this was not strongly linked to shifts in soil moisture or DOC. Our results suggest that exotic grasses in tropical regions are likely to drastically reduce soil C storage, but that N deposition may have an opposite effect via suppression of enzyme activities. However, soil NO3 (-) accumulation here was higher in urban forests than grasslands, potentially related to of aboveground N interception. Net urban effects on C storage across tropical landscapes will likely vary depending on the mosaic of grass cover, rates of N deposition, and responses by local decomposer communities. © 2015 John Wiley & Sons Ltd.
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NASA Technical Reports Server (NTRS)
Duraj, S. A.; Duffy, N. V.; Hepp, A. F.; Cowen, J. E.; Hoops, M. D.; Brothrs, S. M.; Baird, M. J.; Fanwick, P. E.; Harris, J. D.; Jin, M. H.-C.
2009-01-01
Ten dithiocarbamate complexes of indium(III) and gallium(III) have been prepared and characterized by elemental analysis, infrared spectra and melting point. Each complex was decomposed thermally and its decomposition products separated and identified with the combination of gas chromatography/mass spectrometry. Their potential utility as photovoltaic materials precursors was assessed. Bis(dibenzyldithiocarbamato)- and bis(diethyldithiocarbamato)copper(II), Cu(S2CN(CH2C6H5)2)2 and Cu(S2CN(C2H5)2)2 respectively, have also been examined for their suitability as precursors for copper sulfides for the fabrication of photovoltaic materials. Each complex was decomposed thermally and the products analyzed by GC/MS, TGA and FTIR. The dibenzyl derivative complex decomposed at a lower temperature (225-320 C) to yield CuS as the product. The diethyl derivative complex decomposed at a higher temperature (260-325 C) to yield Cu2S. No Cu containing fragments were noted in the mass spectra. Unusual recombination fragments were observed in the mass spectra of the diethyl derivative. Tris(bis(phenylmethyl)carbamodithioato-S,S'), commonly referred to as tris(N,N-dibenzyldithiocarbamato)indium(III), In(S2CNBz2)3, was synthesized and characterized by single crystal X-ray crystallography. The compound crystallizes in the triclinic space group P1(bar) with two molecules per unit cell. The material was further characterized using a novel analytical system employing the combined powers of thermogravimetric analysis, gas chromatography/mass spectrometry, and Fourier transform infrared (FT-IR) spectroscopy to investigate its potential use as a precursor for the chemical vapor deposition (CVD) of thin film materials for photovoltaic applications. Upon heating, the material thermally decomposes to release CS2 and benzyl moieties in to the gas phase, resulting in bulk In2S3. Preliminary spray CVD experiments indicate that In(S2CNBz2)3 decomposed on a Cu substrate reacts to produce stoichiometric CuInS2 films.
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Purification and characterization of two enone reductases from Saccharomyces cerevisiae.
Wanner, P; Tressl, R
1998-07-01
Two enone reductases catalyzing irreversibly the enantioselective reduction of alpha,beta-unsaturated carbonyls have been purified 165-fold and 257-fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, blue Sepharose CL-6B chromatography and Superdex 200 preparation-grade filtration. One enzyme (E I) was NADPH-dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation-grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE. Both enzymes were most active at pH 4.8 and accepted 1-octen-3-one, 1-hexen-3-one, 3-alken-2-ones, 4-alken-3-ones, 2-cyclohexen-1-ones, 2-alkenals, 2,4-alkadienals, 2-phenyl-2-alkenals, and 2-alkyl-2-alkenals as substrates. Both enzymes showed their highest activities with 1-hexen-3-one and 1-octen-3-one, respectively. The Km values for 1-octen-3-one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis- and trans-2-phenyl-2-butenal into (R)-2-phenylbutanal.
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Thermally Regenerative Battery with Intercalatable Electrodes and Selective Heating Means
NASA Technical Reports Server (NTRS)
Sharma, Pramod K. (Inventor); Narayanan, Sekharipuram R. (Inventor); Hickey, Gregory S. (Inventor)
2000-01-01
The battery contains at least one electrode such as graphite that intercalates a first species from the electrolyte disposed in a first compartment such as bromine to form a thermally decomposable complex during discharge. The other electrode can also be graphite which supplies another species such as lithium to the electrolyte in a second electrode compartment. The thermally decomposable complex is stable at room temperature but decomposes at elevated temperatures such as 50 C. to 150 C. The electrode compartments are separated by a selective ion permeable membrane that is impermeable to the first species. Charging is effected by selectively heating the first electrode.
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Wang, Xiuran; Peng, Zhongqi; Sun, Xiaoling; Liu, Dongbo; Chen, Shan; Li, Fan; Xia, Hongmei; Lu, Tiancheng
2012-01-01
Sporocytophaga sp. JL-01 is a sliding cellulose degrading bacterium that can decompose filter paper (FP), carboxymethyl cellulose (CMC) and cellulose CF11. In this paper, the morphological characteristics of S. sp. JL-01 growing in FP liquid medium was studied by Scanning Electron Microscope (SEM), and one of the FPase components of this bacterium was analyzed. The results showed that the cell shapes were variable during the process of filter paper cellulose decomposition and the rod shape might be connected with filter paper decomposing. After incubating for 120 h, the filter paper was decomposed significantly, and it was degraded absolutely within 144 h. An FPase1 was purified from the supernatant and its characteristics were analyzed. The molecular weight of the FPase1 was 55 kDa. The optimum pH was pH 7.2 and optimum temperature was 50°C under experiment conditions. Zn(2+) and Co(2+) enhanced the enzyme activity, but Fe(3+) inhibited it.
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Shah, Firoz; Nicolás, César; Bentzer, Johan; Ellström, Magnus; Smits, Mark; Rineau, Francois; Canbäck, Björn; Floudas, Dimitrios; Carleer, Robert; Lackner, Gerald; Braesel, Jana; Hoffmeister, Dirk; Henrissat, Bernard; Ahrén, Dag; Johansson, Tomas; Hibbett, David S; Martin, Francis; Persson, Per; Tunlid, Anders
2016-03-01
Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
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Han, Xu; Cheng, Zhihui; Meng, Huanwen
2017-07-01
Garlic (Allium sativum L.) stalk is a byproduct of garlic production that is normally thought of as waste but is now considered a useful biological resource. It is necessary to utilize this resource efficiently and reasonably to reduce environmental pollution and achieve sustainable agricultural development. The effect of garlic stalk decomposed for different durations was investigated in this study using wheat (Triticum aestivum L.) and lettuce (Lactuca sativa var. crispa L.) as test plants. Garlic stalk in early stages of decomposition inhibited the shoot and root lengths of wheat and lettuce, but it promoted the shoot and root lengths in later stages; longer durations of garlic stalk decomposition significantly increased the shoot and root fresh weights of wheat and lettuce, whereas shorter decomposing durations significantly decreased the shoot and root fresh weights; and garlic stalk at different decomposition durations increased the activities of urease, sucrase and alkaline phosphatase in soil where wheat or lettuce was planted. Garlic stalk decomposed for 30 or 40 days could promote the growth of wheat and lettuce plants as well as soil enzyme activities. These results may provide a scientific basis for the study and application of garlic stalk. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hahnke, Richard L.; Stackebrandt, Erko; Meier-Kolthoff, Jan P.
Flavobacterium rivuli Ali et al. 2009 emend. Dong et al. 2013 is one of about 100 species in the genus Flavobacterium (family Flavobacteriacae, phylum Bacteroidetes) with a validly published name, and has been isolated from the spring of a hard water rivulet in Northern Germany. Including all type strains of the genus Myroides and Flavobacterium into the 16S rRNA gene sequence phylogeny revealed a clustering of members of the genus Myroides as a monophyletic group within the genus Flavobacterium. Furthermore, F. rivuli WB 3.3-2T and its next relatives seem more closely related to the genus Myroides than to the typemore » species of the genus Flavobacterium, F. aquatile. The 4,489,248 bp long genome with its 3,391 protein-coding and 65 RNA genes is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The genome of F. rivuli has almost as many genes encoding carbohydrate active enzymes (151 CAZymes) as genes encoding peptidases (177). Peptidases comprised mostly metallo (M) and serine (S) peptidases. Among CAZymes, 30 glycoside hydrolase families, 10 glycosyl transferase families, 7 carbohydrate binding module families and 7 carbohydrate esterase families were identified. Furthermore, we found four polysaccharide utilization loci (PUL) and one large CAZy rich gene cluster that might enable strain WB 3.3-2T to decompose plant and algae derived polysaccharides. In conclusion, based on these results we propose F. rivuli as an interesting candidate for further physiological studies and the role of Bacteroidetes in the decomposition of complex polymers in the environment.« less
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Hahnke, Richard L.; Stackebrandt, Erko; Meier-Kolthoff, Jan P.; ...
2015-07-30
Flavobacterium rivuli Ali et al. 2009 emend. Dong et al. 2013 is one of about 100 species in the genus Flavobacterium (family Flavobacteriacae, phylum Bacteroidetes) with a validly published name, and has been isolated from the spring of a hard water rivulet in Northern Germany. Including all type strains of the genus Myroides and Flavobacterium into the 16S rRNA gene sequence phylogeny revealed a clustering of members of the genus Myroides as a monophyletic group within the genus Flavobacterium. Furthermore, F. rivuli WB 3.3-2T and its next relatives seem more closely related to the genus Myroides than to the typemore » species of the genus Flavobacterium, F. aquatile. The 4,489,248 bp long genome with its 3,391 protein-coding and 65 RNA genes is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The genome of F. rivuli has almost as many genes encoding carbohydrate active enzymes (151 CAZymes) as genes encoding peptidases (177). Peptidases comprised mostly metallo (M) and serine (S) peptidases. Among CAZymes, 30 glycoside hydrolase families, 10 glycosyl transferase families, 7 carbohydrate binding module families and 7 carbohydrate esterase families were identified. Furthermore, we found four polysaccharide utilization loci (PUL) and one large CAZy rich gene cluster that might enable strain WB 3.3-2T to decompose plant and algae derived polysaccharides. In conclusion, based on these results we propose F. rivuli as an interesting candidate for further physiological studies and the role of Bacteroidetes in the decomposition of complex polymers in the environment.« less
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Rineau, F; Shah, F; Smits, M M; Persson, P; Johansson, T; Carleer, R; Troein, C; Tunlid, A
2013-01-01
The majority of nitrogen in forest soils is found in organic matter–protein complexes. Ectomycorrhizal fungi (EMF) are thought to have a key role in decomposing and mobilizing nitrogen from such complexes. However, little is known about the mechanisms governing these processes, how they are regulated by the carbon in the host plant and the availability of more easily available forms of nitrogen sources. Here we used spectroscopic analyses and transcriptome profiling to examine how the presence or absence of glucose and/or ammonium regulates decomposition of litter material and nitrogen mobilization by the ectomycorrhizal fungus Paxillus involutus. We found that the assimilation of nitrogen and the decomposition of the litter material are triggered by the addition of glucose. Glucose addition also resulted in upregulation of the expression of genes encoding enzymes involved in oxidative degradation of polysaccharides and polyphenols, peptidases, nitrogen transporters and enzymes in pathways of the nitrogen and carbon metabolism. In contrast, the addition of ammonium to organic matter had relatively minor effects on the expression of transcripts and the decomposition of litter material, occurring only when glucose was present. On the basis of spectroscopic analyses, three major types of chemical modifications of the litter material were observed, each correlated with the expression of specific sets of genes encoding extracellular enzymes. Our data suggest that the expression of the decomposition and nitrogen assimilation processes of EMF can be tightly regulated by the host carbon supply and that the availability of inorganic nitrogen as such has limited effects on saprotrophic activities. PMID:23788332
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Presley, Gerald N.; Ndimba, Bongani K.; Schilling, Jonathan S.
2018-01-01
Sweet sorghum is a promising crop for a warming, drying African climate, and basic information is lacking on conversion pathways for its lignocellulosic residues (bagasse). Brown rot wood-decomposer fungi use carbohydrate-selective pathways that, when assessed on sorghum, a grass substrate, can yield information relevant to both plant biomass conversion and fungal biology. In testing sorghum decomposition by brown rot fungi ( Gloeophyllum trabeum , Serpula lacrymans ), we found that G. trabeum readily degraded sorghum, removing xylan prior to removing glucan. Serpula lacrymans , conversely, caused little decomposition. Ergosterol (fungal biomarker) and protein levels were similar for both fungi, butmore » S. lacrymans produced nearly 4x lower polysaccharide-degrading enzyme specific activity on sorghum than G. trabeum , perhaps a symptom of starvation. Linking this information to genome comparisons including other brown rot fungi known to have a similar issue regarding decomposing grasses (Postia placenta, Fomitopsis pinicola) suggested that a lack of CE 1 feruloyl esterases as well as low xylanase activity in S. lacrymans (3x lower than in G. trabeum ) may hinder S. lacrymans , P. placenta, and F. pinicola when degrading grass substrates. These results indicate variability in brown rot mechanisms, which may stem from a differing ability to degrade certain lignin-carbohydrate complexes.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Presley, Gerald N.; Ndimba, Bongani K.; Schilling, Jonathan S.
Sweet sorghum is a promising crop for a warming, drying African climate, and basic information is lacking on conversion pathways for its lignocellulosic residues (bagasse). Brown rot wood-decomposer fungi use carbohydrate-selective pathways that, when assessed on sorghum, a grass substrate, can yield information relevant to both plant biomass conversion and fungal biology. In testing sorghum decomposition by brown rot fungi ( Gloeophyllum trabeum , Serpula lacrymans ), we found that G. trabeum readily degraded sorghum, removing xylan prior to removing glucan. Serpula lacrymans , conversely, caused little decomposition. Ergosterol (fungal biomarker) and protein levels were similar for both fungi, butmore » S. lacrymans produced nearly 4x lower polysaccharide-degrading enzyme specific activity on sorghum than G. trabeum , perhaps a symptom of starvation. Linking this information to genome comparisons including other brown rot fungi known to have a similar issue regarding decomposing grasses (Postia placenta, Fomitopsis pinicola) suggested that a lack of CE 1 feruloyl esterases as well as low xylanase activity in S. lacrymans (3x lower than in G. trabeum ) may hinder S. lacrymans , P. placenta, and F. pinicola when degrading grass substrates. These results indicate variability in brown rot mechanisms, which may stem from a differing ability to degrade certain lignin-carbohydrate complexes.« less
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NASA Astrophysics Data System (ADS)
Sun, Yong; Zhou, Deqing; Zhao, Feng
2011-03-01
The effects of Fe2+ on the trimethylamine N-oxide (TMAO) demethylating activity of the Harpadon nehereus kidney extract were studied in this research. The activity of the kidney extract was presumably inhibited by ethylene diamine tetra-acetic acid (EDTA), which indicates that the kidney extract contains an enzyme or enzyme system with metal cations as activator. Activity of the kidney extract was enhanced significantly when Fe2+ was added into the model system in vitro. As the concentration of Fe2+ increased, the decomposing rate of TMAO increased rapidly until TMAO decomposed completely. The activity of the kidney extract was also enhanced by reductant such as ascorbic acid. Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) was employed to determine the content of total iron in a number of fishery products. Significant positive correlation between the contents of total iron and endogenous formaldehyde (FA) was found, especially in marine products.
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Pozdnyakova, Natalia; Dubrovskaya, Ekaterina; Chernyshova, Marina; Makarov, Oleg; Golubev, Sergey; Balandina, Svetlana; Turkovskaya, Olga
2018-05-01
The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization. A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization. Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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Fenoy, Encarnación; Casas, J Jesús; Díaz-López, Manuel; Rubio, Juan; Guil-Guerrero, J Luís; Moyano-López, Francisco J
2016-11-01
Abiotic factors, substrate chemistry and decomposers community composition are primary drivers of leaf litter decomposition. In soil, much of the variation in litter decomposition is explained by climate and substrate chemistry, but with a significant contribution of the specialisation of decomposer communities to degrade specific substrates (home-field advantage, HFA). In streams, however, HFA effects on litter decomposition have not been explicitly tested. We evaluated responses of microbial decomposition and β-glucosidase activity to abiotic factors, substrate and decomposer assemblages, using a reciprocal litter transplant experiment: 'ecosystem type' (mountain vs lowland streams) × 'litter chemistry' (alder vs reed). Temperature, pH and ionic concentration were higher in lowland streams. Decomposition for both species was faster in lowland streams. Decomposition of reed was more accelerated in lowland compared with mountain streams than that of alder, suggesting higher temperature sensitivity of decomposition in reed. Q10 (5°C-15°C) values of β-glucosidase activity were over 2. The alkaline pH and high ionic concentration of lowland streams depleted enzyme activity. We found similar relationships of decomposition or enzyme activity with abiotic factors for both species, suggesting limited support to the HFA hypothesis. Overall, our results suggest a prime role of temperature interacting with substrate chemistry on litter decomposition. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Lignocellulose-degrading enzymes from termites and their symbiotic microbiota.
Ni, Jinfeng; Tokuda, Gaku
2013-11-01
Lignocellulose-the dry matter of plants, or "plant biomass"-digestion is of increasing interest in organismal metabolism research, specifically the conversion of biomass into biofuels. Termites efficiently decompose lignocelluloses, and studies on lignocellulolytic systems may elucidate mechanisms of efficient lignocellulose degradation in termites as well as offer novel enzyme sources, findings which have significant potential industrial applications. Recent progress in metagenomic and metatranscriptomic research has illuminated the diversity of lignocellulolytic enzymes within the termite gut. Here, we review state-of-the-art research on lignocellulose-degrading systems in termites, specifically cellulases, xylanases, and lignin modification enzymes produced by termites and their symbiotic microbiota. We also discuss recent investigations into heterologous overexpression of lignocellulolytic enzymes from termites and their symbionts. Copyright © 2013 Elsevier Inc. All rights reserved.
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Schneider, Thomas; Keiblinger, Katharina M; Schmid, Emanuel; Sterflinger-Gleixner, Katja; Ellersdorfer, Günther; Roschitzki, Bernd; Richter, Andreas; Eberl, Leo; Zechmeister-Boltenstern, Sophie; Riedel, Kathrin
2012-01-01
Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the ‘active building blocks' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes. PMID:22402400
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Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; Takasuka, Taichi E.; Doering, Drew T.; Adams, Aaron S.; Blodgett, Joshua A. V.; Clardy, Jon; Raffa, Kenneth F.; Fox, Brian G.
2014-01-01
Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass. PMID:24837391
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Titov, V Iu; Petrenko, Iu M; Vanin, A F; Stepuro, I I
2010-01-01
The capacity of nitrite, S-nitrosothiols (RS-NO), dinitrosyl iron complexes (DNICs) with thiol-containing ligands, and nitrosoamines to inhibit catalase has been used for the selective determination of these compounds in purely chemical systems and biological liquids: cow milk and colostram. The limiting sensitivity of the method is 50 nM. A comparison of the results of the determinations of RS-NO, DNIC, and nitrite by the catalase method and the Greese method conventionally used for nitrite detection showed that, firstly, Greese reagents decompose DNIC and RS-NO to form nitrite. Therefore, the Greese method cannot be used for nitrite determination in solutions of these substances. Secondly, Greese reagents interact with complexes of mercury ions with RS-NO, inducing the release of nitrosonium ions from the complex followed by the hydrolysis of nitrosonium to nitrite. Thus, the proposition about the spontaneous decay of the complexes of mercury ions with RS-NO is incorrect. Keeping in mind a high sensitivity of the method, the use of catalase as an enzyme detector of nitrosocompounds allows one to detect these compounds in neutral medium without prior purification of the object, thereby preventing artificial effects due to noncontrolled modifications of the compounds under study.
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NASA Astrophysics Data System (ADS)
Zak, Dominik; Roth, Cyril; Gelbrecht, Jörg; Fenner, Nathalie; Reuter, Hendrik
2015-04-01
Recently, more than 30,000 ha of drained minerotrophic peatlands (= fens) in NE Germany were rewetted to restore their ecological functions. Due to an extended drainage history, a re-establishment of their original state is not expected in the short-term. Elevated concentrations of dissolved organic carbon, ammonium and phosphate have been measured in the soil porewater of the upper degraded peat layers of rewetted fens at levels of one to three orders higher than the values in pristine systems; an indicator of increased microbial activity in the upper degraded soil layers. On the other hand there is evidence that the substrate availability within the degraded peat layer is lowered since the organic matter has formerly been subject to intense decomposition over the decades of drainage and intense agricultural use of the areas. Previously however, it was suggested that inhibition of hydrolytic enzymes by polyphenolic substances is suspended during aeration of peat soils mainly due to the decomposition of the inhibiting polyphenols by oxidising enzymes such as phenol oxidase. Accordingly we hypothesised a lack of enzyme inhibiting polyphenols in degraded peat soils of rewetted fens compared to less decomposed peat of more natural fens. We collected both peat samples at the soil surface (0-20 cm) and fresh roots of dominating vascular plants and mosses (as peat parent material) from five formerly drained rewetted sites and five more natural sites of NE Germany and NW Poland. Less decomposed peat and living roots were used to obtain an internal standard for polyphenol analysis and to run enzyme inhibition tests. For all samples we determined the total phenolic contents and in addition we distinguished between the contents of hydrolysable and condensed tannic substances. From a methodical perspective the advantage of internal standards compared to the commercially available standards cyanidin chloride and tannic acid became apparent. Quantification with cyanidin or tannic acid led to a considerable underestimation (up to 90%) of polyphenolic concentrations in peat soils. As hypothesised we found that highly degraded peat contains far lower levels of total polyphenolics (factor 8) and condensed tannins (factor 50) than less decomposed peat. In addition we detected large differences between different plant species with highest polyphenolic contents for the roots of Carex appropinquata that were more than 10-fold higher than Sphagnum spp. (450 mg/g dry mass vs. 39 mg/g dry mass). Despite these differences, we did not find a significant correlation between enzyme activities and peat degradation state, indicating that there is no simple linear relationship between polyphenolic contents and microbial activity.
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Quantitative inhibition of soil C and N cycling by ectomycorrhizal fungi under field condition
NASA Astrophysics Data System (ADS)
Averill, C.; Hawkes, C.
2014-12-01
Ectomycorrhizal (ECM) ecosystems store more carbon than non-ectomycorrhizal ecosystems at global scale. Recent theoretical and empirical work suggests the presence of ECM fungi allows plants to compete directly with decomposers for soil nitrogen (N) via exo-enzyme synthesis. Experimental ECM exclusion often results in a release from competition of saprotrophic decomposers, allowing for increased C-degrading enzyme production, increased microbial biomass, and eventually declines in soil C stocks. Our knowledge of this phenomenon is limited, however, to the presence or absence of ECM fungi. It remains unknown if competitive repression of saprotrophic microbes and soil C cycling by ECM fungi varies with ECM abundance. This is particularly relevant to global change experiments when manipulations alter plant C allocation to ECM symbionts. To test if variation in ECM abundance alters the competitive inhibition of saprotrophic soil microbes (quantitative inhibition) we established experimental ECM exclusion treatments along an ECM abundance gradient. We dug trenches to experimentally exclude ECM fungi, allowing us to test for competitive release of soil saprotrophs from competition. To control for disturbance we placed in-growth bags both inside and outside of trenches. Consistent with the quantitative inhibition hypothesis, sites with more ECM fungi had significantly less microbial biomass per unit soil C and lower rates of N mineralization. Consistent with a release from competition, C-degrading enzyme activities were higher and gross proteolytic rates were lower per unit microbial biomass inside compared to outside trenches. We interpret this to reflect increased microbial investment in C-acquisition and decreased investment in N-acquisition in the absence of ECM fungi. Furthermore, the increase in C-degrading enzymes per unit microbial biomass was significantly greater in sites with the most abundant ECM fungi. Based on these results, ECM-saprotroph competition does appear to slow soil C cycling and the effect is quantitative. Soil C cycling is at least partly controlled by interactions between ECM fungi and soil saprotrophs. Environmental change that alters ECM abundance may thus alter soil C stocks by ameliorating or exacerbating plant-decomposer competition for nitrogen.
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Into the decomposed body-forensic digital autopsy using multislice-computed tomography.
Thali, M J; Yen, K; Schweitzer, W; Vock, P; Ozdoba, C; Dirnhofer, R
2003-07-08
It is impossible to obtain a representative anatomical documentation of an entire body using classical X-ray methods, they subsume three-dimensional bodies into a two-dimensional level. We used the novel multislice-computed tomography (MSCT) technique in order to evaluate a case of homicide with putrefaction of the corpse before performing a classical forensic autopsy. This non-invasive method showed gaseous distension of the decomposing organs and tissues in detail as well as a complex fracture of the calvarium. MSCT also proved useful in screening for foreign matter in decomposing bodies, and full-body scanning took only a few minutes. In conclusion, we believe postmortem MSCT imaging is an excellent vizualisation tool with great potential for forensic documentation and evaluation of decomposed bodies.
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Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers.
Lu, Haiyun; Rusling, James F; Hu, Naifei
2007-12-27
Films constructed layer-by-layer on electrodes with architecture {protein/hyaluronic acid (HA)}n containing myoglobin (Mb) or horseradish peroxidase (HRP) were protected against protein damage by H2O2 by using outer catalase layers. Peroxidase activity for substrate oxidation requires activation by H2O2, but {protein/HA}n films without outer catalase layers are damaged slowly and irreversibly by H2O2. The rate and extent of damage were decreased dramatically by adding outer catalase layers to decompose H2O2. Comparative studies suggest that protection results from catalase decomposing a fraction of the H2O2 as it enters the film, rather than by an in-film diffusion barrier. The outer catalase layers controlled the rate of H2O2 entry into inner regions of the film, and they biased the system to favor electrocatalytic peroxide reduction over enzyme damage. Catalase-protected {protein/HA}n films had an increased linear concentration range for H2O2 detection. This approach offers an effective way to protect biosensors from damage by H2O2.
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Role of Reactive Mn Complexes in a Litter Decomposition Model System
NASA Astrophysics Data System (ADS)
Nico, P. S.; Keiluweit, M.; Bougoure, J.; Kleber, M.; Summering, J. A.; Maynard, J. J.; Johnson, M.; Pett-Ridge, J.
2012-12-01
The search for controls on litter decomposition rates and pathways has yet to return definitive characteristics that are both statistically robust and can be understood as part of a mechanistic or numerical model. Herein we focus on Mn, an element present in all litter that is likely an active chemical agent of decomposition. Berg and co-workers (2010) found a strong correlation between Mn concentration in litter and the magnitude of litter degradation in boreal forests, suggesting that litter decomposition proceeds more efficiently in the presence of Mn. Although there is much circumstantial evidence for the potential role of Mn in lignin decomposition, few reports exist on mechanistic details of this process. For the current work, we are guided by the hypothesis that the dependence of decomposition on Mn is due to Mn (III)-oxalate complexes act as a 'pretreatment' for structurally intact ligno-carbohydrate complexes (LCC) in fresh plant cell walls (e.g. in litter, root and wood). Manganese (III)-ligand complexes such as Mn (III)-oxalate are known to be potent oxidizers of many different organic and inorganic compounds. In the litter system, the unique property of these complexes may be that they are much smaller than exo-enzymes and therefore more easily able to penetrate LCC complexes in plant cell walls. By acting as 'diffusible oxidizers' and reacting with the organic matrix of the cell wall, these compounds can increase the porosity of fresh litter thereby facilitating access of more specific lignin- and cellulose decomposing enzymes. This possibility was investigated by reacting cell walls of single Zinnia elegans tracheary elements with Mn (III)-oxalate complexes in a continuous flow reactor. The uniformity of these individual plant cells allowed us to examine Mn (III)-induced changes in cell wall chemistry and ultrastructure on the micro-scale using fluorescence and electron microscopy as well as IR and X-ray spectromicroscopy. This presentation will discuss the chemical changes induced by reaction of Mn (III)-complexes with the Zinnia cells, the impact of such reactions on cell integrity, and potential implications for soil C cycling.
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Schiøtt, Morten; Rogowska-Wrzesinska, Adelina; Roepstorff, Peter; Boomsma, Jacobus J
2010-12-31
Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth. We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade. Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.
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Hildén, Kristiina; Mäkelä, Miia R; Steffen, Kari T; Hofrichter, Martin; Hatakka, Annele; Archer, David B; Lundell, Taina K
2014-11-01
Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer. Copyright © 2014 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Drake, J. E.; Darby, B. A.; Giasson, M.-A.; Kramer, M. A.; Phillips, R. P.; Finzi, A. C.
2012-06-01
Healthy plant roots release a wide range of chemicals into soils. This process, termed root exudation, is thought to increase the activity of microbes and the exo-enzymes they synthesize, leading to accelerated rates of carbon (C) mineralization and nutrient cycling in rhizosphere soils relative to bulk soils. The causal role of exudation, however, is difficult to isolate with in-situ observations, given the complex nature of the rhizosphere environment. We investigated the potential effects of root exudation on microbial and exo-enzyme activity using a theoretical model of decomposition and a field experiment, with a specific focus on the stoichiometric constraint of nitrogen (N) availability. The field experiment isolated the effect of exudation by pumping solutions of exudate mimics through microlysimeter "root simulators" into intact forest soils over two 50-day periods. Using a combined model-experiment approach, we tested two hypotheses: (1) exudation alone is sufficient to stimulate microbial and exo-enzyme activity in rhizosphere soils, and (2) microbial response to C-exudates (carbohydrates and organic acids) is constrained by N-limitation. Experimental delivery of exudate mimics containing C and N significantly increased microbial respiration, microbial biomass, and the activity of exo-enzymes that decompose labile components of soil organic matter (SOM, e.g., cellulose, amino sugars), while decreasing the activity of exo-enzymes that degrade recalcitrant SOM (e.g., polyphenols, lignin). However, delivery of C-only exudates had no effect on microbial biomass or overall exo-enzyme activity, and only increased microbial respiration. The theoretical decomposition model produced complementary results; the modeled microbial response to C-only exudates was constrained by limited N supply to support the synthesis of N-rich microbial biomass and exo-enzymes, while exuding C and N together elicited an increase in modeled microbial biomass, exo-enzyme activity, and decomposition. Thus, hypothesis (2) was supported, while hypothesis (1) was only supported when C and N compounds were exuded together. This study supports a cause-and-effect relationship between root exudation and enhanced microbial activity, and suggests that exudate stoichiometry is an important and underappreciated driver of microbial activity in rhizosphere soils.
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Molecular Docking of Potential Inhibitors of Broccoli Myrosinase.
Román, J; Castillo, A; Mahn, A
2018-05-30
Glucosinolates are secondary metabolites occurring in Brassicaceae plants whose hydrolysis may yield isothiocyanates, widely recognized as health-promoting compounds. Myrosinase catalyzes this conversion. The chemical mechanism involves an unstable intermediary (thiohydroxamate- O -sulfonate) that spontaneously decomposes into isothiocyanates or other non-bioactive compounds depending on pH and cofactors. At acidic pH, non-bioactive compounds such as nitriles and thiocyanates are formed, while at neutral pH isothiocyanates are obtained. Broccoli myrosinase has been poorly studied so far. Recently, its amino acidic sequence was elucidated, and a structural model was built. The aim of this work was to study the molecular interaction of broccoli myrosinase with different ligands at acidic pH to propose possible inhibitors that prevent formation of undesirable compounds at acidic pH, and that at neutral pH dissociate from the enzyme, allowing formation of isothiocyanates. The interaction between broccoli myrosinase and 40 ligands was studied by molecular docking simulations. Both the enzyme and each inhibitor were set at pH 3.0. Amygdaline and arbutin showed the highest affinity to broccoli myrosinase in this condition. The residues that stabilize the complexes agree with those that stabilize the substrate (Gln207, Glu429, Tyr352, and Ser433). Accordingly, amygdaline and arbutin would perform as competitive inhibitors of myrosinase at pH 3.0.
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[Isolation and identification of rumen bacteria for cellulolytic enzyme production].
Aihemaiti, Maierhaba; Zhen, Fan; Li, Yuezhong; Aibaidoula, Gulisimayi; Yimit, Wusiman
2013-05-04
We screened aerobic bacteria with cellulolytic activity from ruminal fluid of sheep, cattle and camel in Xinjiang. Fresh ruminal fluid was inoculated on sterilized sodium carboxymethylcellulose agar plates. Highly cellulolytic aerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media. The combination of morphological and biochemical test with 16SrDNA sequence analysis were used to classify the strains. Enzymatic activities of four strains with strong cellulose-decomposing abilities were studied under different culture conditions. Out 84 isolated cellulolytic strains, 40 exhibited strong abilities in decomposing cellulose. They are including 37 Gram-negative isolates and 3 Gram-positive strains. Identification of these 40 strains shows that they belong to 11 species of 6 genera, 16 strains in Stenotrophomonas maltophilia, 10 Ochrobactrum, 5 Sphingobacterium, 3 Microbacterium, 3 Paracoccus and 2 Pseudomonas. The results of the enzymatic studies of four strains with strong cellulolytic abilities indicates that the strains have the best enzyme producing property when straw powder was chosen as the carbon source; the pH at 5.5 -6.0 and temperature at 37 degrees C. The strains with highly cellulolytic abilities isolated from ruminal fluid show strong abilities in cellulose decomposition.
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Sun, Hongwei; Li, Guiying; Nie, Xin; Shi, Huixian; Wong, Po-Keung; Zhao, Huijun; An, Taicheng
2014-08-19
A systematic approach was developed to understand, in-depth, the mechanisms involved during the inactivation of bacterial cells using photoelectrocatalytic (PEC) processes with Escherichia coli K-12 as the model microorganism. The bacterial cells were found to be inactivated and decomposed primarily due to attack from photogenerated H2O2. Extracellular reactive oxygen species (ROSs), such as H2O2, may penetrate into the bacterial cell and cause dramatically elevated intracellular ROSs levels, which would overwhelm the antioxidative capacity of bacterial protective enzymes such as superoxide dismutase and catalase. The activities of these two enzymes were found to decrease due to the ROSs attacks during PEC inactivation. Bacterial cell wall damage was then observed, including loss of cell membrane integrity and increased permeability, followed by the decomposition of cell envelope (demonstrated by scanning electronic microscope images). One of the bacterial building blocks, protein, was found to be oxidatively damaged due to the ROSs attacks, as well. Leakage of cytoplasm and biomolecules (bacterial building blocks such as proteins and nucleic acids) were evident during prolonged PEC inactivation process. The leaked cytoplasmic substances and cell debris could be further degraded and, ultimately, mineralized with prolonged PEC treatment.
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Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J
2014-07-23
Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.
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2015-01-01
Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR. PMID:24977791
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NASA Astrophysics Data System (ADS)
Grandner, Jessica Marie
Computational methods were used to determine the mechanisms and selectivities of organometallic-catalyzed reactions. The first half of the dissertation focuses on the study of metathesis catalysts in collaboration with the Grubbs group at CalTech. Chapter 1 describes the studies of the decomposition modes of several ruthenium-based metathesis catalysts. These studies were performed to better understand the decomposition of such catalysts in order to prevent decomposition (Chapter 1.2) or utilize decomposed catalysts for alternative reactions (Chapter 1.1). Chapter 2.1 describes the computational investigation of the origins of stereoretentive metathesis with ruthenium-based metathesis catalysts. These findings were then used to computationally design E-selective metathesis catalysts (Chapter 2.2). While the first half of the dissertation was centered around ruthenium catalysts, the second half of the dissertation pertains to iron-catalyzed reaction, in particular, iron-catalyzed reactions by P450 enzymes. The elements of Chapter 3 concentrate on the stereo- and chemo-selectivity of P450-catalyzed C-H hydroxylations. By combining multiple computational methods, the inherent activity of the iron-oxo catalyst and the influence of the active site on such reactions were illuminated. These discoveries allow for the engineering of new substrates and mutant enzymes for tailored C-H hydroxylation. While the mechanism of C-H hydroxylations catalyzed by P450 enzymes has been well studied, there are several P450-catalyzed transformations for which the mechanism is unknown. The components of Chapter 4 describe the use of computations to determine the mechanisms of complex, multi-step reactions catalyzed by P450s. The determination of these mechanisms elucidates how these enzymes react with various functional groups and substrate architectures and allows for a better understanding of how drug-like compounds may be broken down by human P450s.
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Chitinolytic functions in actinobacteria: ecology, enzymes, and evolution.
Lacombe-Harvey, Marie-Ève; Brzezinski, Ryszard; Beaulieu, Carole
2018-06-21
Actinobacteria, a large group of Gram-positive bacteria, secrete a wide range of extracellular enzymes involved in the degradation of organic compounds and biopolymers including the ubiquitous aminopolysaccharides chitin and chitosan. While chitinolytic enzymes are distributed in all kingdoms of life, actinobacteria are recognized as particularly good decomposers of chitinous material and several members of this taxon carry impressive sets of genes dedicated to chitin and chitosan degradation. Degradation of these polymers in actinobacteria is dependent on endo- and exo-acting hydrolases as well as lytic polysaccharide monooxygenases. Actinobacterial chitinases and chitosanases belong to nine major families of glycosyl hydrolases that share no sequence similarity. In this paper, the distribution of chitinolytic actinobacteria within different ecosystems is examined and their chitinolytic machinery is described and compared to those of other chitinolytic organisms.
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Decomposition of Rotor Hopfield Neural Networks Using Complex Numbers.
Kobayashi, Masaki
2018-04-01
A complex-valued Hopfield neural network (CHNN) is a multistate model of a Hopfield neural network. It has the disadvantage of low noise tolerance. Meanwhile, a symmetric CHNN (SCHNN) is a modification of a CHNN that improves noise tolerance. Furthermore, a rotor Hopfield neural network (RHNN) is an extension of a CHNN. It has twice the storage capacity of CHNNs and SCHNNs, and much better noise tolerance than CHNNs, although it requires twice many connection parameters. In this brief, we investigate the relations between CHNN, SCHNN, and RHNN; an RHNN is uniquely decomposed into a CHNN and SCHNN. In addition, the Hebbian learning rule for RHNNs is decomposed into those for CHNNs and SCHNNs.
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Xiao, Xiaopeng; Mazza, Lorenzo; Yu, Yongqiang; Cai, Minmin; Zheng, Longyu; Tomberlin, Jeffery K; Yu, Jeffrey; van Huis, Arnold; Yu, Ziniu; Fasulo, Salvatore; Zhang, Jibin
2018-07-01
A chicken manure management process was carried out through co-conversion of Hermetia illucens L. larvae (BSFL) with functional bacteria for producing larvae as feed stuff and organic fertilizer. Thirteen days co-conversion of 1000 kg of chicken manure inoculated with one million 6-day-old BSFL and 10 9 CFU Bacillus subtilis BSF-CL produced aging larvae, followed by eleven days of aerobic fermentation inoculated with the decomposing agent to maturity. 93.2 kg of fresh larvae were harvested from the B. subtilis BSF-CL-inoculated group, while the control group only harvested 80.4 kg of fresh larvae. Chicken manure reduction rate of the B. subtilis BSF-CL-inoculated group was 40.5%, while chicken manure reduction rate of the control group was 35.8%. The weight of BSFL increased by 15.9%, BSFL conversion rate increased by 12.7%, and chicken manure reduction rate increased by 13.4% compared to the control (no B. subtilis BSF-CL). The residue inoculated with decomposing agent had higher maturity (germination index >92%), compared with the no decomposing agent group (germination index ∼86%). The activity patterns of different enzymes further indicated that its production was more mature and stable than that of the no decomposing agent group. Physical and chemical production parameters showed that the residue inoculated with the decomposing agent was more suitable for organic fertilizer than the no decomposing agent group. Both, the co-conversion of chicken manure by BSFL with its synergistic bacteria and the aerobic fermentation with the decomposing agent required only 24 days. The results demonstrate that co-conversion process could shorten the processing time of chicken manure compared to traditional compost process. Gut bacteria could enhance manure conversion and manure reduction. We established efficient manure co-conversion process by black soldier fly and bacteria and harvest high value-added larvae mass and biofertilizer. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Opaque for the Reader but Transparent for the Brain: Neural Signatures of Morphological Complexity
ERIC Educational Resources Information Center
Meinzer, Marcus; Lahiri, Aditi; Flaisch, Tobias; Hannemann, Ronny; Eulitz, Carsten
2009-01-01
Within linguistics, words with a complex internal structure are commonly assumed to be decomposed into their constituent morphemes (e.g., un-help-ful). Nevertheless, an ongoing debate concerns the brain structures that subserve this process. Using functional magnetic resonance imaging, the present study varied the internal complexity of derived…
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Management intensity alters decomposition via biological pathways
Wickings, Kyle; Grandy, A. Stuart; Reed, Sasha; Cleveland, Cory
2011-01-01
Current conceptual models predict that changes in plant litter chemistry during decomposition are primarily regulated by both initial litter chemistry and the stage-or extent-of mass loss. Far less is known about how variations in decomposer community structure (e.g., resulting from different ecosystem management types) could influence litter chemistry during decomposition. Given the recent agricultural intensification occurring globally and the importance of litter chemistry in regulating soil organic matter storage, our objectives were to determine the potential effects of agricultural management on plant litter chemistry and decomposition rates, and to investigate possible links between ecosystem management, litter chemistry and decomposition, and decomposer community composition and activity. We measured decomposition rates, changes in litter chemistry, extracellular enzyme activity, microarthropod communities, and bacterial versus fungal relative abundance in replicated conventional-till, no-till, and old field agricultural sites for both corn and grass litter. After one growing season, litter decomposition under conventional-till was 20% greater than in old field communities. However, decomposition rates in no-till were not significantly different from those in old field or conventional-till sites. After decomposition, grass residue in both conventional- and no-till systems was enriched in total polysaccharides relative to initial litter, while grass litter decomposed in old fields was enriched in nitrogen-bearing compounds and lipids. These differences corresponded with differences in decomposer communities, which also exhibited strong responses to both litter and management type. Overall, our results indicate that agricultural intensification can increase litter decomposition rates, alter decomposer communities, and influence litter chemistry in ways that could have important and long-term effects on soil organic matter dynamics. We suggest that future efforts to more accurately predict soil carbon dynamics under different management regimes may need to explicitly consider how changes in litter chemistry during decomposition are influenced by the specific metabolic capabilities of the extant decomposer communities.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sengupta, S.; Soda, H.; McLean, A.
2000-01-01
A ternary eutectic alloy with a composition of 57.2 pct Bi, 24.8 pct In, and 18 pct Sn was continuously cast into wire of 2 mm diameter with casting speeds of 14 and 79 mm/min using the Ohno Continuous Casting (OCC) process. The microstructures obtained were compared with those of statically cast specimens. Extensive segregation of massive Bi blocks, Bi complex structures, and tin-rich dendrites was found in specimens that were statically cast. Decomposition of {radical}Sn by a eutectoid reaction was confirmed based on microstructural evidence. Ternary eutectic alloy with a cooling rate of approximately 1 C/min formed a doublemore » binary eutectic. The double binary eutectic consisted of regions of BiIn and decomposed {radical}Sn in the form of a dendrite cell structure and regions of Bi and decomposed {radical}Sn in the form of a complex-regular cell. The Bi complex-regular cells, which are a ternary eutectic constituent, existed either along the boundaries of the BiIn-decomposed {radical}Sn dendrite cells or at the front of elongated dendrite cell structures. In the continuously cast wires, primary Sn dendrites coupled with a small Bi phase were uniformly distributed within the Bi-In alloy matrix. Neither massive Bi phase, Bi complex-regular cells, no BiIn eutectic dendrite cells were observed, resulting in a more uniform microstructure in contrast to the heavily segregated structures of the statically cast specimens.« less
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A Modified Demonstration of the Catalytic Decomposition of Hydrogen Peroxide
NASA Astrophysics Data System (ADS)
Trujillo, Carlos Alexander
2005-06-01
A safer and cheaper version of the popular catalyzed decomposition of hydrogen peroxide demonstration commonly called the “Elephants’ Toothpaste” is presented. Hydrogen peroxide is decomposed in the presence of a surfactant by the enzyme catalase producing foam. Catalase has a higher activity compared with the traditional iodide and permits the use of diluted hydrogen peroxide solutions. The demonstration can be made with household products with similar amazing effects.
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[Polyelectrolyte microcapsules as systems for delivery of biologically active substances].
Borodina, T N; Rumsh, L D; Kunizhev, S M; Sukhorukov, G B; Vorozhtsov, G N; Fel'dman, B M; Markvicheva, E A
2007-01-01
Novel biodegradable microcapsules for delivery of biologically active substances (BAS) were prepared by layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, namely sodium alginate (Alg) and poly-L-lysine (PLL). To immobilize these BAS, porous spherical CaCO3 microparticles were used as templates. The templates (cores) were coated with several layers of oppositely charged polyelectrolytes forming shell on a core surface. The core-shell microparticles were converted into hollow microcapsules by a core dissolution after an EDTA treatment. Mild conditions for microcapsule fabrication allow to perform an entrapment of various biomolecules while keeping their bioactivity. Biocompatibility and biodegradable capability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin (Chym) entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme was found to keep about 86% of the activity compared to a native Chym. The obtained microcapsules were stable at an acidic medium while they could be easily decomposed by trypsin treatment at an slightly alkaline medium. Chym was shown to be active after being released from the microcapsules decomposed by trypsin treatment. Thus, the microcapsules prepared by the LbL - technique can be used for the development of new type of BAS delivery systems in humans and animals.
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Vertebrate Decomposition Is Accelerated by Soil Microbes
Lauber, Christian L.; Metcalf, Jessica L.; Keepers, Kyle; Ackermann, Gail; Carter, David O.
2014-01-01
Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors, including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either (i) soil with an intact microbial community or (ii) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e., gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2 to 3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of active and advanced decay were significantly different between soil types (sterile versus untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil- versus carcass-associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and have important implications for understanding carrion ecology. PMID:24907317
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Immobilization of Candida rugosa lipase by adsorption-crosslinking onto corn husk
NASA Astrophysics Data System (ADS)
Nuraliyah, A.; Wijanarko, A.; Hermansyah, H.
2018-04-01
Corn husk is one of the agricutural waste that has not been used optimally. corn husk waste allows to be used as immobilized support for biocatalyst because it is easy to obtain, available abundant, renewable and easy to decompose. This research was conducted in two phases, namely the adsorption of enzyme immobilization on the support, followed by cross- linking between the enzyme and support through the addition of glutaraldehyde. The optimum conditions for cross-linked adsorption immobilization using support of corn husk were achieved at concentrations of 0,75 mg / ml at 4 hour reaction time. The biggest unit activity value is obtained at 2,37 U / g support through 0.5% glutaraldehyde addition.
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Mechanism of inhibition of catalase by nitro and nitroso compounds.
Titov, V Yu; Petrenko, Yu M; Vanin, A F
2008-01-01
Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested--nitroarginine, nitroglycerol, nitrophenol, and furazolidone--gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.
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Gao, Yanzheng; Li, Hui; Gong, Shuaishuai
2012-01-01
Plant contamination by polycyclic aromatic hydrocarbons (PAHs) is crucial to food safety and human health. Enzyme inhibitors are commonly utilized in agriculture to control plant metabolism of organic components. This study revealed that the enzyme inhibitor ascorbic acid (AA) significantly reduced the activities of peroxidase (POD) and polyphenol oxidase (PPO), thus enhancing the potential risks of PAH contamination in tall fescue (Festuca arundinacea Schreb.). POD and PPO enzymes in vitro effectively decomposed naphthalene (NAP), phenanthrene (PHE) and anthracene (ANT). The presence of AA reduced POD and PPO activities in plants, and thus was likely responsible for enhanced PAH accumulation in tall fescue. This conclusion is supported by the significantly enhanced uptake of PHE in plants in the presence of AA, and the positive correlation between enzyme inhibition efficiencies and the rates of metabolism of PHE in tall fescue roots. This study provides a new perspective, that the common application of enzyme inhibitors in agricultural production could increase the accumulation of organic contaminants in plants, hence enhancing risks to food safety and quality. PMID:23185628
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Morphological Decomposition Based on the Analysis of Orthography
ERIC Educational Resources Information Center
Rastle, Kathleen; Davis, Matthew H.
2008-01-01
Recent theories of morphological processing have been dominated by the notion that morphologically complex words are decomposed into their constituents on the basis of their semantic properties. In this article we argue that the weight of evidence now suggests that the recognition of morphologically complex words begins with a rapid morphemic…
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NASA Astrophysics Data System (ADS)
Hammer, Edith C.; Aleklett, Kristin; Arellano Caicedo, Carlos G.; Bengtsson, Martin; Micaela Mafla Endara, Paola; Ohlsson, Pelle
2017-04-01
From the point of view of microorganisms, the soil environment is an enormously complex labyrinth with paths and dead-end streets, where resources and shelters are unevenly distributed. We study foraging strategies of soil organisms, especially fungi, and the possibility of physio-spatial stabilization of organic matter by "hiding" in occluded soil spaces. We manipulate growth habitat microstructure with lab-on-a-chip techniques, where we designed complex environments with channels and obstacle at dimensions of the size of hyphae, and construct them in the transparent, gas-permeable polymer PDMS. We fill those with different nutrient solutions or combine with mineral nutrient gradients, and inoculate them with soil organisms. We analyze organisms and substrates with microscopy, fluorescence microscopy and analytical chemistry. We compared different soil litter decomposers and an arbuscular mycorrhizal fungus for their ability to forage through complex air-gap structures and attempt to classify them into functional traits concerning their mycelium directionality, space-exploring approach and ability to grow through acute angles and narrow constrictions. We identified structures which are very difficult to penetrate for most species, and compounds located behind such features may thus be spatially unavailable for decomposers. We discuss our approach in comparison to soil pore space tomographic analyses and findings we made in the pore space of colonized wood biochar.
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Does Learning a Complex Task Have To Be Complex?: A Study in Learning Decomposition.
ERIC Educational Resources Information Center
Lee, Frank J.; Anderson, John R.
2001-01-01
Decomposed the learning in the Kanfer-Ackerman Air-Traffic Controller Task (P. Ackerman, 1988) down to learning at the keyboard level. Reanalyzed the Ackerman data to show that learning in this complex task reflects learning at the keystroke level. Conducted an eye-tracking experiment with 10 adults that showed that learning at the key stroke…
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Interplay between morphology and frequency in lexical access: The case of the base frequency effect
Vannest, Jennifer; Newport, Elissa L.; Newman, Aaron J.; Bavelier, Daphne
2011-01-01
A major issue in lexical processing concerns storage and access of lexical items. Here we make use of the base frequency effect to examine this. Specifically, reaction time to morphologically complex words (words made up of base and suffix, e.g., agree+able) typically reflects frequency of the base element (i.e., total frequency of all words in which agree appears) rather than surface word frequency (i.e., frequency of agreeable itself). We term these complex words decomposable. However, a class of words termed whole-word do not show such sensitivity to base frequency (e.g., serenity). Using an event-related MRI design, we exploited the fact that processing low-frequency words increases BOLD activity relative to high frequency ones, and examined effects of base frequency on brain activity for decomposable and whole-word items. Morphologically complex words, half high and half low base frequency, were compared to matched high and low frequency simple monomorphemic words using a lexical decision task. Morphologically complex words increased activation in left inferior frontal and left superior temporal cortices versus simple words. The only area to mirror the behavioral distinction between decomposable and whole-word types was the thalamus. Surprisingly, most frequency-sensitive areas failed to show base frequency effects. This variety of responses to frequency and word type across brain areas supports an integrative view of multiple variables during lexical access, rather than a dichotomy between memory-based access and on-line computation. Lexical access appears best captured as interplay of several neural processes with different sensitivities to various linguistic factors including frequency and morphological complexity. PMID:21167136
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Formation of ethyl ferulate by rice koji enzyme in sake and mirin mash conditions.
Hashizume, Katsumi; Ito, Toshihiko; Ishizuka, Takahiro; Takeda, Naoki
2013-08-01
Formation mechanism of ethyl ferulate (EF) in sake and mirin mash conditions was investigated to understand EF level control in the manufacturing process. Rice koji formed EF from ferulic acid (FA) and ethanol and decomposed EF to FA. This did not occur in sake yeast and chemical esterification was rare. Esterification of FA and hydrolysis of EF by rice koji might be due to feruloyl esterase(s). The rice koji enzyme showed normal Michaelis-Menten kinetics for FA in ethyl esterification and for EF in hydrolysis, but not for ethanol in the esterification reaction. Substrate specificity of the rice koji enzyme for hydroxycinnamic acids suggested that the main enzyme involved might be similar to type A feruloyl esterase. We studied the rice koji enzyme properties, short-term digestion of steamed rice grains with exogenous ethanol and small scale mirin making with pH adjustment. Our results suggested differences in the esterification and hydrolysis properties of the enzyme, in particular, different pH dependencies and different behaviors under high ethanol conditions; these factors might cause the differing EF levels in sake and mirin mashes. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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[Acatalasemia and type 2 diabetes mellitus].
Góth, László; Nagy, Teréz; Káplár, Miklós
2015-03-08
The catalase enzyme decomposes the toxic concentrations of hydrogen peroxide into oxygen and water. Hydrogen peroxide is a highly reactive small molecule and its excessive concentration may cause significant damages to proteins, deoxyribonucleic acid, ribonucleic acid and lipids. Acatalasemia refers to inherited deficiency of the catalase enzyme. In this review the authors discuss the possible role of the human catalase enzyme, the metabolism of hydrogen peroxide, and the phenomenon of hydrogen peroxide paradox. In addition, they review data obtained from Hungarian acatalasemic patients indicating an increased frequency of type 2 diabetes mellitus, especially in female patients, and an early onset of type 2 diabetes in these patients. There are 10 catalase gene variants which appear to be responsible for decreased blood catalase activity in acatalasemic patients with type 2 diabetes. It is assumed that low levels of blood catalase may cause an increased concentration of hydrogen peroxide which may contribute to the pathogenesis of type 2 diabetes mellitus.
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Mohr, S; Hallak, H; de Boitte, A; Lapetina, E G; Brüne, B
1999-04-02
S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.
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Marine-derived fungi: diversity of enzymes and biotechnological applications
Bonugli-Santos, Rafaella C.; dos Santos Vasconcelos, Maria R.; Passarini, Michel R. Z.; Vieira, Gabriela A. L.; Lopes, Viviane C. P.; Mainardi, Pedro H.; dos Santos, Juliana A.; de Azevedo Duarte, Lidia; Otero, Igor V. R.; da Silva Yoshida, Aline M.; Feitosa, Valker A.; Pessoa, Adalberto; Sette, Lara D.
2015-01-01
The ocean is considered to be a great reservoir of biodiversity. Microbial communities in marine environments are ecologically relevant as intermediaries of energy, and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. In this sense, marine-derived fungi can be considered as a source of enzymes of industrial and/or environmental interest. Fungal strains isolated from different substrates, such as invertebrates, decaying wood, seawater, sediments, and mangrove detritus, have been reported to be producers of hydrolytic and/or oxidative enzymes, with alginate lyase, amylase, cellulase, chitinase, glucosidase, inulinase, keratinase, ligninase, lipase, nuclease, phytase, protease, and xylanase being among the enzymes produced by fungi of marine origin. These enzymes present temperature and pH optima ranging from 35 to 70∘C, and 3.0 to 11.0, respectively. High-level production in bioreactors is mainly performed using submerged-state fermentation. Certain marine-derived fungal strains present enzymes with alkaline and cold-activity characteristics, and salinity is considered an important condition in screening and production processes. The adaptability of marine-derived fungi to oceanic conditions can be considered an attractive point in the field of fungal marine biotechnology. In this review, we focus on the advances in discovering enzymes from marine-derived fungi and their biotechnological relevance. PMID:25914680
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The Roles of Glutathione Peroxidases during Embryo Development
Ufer, Christoph; Wang, Chi Chiu
2011-01-01
Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency – in contrast to all other GPx family members – leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4. PMID:21847368
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The Roles of Glutathione Peroxidases during Embryo Development.
Ufer, Christoph; Wang, Chi Chiu
2011-01-01
Embryo development relies on the complex interplay of the basic cellular processes including proliferation, differentiation, and apoptotic cell death. Precise regulation of these events is the basis for the establishment of embryonic structures and the organ development. Beginning with fertilization of the oocyte until delivery the developing embryo encounters changing environmental conditions such as varying levels of oxygen, which can give rise to reactive oxygen species (ROS). These challenges are met by the embryo with metabolic adaptations and by an array of anti-oxidative mechanisms. ROS can be deleterious by modifying biological molecules including lipids, proteins, and nucleic acids and may induce abnormal development or even embryonic lethality. On the other hand ROS are vital players of various signaling cascades that affect the balance between cell growth, differentiation, and death. An imbalance or dysregulation of these biological processes may generate cells with abnormal growth and is therefore potentially teratogenic and tumorigenic. Thus, a precise balance between processes generating ROS and those decomposing ROS is critical for normal embryo development. One tier of the cellular protective system against ROS constitutes the family of selenium-dependent glutathione peroxidases (GPx). These enzymes reduce hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. Of special interest within this protein family is the moonlighting enzyme glutathione peroxidase 4 (Gpx4). This enzyme is a scavenger of lipophilic hydroperoxides on one hand, but on the other hand can be transformed into an enzymatically inactive cellular structural component. GPx4 deficiency - in contrast to all other GPx family members - leads to abnormal embryo development and finally produces a lethal phenotype in mice. This review is aimed at summarizing the current knowledge on GPx isoforms during embryo development and tumor development with an emphasis on GPx4.
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Mycorrhiza-mediated competition between plants and decomposers drives soil carbon storage.
Averill, Colin; Turner, Benjamin L; Finzi, Adrien C
2014-01-23
Soil contains more carbon than the atmosphere and vegetation combined. Understanding the mechanisms controlling the accumulation and stability of soil carbon is critical to predicting the Earth's future climate. Recent studies suggest that decomposition of soil organic matter is often limited by nitrogen availability to microbes and that plants, via their fungal symbionts, compete directly with free-living decomposers for nitrogen. Ectomycorrhizal and ericoid mycorrhizal (EEM) fungi produce nitrogen-degrading enzymes, allowing them greater access to organic nitrogen sources than arbuscular mycorrhizal (AM) fungi. This leads to the theoretical prediction that soil carbon storage is greater in ecosystems dominated by EEM fungi than in those dominated by AM fungi. Using global data sets, we show that soil in ecosystems dominated by EEM-associated plants contains 70% more carbon per unit nitrogen than soil in ecosystems dominated by AM-associated plants. The effect of mycorrhizal type on soil carbon is independent of, and of far larger consequence than, the effects of net primary production, temperature, precipitation and soil clay content. Hence the effect of mycorrhizal type on soil carbon content holds at the global scale. This finding links the functional traits of mycorrhizal fungi to carbon storage at ecosystem-to-global scales, suggesting that plant-decomposer competition for nutrients exerts a fundamental control over the terrestrial carbon cycle.
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Decomposition of recalcitrant carbon under experimental warming in boreal forest
Allison, Steven D.; Treseder, Kathleen K.
2017-01-01
Over the long term, soil carbon (C) storage is partly determined by decomposition rate of carbon that is slow to decompose (i.e., recalcitrant C). According to thermodynamic theory, decomposition rates of recalcitrant C might differ from those of non-recalcitrant C in their sensitivities to global warming. We decomposed leaf litter in a warming experiment in Alaskan boreal forest, and measured mass loss of recalcitrant C (lignin) vs. non-recalcitrant C (cellulose, hemicellulose, and sugars) throughout 16 months. We found that these C fractions responded differently to warming. Specifically, after one year of decomposition, the ratio of recalcitrant C to non-recalcitrant C remaining in litter declined in the warmed plots compared to control. Consistent with this pattern, potential activities of enzymes targeting recalcitrant C increased with warming, relative to those targeting non-recalcitrant C. Even so, mass loss of individual C fractions showed that non-recalcitrant C is preferentially decomposed under control conditions whereas recalcitrant C losses remain unchanged between control and warmed plots. Moreover, overall mass loss was greater under control conditions. Our results imply that direct warming effects, as well as indirect warming effects (e.g. drying), may serve to maintain decomposition rates of recalcitrant C compared to non-recalcitrant C despite negative effects on overall decomposition. PMID:28622366
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Preclinical safety evaluation of intravenously administered mixed micelles.
Teelmann, K; Schläppi, B; Schüpbach, M; Kistler, A
1984-01-01
Mixed micelles, with their main constituents lecithin and glycocholic acid, form a new principle for the parenteral administration of compounds which are poorly water-soluble. Their composition of mainly physiological substances as well as their comparatively good stability substantiate their attractivity in comparison to existing solvents. A decomposition due to physical influences such as heat or storage for several years will almost exclusively affect the lecithin component in the form of hydrolysis into free fatty acids and lysolecithin. Their toxicity was examined experimentally in various studies using both undecomposed and artificially decomposed mixed micelles. In these studies the mixed micelles were locally and systemically well tolerated and proved to be neither embryotoxic, teratogenic nor mutagenic. Only when comparatively high doses of the undecomposed mixed micelles were administered, corresponding to approximately 30 to 50 times the anticipated clinical injection volume (of e.g. diazepam mixed micelles), did some vomitus (dogs), slight liver enzyme elevation (rats and dogs), and slightly increased liver weights (dogs) occur. After repeated injections of the artificially decomposed formulation (approximately 25% of lecithin hydrolyzed to free fatty acids and lysolecithin) effects such as intravascular haemolysis, liver enzyme elevations and intrahepatic cholestasis (dogs only) were observed but only when doses exceeding a threshold of approximately 40 to 60 mg lysolecithin/kg body weight were administered. All alterations were reversible after cessation of treatment.
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Challenges of including nitrogen effects on decomposition in earth system models
NASA Astrophysics Data System (ADS)
Hobbie, S. E.
2011-12-01
Despite the importance of litter decomposition for ecosystem fertility and carbon balance, key uncertainties remain about how this fundamental process is affected by nitrogen (N) availability. Nevertheless, resolving such uncertainties is critical for mechanistic inclusion of such processes in earth system models, towards predicting the ecosystem consequences of increased anthropogenic reactive N. Towards that end, we have conducted a series of experiments examining nitrogen effects on litter decomposition. We found that both substrate N and externally supplied N (regardless of form) accelerated the initial decomposition rate. Faster initial decomposition rates were linked to the higher activity of carbohydrate-degrading enzymes associated with externally supplied N and the greater relative abundances of Gram negative and Gram positive bacteria associated with green leaves and externally supplied organic N (assessed using phospholipid fatty acid analysis, PLFA). By contrast, later in decomposition, externally supplied N slowed decomposition, increasing the fraction of slowly decomposing litter and reducing lignin-degrading enzyme activity and relative abundances of Gram negative and Gram positive bacteria. Our results suggest that elevated atmospheric N deposition may have contrasting effects on the dynamics of different soil carbon pools, decreasing mean residence times of active fractions comprising very fresh litter, while increasing those of more slowly decomposing fractions including more processed litter. Incorporating these contrasting effects of N on decomposition processes into models is complicated by lingering uncertainties about how these effects generalize across ecosystems and substrates.
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NASA Astrophysics Data System (ADS)
Mao, J.; Chen, N.; Harmon, M. E.; Li, Y.; Cao, X.; Chappell, M.
2012-12-01
Advanced 13C solid-state NMR techniques were employed to study the chemical structural changes of litter decomposition across broad spatial and long time scales. The fresh and decomposed litter samples of four species (Acer saccharum (ACSA), Drypetes glauca (DRGL), Pinus resinosa (PIRE), and Thuja plicata (THPL)) incubated for up to 10 years at four sites under different climatic conditions (from Arctic to tropical forest) were examined. Decomposition generally led to an enrichment of cutin and surface wax materials, and a depletion of carbohydrates causing overall composition to become more similar compared with original litters. However, the changes of main constituents in the four litters were inconsistent with the four litters following different pathways of decomposition at the same site. As decomposition proceeded, waxy materials decreased at the early stage and then gradually increased in PIRE; DRGL showed a significant depletion of lignin and tannin while the changes of lignin and tannin were relative small and inconsistent for ACSA and THPL. In addition, the NCH groups, which could be associated with either fungal cell wall chitin or bacterial wall petidoglycan, were enriched in all litters except THPL. Contrary to the classic lignin-enrichment hypothesis, DRGL with low-quality C substrate had the highest degree of composition changes. Furthermore, some samples had more "advanced" compositional changes in the intermediate stage of decomposition than in the highly-decomposed stage. This pattern might be attributed to the formation of new cross-linking structures, that rendered substrates more complex and difficult for enzymes to attack. Finally, litter quality overrode climate and time factors as a control of long-term changes of chemical composition.
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NASA Astrophysics Data System (ADS)
Keiluweit, M.; Bougoure, J.; Pett-Ridge, J.; Kleber, M.; Nico, P. S.
2011-12-01
Lignin comprises a dominant proportion of carbon fluxes into the soil (representing up to 50% of plant litter and roots). Two lines of evidence suggest that manganese (Mn) acts as a strong controlling factor on the residence time of lignin in soil ecosystems. First, Mn content is highly correlated with litter decomposition in temperate and boreal forest soil ecosystems and, second, microbial agents of lignin degradation have been reported to rely on reactive Mn(III)-complexes to specifically oxidize lignin. However, few attempts have been made to isolate the mechanisms responsible for the apparent Mn-dependence of lignin decomposition in soils. Here we tested the hypothesis that Mn(III)-oxalate complexes may act as a perforating 'pretreatment' for structurally intact plant cell walls. We propose that these diffusible oxidizers are small enough to penetrate and react with non-porous ligno-cellulose in cell walls. This process was investigated by reacting single Zinnia elegans tracheary elements with Mn(III)-oxalate complexes in a continuous flow-through microreactor. The uniformity of cultured tracheary elements allowed us to examine Mn(III)-induced changes in cell wall chemistry and ultrastructure on the micro-scale using fluorescence and electron microscopy as well as synchrotron-based infrared and X-ray spectromicroscopy. Our results show that Mn(III)-complexes substantially oxidize specific lignin components of the cell wall, solubilize decomposition products, severely undermine the cell wall integrity, and cause cell lysis. We conclude that Mn(III)-complexes induce oxidative damage in plant cell walls that renders ligno-cellulose substrates more accessible for microbial lignin- and cellulose-decomposing enzymes. Implications of our results for the rate limiting impact of soil Mn speciation and availability on litter decomposition in forest soils will be discussed.
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Anh, Dau Hung; Ullrich, René; Benndorf, Dirk; Svatoś, Aleś; Muck, Alexander; Hofrichter, Martin
2007-01-01
Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies. PMID:17601809
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Anh, Dau Hung; Ullrich, René; Benndorf, Dirk; Svatos, Ales; Muck, Alexander; Hofrichter, Martin
2007-09-01
Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.
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Stoichiometric network theory for nonequilibrium biochemical systems.
Qian, Hong; Beard, Daniel A; Liang, Shou-dan
2003-02-01
We introduce the basic concepts and develop a theory for nonequilibrium steady-state biochemical systems applicable to analyzing large-scale complex isothermal reaction networks. In terms of the stoichiometric matrix, we demonstrate both Kirchhoff's flux law sigma(l)J(l)=0 over a biochemical species, and potential law sigma(l) mu(l)=0 over a reaction loop. They reflect mass and energy conservation, respectively. For each reaction, its steady-state flux J can be decomposed into forward and backward one-way fluxes J = J+ - J-, with chemical potential difference deltamu = RT ln(J-/J+). The product -Jdeltamu gives the isothermal heat dissipation rate, which is necessarily non-negative according to the second law of thermodynamics. The stoichiometric network theory (SNT) embodies all of the relevant fundamental physics. Knowing J and deltamu of a biochemical reaction, a conductance can be computed which directly reflects the level of gene expression for the particular enzyme. For sufficiently small flux a linear relationship between J and deltamu can be established as the linear flux-force relation in irreversible thermodynamics, analogous to Ohm's law in electrical circuits.
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D’haeseleer, Patrik; Khudyakov, Jane; Burd, Helcio; Hadi, Masood; Simmons, Blake A.; Singer, Steven W.; Thelen, Michael P.; VanderGheynst, Jean S.
2013-01-01
High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production. PMID:24205054
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Reactions in trifluoroacetic acid (CF 3COOH) induced by low energy electron attachment
NASA Astrophysics Data System (ADS)
Langer, Judith; Stano, Michal; Gohlke, Sascha; Foltin, Victor; Matejcik, Stefan; Illenberger, Eugen
2006-02-01
Dissociative electron attachment to trifluoroacetic acid (CF 3COOH) is characterized by an intense low energy shape resonance located near 1 eV and a comparatively weaker core excited resonance located near 7 eV. The shape resonance decomposes into the fragment ions CF 3COO -, CF 2COO -, and CF2-. The underlying reactions include simple bond cleavage but also more complex sequences involving multiple bond cleavages, rearrangement in the precursor ion and formation of new molecules (HF, CO 2). The core excited resonance additionally decomposes into F -, CF3- and probably metastable CO2-.
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Yamamoto, Shuji; Suzuki, Kei; Araki, Yoko; Mochihara, Hiroki; Hosokawa, Tetsuya; Kubota, Hiroko; Chiba, Yusuke; Rubaba, Owen; Tashiro, Yosuke; Futamata, Hiroyuki
2014-01-01
The relationship between the bacterial communities in anolyte and anode biofilms and the electrochemical properties of microbial fuel cells (MFCs) was investigated when a complex organic waste-decomposing solution was continuously supplied to MFCs as an electron donor. The current density increased gradually and was maintained at approximately 100 to 150 mA m−2. Polarization curve analyses revealed that the maximum power density was 7.4 W m−3 with an internal resistance of 110 Ω. Bacterial community structures in the organic waste-decomposing solution and MFCs differed from each other. Clonal analyses targeting 16S rRNA genes indicated that bacterial communities in the biofilms on MFCs developed to specific communities dominated by novel Geobacter. Multidimensional scaling analyses based on DGGE profiles revealed that bacterial communities in the organic waste-decomposing solution fluctuated and had no dynamic equilibrium. Bacterial communities on the anolyte in MFCs had a dynamic equilibrium with fluctuations, while those of the biofilm converged to the Geobacter-dominated structure. These bacterial community dynamics of MFCs differed from those of control-MFCs under open circuit conditions. These results suggested that bacterial communities in the anolyte and biofilm have a gentle symbiotic system through electron flow, which resulted in the advance of current density from complex organic waste. PMID:24789988
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Yamamoto, Shuji; Suzuki, Kei; Araki, Yoko; Mochihara, Hiroki; Hosokawa, Tetsuya; Kubota, Hiroko; Chiba, Yusuke; Rubaba, Owen; Tashiro, Yosuke; Futamata, Hiroyuki
2014-01-01
The relationship between the bacterial communities in anolyte and anode biofilms and the electrochemical properties of microbial fuel cells (MFCs) was investigated when a complex organic waste-decomposing solution was continuously supplied to MFCs as an electron donor. The current density increased gradually and was maintained at approximately 100 to 150 mA m(-2). Polarization curve analyses revealed that the maximum power density was 7.4 W m(-3) with an internal resistance of 110 Ω. Bacterial community structures in the organic waste-decomposing solution and MFCs differed from each other. Clonal analyses targeting 16S rRNA genes indicated that bacterial communities in the biofilms on MFCs developed to specific communities dominated by novel Geobacter. Multidimensional scaling analyses based on DGGE profiles revealed that bacterial communities in the organic waste-decomposing solution fluctuated and had no dynamic equilibrium. Bacterial communities on the anolyte in MFCs had a dynamic equilibrium with fluctuations, while those of the biofilm converged to the Geobacter-dominated structure. These bacterial community dynamics of MFCs differed from those of control-MFCs under open circuit conditions. These results suggested that bacterial communities in the anolyte and biofilm have a gentle symbiotic system through electron flow, which resulted in the advance of current density from complex organic waste.
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Speelman, Amy L; Lehnert, Nicolai
2014-04-15
Researchers have completed extensive studies on heme and non-heme iron-nitrosyl complexes, which are labeled {FeNO}(7) in the Enemark-Feltham notation, but they have had very limited success in producing corresponding, one-electron reduced, {FeNO}(8) complexes where a nitroxyl anion (NO(-)) is formally bound to an iron(II) center. These complexes, and their protonated iron(II)-NHO analogues, are proposed key intermediates in nitrite (NO2(-)) and nitric oxide (NO) reducing enzymes in bacteria and fungi. In addition, HNO is known to have a variety of physiological effects, most notably in the cardiovascular system. HNO may also serve as a signaling molecule in mammals. For these functions, iron-containing proteins may mediate the production of HNO and serve as receptors for HNO in vivo. In this Account, we highlight recent key advances in the preparation, spectroscopic characterization, and reactivity of ferrous heme and non-heme nitroxyl (NO(-)/HNO) complexes that have greatly enhanced our understanding of the potential biological roles of these species. Low-spin (ls) heme {FeNO}(7) complexes (S = 1/2) can be reversibly reduced to the corresponding {FeNO}(8) species, which are stable, diamagnetic compounds. Because the reduction is ligand (NO) centered in these cases, it occurs at extremely negative redox potentials that are at the edge of the biologically feasible range. Interestingly, the electronic structures of ls-{FeNO}(7) and ls-{FeNO}(8) species are strongly correlated with very similar frontier molecular orbitals (FMOs) and thermodynamically strong Fe-NO bonds. In contrast, high-spin (hs) non-heme {FeNO}(7) complexes (S = 3/2) can be reduced at relatively mild redox potentials. Here, the reduction is metal-centered and leads to a paramagnetic (S = 1) {FeNO}(8) complex. The increased electron density at the iron center in these species significantly decreases the covalency of the Fe-NO bond, making the reduced complexes highly reactive. In the absence of steric bulk, monomeric high-spin {FeNO}(8) complexes decompose rapidly. Notably, in a recently prepared, dimeric [{FeNO}(7)]2 species, we observed that reduction leads to rapid N-N bond formation and N2O generation, which directly models the reactivity of flavodiiron NO reductases (FNORs). We have also made key progress in the preparation and stabilization of corresponding HNO complexes, {FeNHO}(8), using both heme and non-heme ligand sets. In both cases, we have taken advantage of sterically bulky coligands to stabilize these species. ls-{FeNO}(8) complexes are basic and easily form corresponding ls-{FeNHO}(8) species, which, however, decompose rapidly via disproportionation and H2 release. Importantly, we recently showed that we can suppress this reaction via steric protection of the bound HNO ligand. As a result, we have demonstrated that ls-{FeNHO}(8) model complexes are stable and amenable to spectroscopic characterization. Neither ls-{FeNO}(8) nor ls-{FeNHO}(8) model complexes are active for N-N coupling, and hence, seem unsuitable as reactive intermediates in nitric oxide reductases (NORs). Hs-{FeNO}(8) complexes are more basic than their hs-{FeNO}(7) precursors, but their electronic structure and reactivity is not as well characterized.
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Prokaryotic carbonic anhydrases of Earth's environment.
Kumar, R Siva Sai; Ferry, James G
2014-01-01
Carbonic anhydrase is a metalloenzyme catalyzing the reversible hydration of carbon dioxide to bicarbonate. Five independently evolved classes have been described for which one or more are found in nearly every cell type underscoring the general importance of this ubiquitous enzyme in Nature. The bulk of research to date has centered on the enzymes from mammals and plants with less emphasis on prokaryotes. Prokaryotic carbonic anhydrases play important roles in the ecology of Earth's biosphere including acquisition of CO2 for photosynthesis and the physiology of aerobic and anaerobic prokaryotes decomposing the photosynthate back to CO2 thereby closing the global carbon cycle. This review focuses on the physiology and biochemistry of carbonic anhydrases from prokaryotes belonging to the domains Bacteria and Archaea that play key roles in the ecology of Earth's biosphere.
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Acquisition of HPLC-Mass Spectrometer
2015-08-18
phenyl alanine. This dithiol is coordinated to the iron and all attempts to decompose the ionic coordination complex 56 to recover strictly the...sulfonation process of an asymmetric deprotonation providing a lithium complex with sparteine. This reaction scheme will also direct stereochemistry of...currently used in ointments for treatment of pain and inflammation. Capsaicin shows promise as an effective anti-cancer nutritional agent and
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Diversity of Riparian Plants among and within Species Shapes River Communities
Jackrel, Sara L.; Wootton, J. Timothy
2015-01-01
Organismal diversity among and within species may affect ecosystem function with effects transmitting across ecosystem boundaries. Whether recipient communities adjust their composition, in turn, to maximize their function in response to changes in donor composition at these two scales of diversity is unknown. We use small stream communities that rely on riparian subsidies as a model system. We used leaf pack experiments to ask how variation in plants growing beside streams in the Olympic Peninsula of Washington State, USA affects stream communities via leaf subsidies. Leaves from red alder (Alnus rubra), vine maple (Acer cinereus), bigleaf maple (Acer macrophyllum) and western hemlock (Tsuga heterophylla) were assembled in leaf packs to contrast low versus high diversity, and deployed in streams to compare local versus non-local leaf sources at the among and within species scales. Leaves from individuals within species decomposed at varying rates; most notably thin leaves decomposed rapidly. Among deciduous species, vine maple decomposed most rapidly, harbored the least algal abundance, and supported the greatest diversity of aquatic invertebrates, while bigleaf maple was at the opposite extreme for these three metrics. Recipient communities decomposed leaves from local species rapidly: leaves from early successional plants decomposed rapidly in stream reaches surrounded by early successional forest and leaves from later successional plants decomposed rapidly adjacent to later successional forest. The species diversity of leaves inconsistently affected decomposition, algal abundance and invertebrate metrics. Intraspecific diversity of leaf packs also did not affect decomposition or invertebrate diversity. However, locally sourced alder leaves decomposed more rapidly and harbored greater levels of algae than leaves sourced from conspecifics growing in other areas on the Olympic Peninsula, but did not harbor greater aquatic invertebrate diversity. In contrast to alder, local intraspecific differences via decomposition, algal or invertebrate metrics were not observed consistently among maples. These results emphasize that biodiversity of riparian subsidies at the within and across species scale have the potential to affect aquatic ecosystems, although there are complex species-specific effects. PMID:26539714
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Diversity of Riparian Plants among and within Species Shapes River Communities.
Jackrel, Sara L; Wootton, J Timothy
2015-01-01
Organismal diversity among and within species may affect ecosystem function with effects transmitting across ecosystem boundaries. Whether recipient communities adjust their composition, in turn, to maximize their function in response to changes in donor composition at these two scales of diversity is unknown. We use small stream communities that rely on riparian subsidies as a model system. We used leaf pack experiments to ask how variation in plants growing beside streams in the Olympic Peninsula of Washington State, USA affects stream communities via leaf subsidies. Leaves from red alder (Alnus rubra), vine maple (Acer cinereus), bigleaf maple (Acer macrophyllum) and western hemlock (Tsuga heterophylla) were assembled in leaf packs to contrast low versus high diversity, and deployed in streams to compare local versus non-local leaf sources at the among and within species scales. Leaves from individuals within species decomposed at varying rates; most notably thin leaves decomposed rapidly. Among deciduous species, vine maple decomposed most rapidly, harbored the least algal abundance, and supported the greatest diversity of aquatic invertebrates, while bigleaf maple was at the opposite extreme for these three metrics. Recipient communities decomposed leaves from local species rapidly: leaves from early successional plants decomposed rapidly in stream reaches surrounded by early successional forest and leaves from later successional plants decomposed rapidly adjacent to later successional forest. The species diversity of leaves inconsistently affected decomposition, algal abundance and invertebrate metrics. Intraspecific diversity of leaf packs also did not affect decomposition or invertebrate diversity. However, locally sourced alder leaves decomposed more rapidly and harbored greater levels of algae than leaves sourced from conspecifics growing in other areas on the Olympic Peninsula, but did not harbor greater aquatic invertebrate diversity. In contrast to alder, local intraspecific differences via decomposition, algal or invertebrate metrics were not observed consistently among maples. These results emphasize that biodiversity of riparian subsidies at the within and across species scale have the potential to affect aquatic ecosystems, although there are complex species-specific effects.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Pace, Sara; Ceballos, Shannon J.; Harrold, Duff
Our aims were to identify thermophilic microbial communities that degrade green waste in the presence of the ionic liquids (IL) tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate and examine preservation methods for IL-tolerant communities. High-solids incubations with stepwise increases in IL concentration were conducted to enrich for thermophilic IL-tolerant communities that decomposed green waste. 16S rRNA sequencing of enriched communities revealed microorganisms capable of tolerating high levels of IL. Furthermore, cryogenic preservation of enriched communities reduced the IL tolerance of the community and decreased the relative abundance of IL-tolerant organisms. The use of cryoprotectants did not have an effect on microbial activitymore » on green waste of the stored community. A successful approach was developed to enrich communities that decompose green waste in thermophilic high-solids environments in the presence of IL. Alternative community storage and revival methods are necessary for maintenance and recovery of IL-tolerant communities. The enriched communities provide a targeted source of enzymes for the bioconversion of IL-pretreated green waste for conversion to biofuels.« less
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Complementary symbiont contributions to plant decomposition in a fungus-farming termite
Hu, Haofu; Li, Cai; Chen, Zhensheng; Otani, Saria; Nygaard, Sanne; Nobre, Tania; Klaubauf, Sylvia; Schindler, Philipp M.; Hauser, Frank; Pan, Hailin; Yang, Zhikai; Sonnenberg, Anton S. M.; de Beer, Z. Wilhelm; Zhang, Yong; Wingfield, Michael J.; Grimmelikhuijzen, Cornelis J. P.; de Vries, Ronald P.; Korb, Judith; Aanen, Duur K.; Wang, Jun; Boomsma, Jacobus J.; Zhang, Guojie
2014-01-01
Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate. PMID:25246537
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A parallelized binary search tree
USDA-ARS?s Scientific Manuscript database
PTTRNFNDR is an unsupervised statistical learning algorithm that detects patterns in DNA sequences, protein sequences, or any natural language texts that can be decomposed into letters of a finite alphabet. PTTRNFNDR performs complex mathematical computations and its processing time increases when i...
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Wieczorek, Andrew S; Martin, Vincent J J
2012-12-15
The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.
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NASA Astrophysics Data System (ADS)
Yokoyama, Kenji; Arishima, Koichi; Sukegawa, Ken
1994-07-01
Photorefractive polymers with the same electro-optic effect were fabricated to investigate the photorefractive effects in different photoconductive systems. The photoconduction in the polymers was varied by the addition of squarylium dye to diethylaminobenzaldehyde-diphenylhydrazone (DEH), by the formation of a charge-transfer complex between tetracyanoquinodimethane and DEH, and by the thermal decomposition of DEH. The largest photorefractive effect was observed in the thermally decomposed polymer among these polymers. A diffraction efficiency of 1.1% and a beam-coupling gain coefficient of 10 cm-1 were achieved in a 34.9 V/μm dc electric field.
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Huang, Yuhong; Busk, Peter Kamp; Lange, Lene
2015-06-01
Specific enzymes from plant-pathogenic microbes demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilization. The secreted lignocellulolytic enzymes of Fusarium species have not been investigated comprehensively, however. In this study we compared cellulose and hemicellulose-degrading enzymes of classical fungal enzyme producers with those of Fusarium species. The results indicated that Fusarium species are robust cellulose and hemicellulose degraders. Wheat bran, carboxymethylcellulose and xylan-based growth media induced a broad spectrum of lignocellulolytic enzymes in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified, respectively, into GH10 subfamily 1, subfamily 3 and GH11 subfamily 1. These xylanases were successfully expressed in the PichiaPink™ system with the following properties: the purified recombinant XYL10A had interesting high specific activity; XYL10B was active at alkaline conditions with both endo-1,4-β-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass. Copyright © 2015 Elsevier Inc. All rights reserved.
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A meta-analysis of soil exoenzyme responses to simulated climate change
NASA Astrophysics Data System (ADS)
Gebhardt, M.; Espinosa, N. J.; Blankinship, J. C.; Gallery, R. E.
2017-12-01
Microorganisms produce extracellular enzymes to decompose plant matter and drive biogeochemical transformations in soils. Climate change factors, such as warming and altered precipitation patterns, can impact enzyme activity through both direct and indirect mechanisms. Although many individual studies have examined how soil exoenzyme activities respond to climate change manipulations, there is disagreement surrounding the direction of these responses. We performed a synthesis of published studies to examine the influence of warming and altered precipitation on microbial exoenzyme activity. We found that warming increased enzyme activity with a more pronounced effect for oxidative relative to hydrolytic enzymes. Reduced precipitation consistently decreased exoenzyme activity. These responses, however, varied by season, biome, and enzyme type. The majority of studies fitting our criteria (e.g., experiments lasting a minimum of one growing season, paired treatments and controls) were located in North America and Europe. Inferences from this analysis therefore exclude many important ecosystems such as hyper-arid, wetlands, and artic systems. Carbon degrading enzyme activities were less sensitive to climate change manipulations when compared to phosphorus and nitrogen degrading enzyme activities. Linking enzyme activity to biogeochemical processes requires concomitant measurements of organic and inorganic carbon pools, mineralogy, nutrients, microbial biomass and community structure, and heterotrophic respiration within individual studies. Furthermore, linking these parameters to climate and environmental factors will require a comprehensive and consistent inclusion of biotic and abiotic variables among researchers and experiments. Globally, soils contain the largest carbon pools. Understanding the impacts of large-scale perturbations on soil enzyme activity will help to constrain predictions on the fate of biogeochemical transformations and improve model projections.
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Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei
2017-06-20
A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.
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Enzymatic mechanisms of soil-carbon response to temperature on Mt. Kilimanjaro
NASA Astrophysics Data System (ADS)
Blagodatskaya, Evgenia; Blagodatskiy, Sergey; Kuzyakov, Yakov
2016-04-01
Short-term acceleration of soil organic matter (SOM) decomposition by increasing temperature contradicts the acclimation observed in long-term studies. We used the unique altitudinal gradient (from colline tropical zone to subalpine zone) on Mt. Kilimanjaro to demonstrate the mechanisms of short- and long-term acclimation of extra- and intracellular enzymes that decompose polymers (cellulose, chitin, phytate) and oxidize monomers (14C-glucose). Basing on Michaelis-Menten kinetics we determined the enzymes affinity to substrate (Km) and mineralization potential of heterotrophic microorganisms (Vmax) 1) for three hydrolytic enzymes: β-1,4-glucosidase, N-acetyl- β -D-glucosaminidase and phosphatase by the application of fluorogenically labeled substrates and 2) for mineralization of 14C-labeled glucose by substrate-dependent respiratory response. Here we show that the amount of available substrate is responsible for temperature sensitivity of hydrolysis of polymers in soil, whereas monomers oxidation to CO2 does not depend on substrate amount and is mainly temperature governed. We also found that substrate affinity of enzymes (which is usually decreases with the temperature) differently responded to warming for the process of depolymerisation versus monomers oxidation. We suggest the mechanism to temperature acclimation based on different temperature sensitivity of enzymes kinetics for hydrolysis of polymers and for monomers oxidation
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Airflow and nanoparticle deposition in a 16-generation tracheobronchial airway model
In order to achieve both manageable simulation and local accuracy of airflow and nanoparticle deposition in a representative human tracheobronchial (TB) region, the complex airway network was decomposed into adjustable triple-bifurcation units, spreading axially and laterally. Gi...
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López-Mondéjar, Ruben; Brabcová, Vendula; Štursová, Martina; Davidová, Anna; Jansa, Jan; Cajthaml, Tomaš; Baldrian, Petr
2018-06-01
Forest soils represent important terrestrial carbon (C) pools where C is primarily fixed in the plant-derived biomass but it flows further through the biomass of fungi and bacteria before it is lost from the ecosystem as CO 2 or immobilized in recalcitrant organic matter. Microorganisms are the main drivers of C flow in forests and play critical roles in the C balance through the decomposition of dead biomass of different origins. Here, we track the path of C that enters forest soil by following respiration, microbial biomass production, and C accumulation by individual microbial taxa in soil microcosms upon the addition of 13 C-labeled biomass of plant, fungal, and bacterial origin. We demonstrate that both fungi and bacteria are involved in the assimilation and mineralization of C from the major complex sources existing in soil. Decomposer fungi are, however, better suited to utilize plant biomass compounds, whereas the ability to utilize fungal and bacterial biomass is more frequent among bacteria. Due to the ability of microorganisms to recycle microbial biomass, we suggest that the decomposer food web in forest soil displays a network structure with loops between and within individual pools. These results question the present paradigms describing food webs as hierarchical structures with unidirectional flow of C and assumptions about the dominance of fungi in the decomposition of complex organic matter.
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Ionic complexation of N 2O 4 by 18-crown-6
NASA Astrophysics Data System (ADS)
Ricard, S.; Audet, P.; Savoie, R.
1988-08-01
An ionic complex has been obtained from N 2O 4 in the presence of the macrocyclic ether 18-crown-6. This crystalline compound has been shown from its Raman spectrum to have the formula NO +·crown·H(NO 3) 2-, with the nitrosonium ion closely associated with the crown ether rather than with the hydrogen dinitrate accompanying ion. This adduct decomposes readily in moist air to give the known complex (HNO 3·H 2O) 2·crown.
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Characterising Complex Enzyme Reaction Data
Rahman, Syed Asad; Thornton, Janet M.
2016-01-01
The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640
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The earthworm gastrointestinal effect on the release of organic bound residues in soils
NASA Astrophysics Data System (ADS)
Du, J. H.
2018-03-01
Earthworm activities promote the release of bound residues and the digestive activities of earthworms contribute to the process. Earthworm digestive effects on bound residues can be divided into physical and chemical effects. Physical effects include gastrointestinal abrasion and mixing. The abrasion of soil and litter residues in earthworm gizzards and intestine can grind the food into fine particles, which increase the contact surface with microbial and promote the desorption of bound residues. Chemical effects are attributed to the secreted surfactant substances and digestive enzymes. The surfactants, especially at levels that lead to micellization, can enhance the desorption process of the organic contaminants that sored in the soil. The enzymes in earthworm digestive tracts can decompose the humus in soil, which may promote the release of organic residues that bind with humus.
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Membrane extraction with thermodynamically unstable diphosphonic acid derivatives
Horwitz, Earl Philip; Gatrone, Ralph Carl; Nash, Kenneth LaVerne
1997-01-01
Thermodynamically-unstable complexing agents which are diphosphonic acids and diphosphonic acid derivatives (or sulphur containing analogs), like carboxyhydroxymethanediphosphonic acid and vinylidene-1,1-diphosphonic acid, are capable of complexing with metal ions, and especially metal ions in the II, III, IV, V and VI oxidation states, to form stable, water-soluble metal ion complexes in moderately alkaline to highly-acidic media. However, the complexing agents can be decomposed, under mild conditions, into non-organic compounds which, for many purposes are environmentally-nondamaging compounds thereby degrading the complex and releasing the metal ion for disposal or recovery. Uses for such complexing agents as well as methods for their manufacture are also described.
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Serezhenkov, Vladimir A; Timoshin, Alexander A; Orlova, Tsvetina R; Mikoyan, Vasak D; Kubrina, Lioudmila N; Poltorakov, Alexander P; Ruuge, Enno K; Sanina, Natalia A; Vanin, Anatoly F
2008-05-01
EPR studies have shown that water-soluble mononitrosyl iron complexes with N-methyl-d-glucamine dithiocarbamate (MNIC-MGD) (3 micromol) injected to intact mice were decomposed virtually completely within 1h. The total content of MNIC-MGD in animal urine did not exceed 30 nmol/ml. In the liver, a small amount of MNIC-MGD were converted into dinitrosyl iron complexes (30 nmol/g of liver tissue). The same was observed in intact rabbits in which MNIC-MGD formation was induced by endogenous or exogenous NO binding to NO traps, viz., iron complexes with MGD. In mice, the content of MNIC-MGD in urine samples did not change after bacterial lipopolysaccharide-induced expression of iNOS. It was supposed that MNIC-MGD decomposition in intact animals was largely due to the release of NO from the complexes and its further transfer to other specific acceptors. In mice with iNOS expression, the main contribution to MNIC-MGD decomposition was made by superoxide ions whose destructive effect is mediated by an oxidative mechanism. This effect could fully compensate the augmented synthesis of MNIC-MGD involving endogenous NO whose production was supported by iNOS. Water-soluble dinitrosyl iron complexes (DNIC) with various thiol-containing ligands and thiosulfate injected to intact mice were also decomposed; however, in this case the effect was less pronounced than in the case of MNIC-MGD. It was concluded that DNIC decomposition was largely due to the oxidative effect of superoxide ions on these complexes.
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Petri net model for analysis of concurrently processed complex algorithms
NASA Technical Reports Server (NTRS)
Stoughton, John W.; Mielke, Roland R.
1986-01-01
This paper presents a Petri-net model suitable for analyzing the concurrent processing of computationally complex algorithms. The decomposed operations are to be processed in a multiple processor, data driven architecture. Of particular interest is the application of the model to both the description of the data/control flow of a particular algorithm, and to the general specification of the data driven architecture. A candidate architecture is also presented.
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McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.
2016-01-01
The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034
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An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.
Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun
2016-01-01
Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.
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Mauracher, Andreas; Schöbel, Harald; Ferreira da Silva, Filipe; Edtbauer, Achim; Mitterdorfer, Christian; Denifl, Stephan; Märk, Tilmann D; Illenberger, Eugen; Scheier, Paul
2009-10-01
Electron attachment to the explosive trinitrotoluene (TNT) embedded in Helium droplets (TNT@He) generates the non-decomposed complexes (TNT)(n)(-), but no fragment ions in the entire energy range 0-12 eV. This strongly contrasts the behavior of single TNT molecules in the gas phase at ambient temperatures, where electron capture leads to a variety of different fragmentation products via different dissociative electron attachment (DEA) reactions. Single TNT molecules decompose by attachment of an electron at virtually no extra energy reflecting the explosive nature of the compound. The complete freezing of dissociation intermediates in TNT embedded in the droplet is explained by the particular mechanisms of DEA in nitrobenzenes, which is characterized by complex rearrangement processes in the transient negative ion (TNI) prior to decomposition. These mechanisms provide the condition for effective energy withdrawal from the TNI into the dissipative environment thereby completely suppressing its decomposition.
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Franco Cairo, João P L; Carazzolle, Marcelo F; Leonardo, Flávia C; Mofatto, Luciana S; Brenelli, Lívia B; Gonçalves, Thiago A; Uchima, Cristiane A; Domingues, Romênia R; Alvarez, Thabata M; Tramontina, Robson; Vidal, Ramon O; Costa, Fernando F; Costa-Leonardo, Ana M; Paes Leme, Adriana F; Pereira, Gonçalo A G; Squina, Fabio M
2016-01-01
Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi . Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi . Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.
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Franco Cairo, João P. L.; Carazzolle, Marcelo F.; Leonardo, Flávia C.; Mofatto, Luciana S.; Brenelli, Lívia B.; Gonçalves, Thiago A.; Uchima, Cristiane A.; Domingues, Romênia R.; Alvarez, Thabata M.; Tramontina, Robson; Vidal, Ramon O.; Costa, Fernando F.; Costa-Leonardo, Ana M.; Paes Leme, Adriana F.; Pereira, Gonçalo A. G.; Squina, Fabio M.
2016-01-01
Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes. PMID:27790186
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Sinsabaugh, R. L.; Carreiro, M.M.; Repert, D.A.
2002-01-01
Decomposition of plant material is a complex process that requires interaction among a diversity of microorganisms whose presence and activity is subject to regulation by a wide range of environmental factors. Analysis of extracellular enzyme activity (EEA) provides a way to relate the functional organization of microdecomposer communities to environmental variables. In this study, we examined EEA in relation to litter composition and nitrogen deposition. Mesh bags containing senescent leaves of Quercus borealis (red oak), Acer rubrum (red maple) and Cornus florida (flowering dogwood) were placed on forest floor plots in southeastern New York. One-third of the plots were sprayed monthly with distilled water. The other plots were sprayed monthly with NH4NO3 solution at dose rates equivalent to 2 or 8 g N m-2 y-1. Mass loss, litter composition, fungal mass, and the activities of eight enzymes were measured on 13 dates for each litter type. Dogwood was followed for one year, maple for two, oak for three, For each litter type and treatment, enzymatic turnover activities were calculated from regressions of LN (%mass remaining) vs. cumulative activity. The decomposition of dogwood litter was more efficient than that of maple and oak. Maple litter had the lowest fungal mass and required the most enzymatic work to decompose, even though its mass loss rate was twice that of oak. Across litter types, N amendment reduced apparent enzymatic efficiencies and shifted EEA away from N acquisition and toward P acquisition, and away from polyphenol oxidation and toward polysaccharide hydrolysis. The effect of these shifts on decomposition rate varied with litter composition: dogwood was stimulated, oak was inhibited and maple showed mixed effects. The results show that relatively small shifts in the activity of one or two critical enzymes can significantly alter decomposition rates.
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Heath, Caroline; Posner, Mareike G; Aass, Hans C; Upadhyay, Abhishek; Scott, David J; Hough, David W; Danson, Michael J
2007-10-01
The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.
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Saccharification efficiencies of multi-enzyme complexes produced by aerobic fungi.
Badhan, Ajay; Huang, Jiangli; Wang, Yuxi; Abbott, D Wade; Di Falco, Marcos; Tsang, Adrian; McAllister, Tim
2018-05-24
In the present study, we have characterized high molecular weight multi-enzyme complexes in two commercial enzymes produced by Trichoderma reesei (Spezyme CP) and Penicillium funiculosum (Accellerase XC). We successfully identified 146-1000 kDa complexes using Blue native polyacrylamide gel electrophoresis (BN-PAGE) to fractionate the protein profile in both preparations. Identified complexes dissociated into lower molecular weight constituents when loaded on SDS PAGE. Unfolding of the secondary structure of multi-enzyme complexes with trimethylamine (pH >10) suggested that they were not a result of unspecific protein aggregation. Cellulase (CMCase) profiles of extracts of BN-PAGE fractionated protein bands confirmed cellulase activity within the multi-enzyme complexes. A microassay was used to identify protein bands that promoted high levels of glucose release from barley straw. Those with high saccharification yield were subjected to LC-MS analysis to identify the principal enzymatic activities responsible. The results suggest that secretion of proteins by aerobic fungi leads to the formation of high molecular weight multi-enzyme complexes that display activity against carboxymethyl cellulose and barley straw. Copyright © 2018. Published by Elsevier B.V.
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Self-reduction of a copper complex MOD ink for inkjet printing conductive patterns on plastics.
Farraj, Yousef; Grouchko, Michael; Magdassi, Shlomo
2015-01-31
Highly conductive copper patterns on low-cost flexible substrates are obtained by inkjet printing a metal complex based ink. Upon heating the ink, the soluble complex, which is composed of copper formate and 2-amino-2-methyl-1-propanol, decomposes under nitrogen at 140 °C and is converted to pure metallic copper. The decomposition process of the complex is investigated and a suggested mechanism is presented. The ink is stable in air for prolonged periods, with no sedimentation or oxidation problems, which are usually encountered in copper nanoparticle based inks.
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Chenthamara, Komal; Zhang, Jian; Atanasova, Lea; Yang, Dongqing; Miao, Youzhi; Grujic, Marica; Pourmehdi, Shadi; Pretzer, Carina; Kopchinskiy, Alexey G.; Hundley, Hope; Wang, Mei; Aerts, Andrea; Salamov, Asaf; Lipzen, Anna; Barry, Kerrie; Grigoriev, Igor V.; Shen, Qirong; Kubicek, Christian P.
2018-01-01
Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass. PMID:29630596
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Shang, Yanan; Wang, Ziyang; Xu, Xing; Gao, Baoyu; Ren, Zhongfei
2018-08-01
Pure bacteria cell (Azospira sp. KJ) and mixed perchlorate reducing bacteria (MPRB) were employed for decomposing the free perchlorate in water as well as the laden perchlorate on surface of quaternary ammonium wheat residuals (QAWR). Results indicated that perchlorate was decomposed by the Azospira sp. KJ prior to nitrate while MPRB was just the reverse. Bio-reduction of laden perchlorate by Azospira sp. KJ was optimal at pH 8.0. In contrast, bio-reduction of laden perchlorate by MPRB was optimal at pH 7.0. Generally, the rate of perchlorate reduction was controlled by the enzyme activity of PRB. In addition, perchlorate recovery (26.0 mg/g) onto bio-regenerated QAWR by MPRB was observed with a small decrease as compared with that (31.1 mg/g) by Azospira sp. KJ at first 48 h. Basically, this study is expected to offer some different ideas on bio-regeneration of perchlorate-saturated adsorbents using biological process, which may provide the economically alternative to conventional methods. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Pace, Sara; Ceballos, Shannon J.; Harrold, Duff; ...
2016-04-22
Our aims were to identify thermophilic microbial communities that degrade green waste in the presence of the ionic liquids (IL) tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate and examine preservation methods for IL-tolerant communities. High-solids incubations with stepwise increases in IL concentration were conducted to enrich for thermophilic IL-tolerant communities that decomposed green waste. 16S rRNA sequencing of enriched communities revealed microorganisms capable of tolerating high levels of IL. Furthermore, cryogenic preservation of enriched communities reduced the IL tolerance of the community and decreased the relative abundance of IL-tolerant organisms. The use of cryoprotectants did not have an effect on microbial activitymore » on green waste of the stored community. A successful approach was developed to enrich communities that decompose green waste in thermophilic high-solids environments in the presence of IL. Alternative community storage and revival methods are necessary for maintenance and recovery of IL-tolerant communities. The enriched communities provide a targeted source of enzymes for the bioconversion of IL-pretreated green waste for conversion to biofuels.« less
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Chemically generated convective transport in microfluidic system
NASA Astrophysics Data System (ADS)
Shklyaev, Oleg; Das, Sambeeta; Altemose, Alicia; Shum, Henry; Balazs, Anna; Sen, Ayusman
High precision manipulation of small volumes of fluid, containing suspended micron sized objects like cells, viruses, and large molecules, is one of the main goals in designing modern lab-on-a-chip devices which can find a variety of chemical and biological applications. To transport the cargo toward sensing elements, typical microfluidic devices often use pressure driven flows. Here, we propose to use enzymatic chemical reactions which decompose reagent into less dense products and generate flows that can transport particles. Density variations that lead to flow in the assigned direction are created between the place where reagent is fed into the solution and the location where it is decomposed by enzymes attached to the surface of the microchannel. When the reagent is depleted, the fluid motion stops and particles sediment to the bottom. We demonstrate how the choice of chemicals, leading to specific reaction rates, can affect the transport properties. In particular, we show that the intensity of the fluid flow, the final location of cargo, and the time for cargo delivery are controlled by the amount and type of reagent in the system.
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Kong, Xiangshi; Jia, Yanyan; Song, Fuqiang; Tian, Kai; Lin, Hong; Bei, Zhanlin; Jia, Xiuqin; Yao, Bei; Guo, Peng; Tian, Xingjun
2018-02-01
Arbuscular mycorrhizal fungi (AMF) play an important role in litter decomposition. This study investigated how soil nutrient level affected the process. Results showed that AMF colonization had no significant effect on litter decomposition under normal soil nutrient conditions. However, litter decomposition was accelerated significantly under lower nutrient conditions. Soil microbial biomass in decomposition system was significantly increased. Especially, in moderate lower nutrient treatment (condition of half-normal soil nutrient), litters exhibited the highest decomposition rate, AMF hypha revealed the greatest density, and enzymes (especially nitrate reductase) showed the highest activities as well. Meanwhile, the immobilization of nitrogen (N) in the decomposing litter remarkably decreased. Our results suggested that the roles AMF played in ecosystem were largely affected by soil nutrient levels. At normal soil nutrient level, AMF exhibited limited effects in promoting decomposition. When soil nutrient level decreased, the promoting effect of AMF on litter decomposition began to appear, especially on N mobilization. However, under extremely low nutrient conditions, AMF showed less influence on decomposition and may even compete with decomposer microorganisms for nutrients.
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NASA Astrophysics Data System (ADS)
Muche, Simon; Levacheva, Irina; Samsonova, Olga; Biernasiuk, Anna; Malm, Anna; Lonsdale, Richard; Popiołek, Łukasz; Bakowsky, Udo; Hołyńska, Małgorzata
2017-01-01
Studies of the stability of a ligand derived from L-glutamic acid and ortho-vanillin and its new [Cu4] complex are presented. The [Cu4] complex contains a heterocubane [CuII4O4] core and pendant carboxylic groups increasing its solubility in water, also under basic conditions. The stability of the complex in different solvents is confirmed with ESI-MS studies and such experiments as successful recrystallization. The complex is stable also under physiological conditions whereas the ligand is partly decomposed to L-glutamic acid and ortho-vanillin.
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Valence bond and enzyme catalysis: a time to break down and a time to build up.
Sharir-Ivry, Avital; Varatharaj, Rajapandian; Shurki, Avital
2015-05-04
Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Wild, Birgit; Schnecker, Jörg; Alves, Ricardo J. Eloy; Barsukov, Pavel; Bárta, Jiří; Čapek, Petr; Gentsch, Norman; Gittel, Antje; Guggenberger, Georg; Lashchinskiy, Nikolay; Mikutta, Robert; Rusalimova, Olga; Šantrůčková, Hana; Shibistova, Olga; Urich, Tim; Watzka, Margarete; Zrazhevskaya, Galina; Richter, Andreas
2014-01-01
Rising temperatures in the Arctic can affect soil organic matter (SOM) decomposition directly and indirectly, by increasing plant primary production and thus the allocation of plant-derived organic compounds into the soil. Such compounds, for example root exudates or decaying fine roots, are easily available for microorganisms, and can alter the decomposition of older SOM (“priming effect”). We here report on a SOM priming experiment in the active layer of a permafrost soil from the central Siberian Arctic, comparing responses of organic topsoil, mineral subsoil, and cryoturbated subsoil material (i.e., poorly decomposed topsoil material subducted into the subsoil by freeze–thaw processes) to additions of 13C-labeled glucose, cellulose, a mixture of amino acids, and protein (added at levels corresponding to approximately 1% of soil organic carbon). SOM decomposition in the topsoil was barely affected by higher availability of organic compounds, whereas SOM decomposition in both subsoil horizons responded strongly. In the mineral subsoil, SOM decomposition increased by a factor of two to three after any substrate addition (glucose, cellulose, amino acids, protein), suggesting that the microbial decomposer community was limited in energy to break down more complex components of SOM. In the cryoturbated horizon, SOM decomposition increased by a factor of two after addition of amino acids or protein, but was not significantly affected by glucose or cellulose, indicating nitrogen rather than energy limitation. Since the stimulation of SOM decomposition in cryoturbated material was not connected to microbial growth or to a change in microbial community composition, the additional nitrogen was likely invested in the production of extracellular enzymes required for SOM decomposition. Our findings provide a first mechanistic understanding of priming in permafrost soils and suggest that an increase in the availability of organic carbon or nitrogen, e.g., by increased plant productivity, can change the decomposition of SOM stored in deeper layers of permafrost soils, with possible repercussions on the global climate. PMID:25089062
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Membrane extraction with thermodynamically unstable diphosphonic acid derivatives
Horwitz, E.P.; Gatrone, R.C.; Nash, K.L.
1997-10-14
Thermodynamically-unstable complexing agents which are diphosphonic acids and diphosphonic acid derivatives (or sulphur containing analogs), like carboxyhydroxymethanediphosphonic acid and vinylidene-1,1-diphosphonic acid, are capable of complexing with metal ions, and especially metal ions in the II, III, IV, V and VI oxidation states, to form stable, water-soluble metal ion complexes in moderately alkaline to highly-acidic media. However, the complexing agents can be decomposed, under mild conditions, into non-organic compounds which, for many purposes are environmentally-nondamaging compounds thereby degrading the complex and releasing the metal ion for disposal or recovery. Uses for such complexing agents as well as methods for their manufacture are also described. 1 fig.
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Extracting metal ions with diphosphonic acid, or derivative thereof
Horwitz, Earl P.; Gatrone, Ralph C.; Nash, Kenneth L.
1994-01-01
Thermodynamically-unstable complexing agents which are diphosphonic acids and diphosphonic acid derivatives (or sulphur containing analogs), like carboxyhydroxymethanediphosphonic acid and vinylidene-1,1-diphosphonic acid, are capable of complexing with metal ions, and especially metal ions in the II, III, IV, V and VI oxidation states, to form stable, water-soluble metal ion complexes in moderately alkaline to highly-acidic media. However, the complexing agents can be decomposed, under mild conditions, into non-organic compounds which, for many purposes are environmentally-nondamaging compounds thereby degrading the complex and releasing the metal ion for disposal or recovery. Uses for such complexing agents as well as methods for their manufacture are also described.
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Extracting metal ions with diphosphonic acid, or derivative thereof
Horwitz, E.P.; Gatrone, R.C.; Nash, K.L.
1994-07-26
Thermodynamically-unstable complexing agents which are diphosphonic acids and diphosphonic acid derivatives (or sulfur containing analogs), like carboxyhydroxymethanediphosphonic acid and vinylidene-1,1-diphosphonic acid, are capable of complexing with metal ions, and especially metal ions in the II, III, IV, V and VI oxidation states, to form stable, water-soluble metal ion complexes in moderately alkaline to highly-acidic media. However, the complexing agents can be decomposed, under mild conditions, into non-organic compounds which, for many purposes are environmentally-nondamaging compounds thereby degrading the complex and releasing the metal ion for disposal or recovery. Uses for such complexing agents as well as methods for their manufacture are also described. 1 fig.
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Schnecker, Jörg; Wild, Birgit; Hofhansl, Florian; Eloy Alves, Ricardo J.; Bárta, Jiří; Čapek, Petr; Fuchslueger, Lucia; Gentsch, Norman; Gittel, Antje; Guggenberger, Georg; Hofer, Angelika; Kienzl, Sandra; Knoltsch, Anna; Lashchinskiy, Nikolay; Mikutta, Robert; Šantrůčková, Hana; Shibistova, Olga; Takriti, Mounir; Urich, Tim; Weltin, Georg; Richter, Andreas
2014-01-01
Enzyme-mediated decomposition of soil organic matter (SOM) is controlled, amongst other factors, by organic matter properties and by the microbial decomposer community present. Since microbial community composition and SOM properties are often interrelated and both change with soil depth, the drivers of enzymatic decomposition are hard to dissect. We investigated soils from three regions in the Siberian Arctic, where carbon rich topsoil material has been incorporated into the subsoil (cryoturbation). We took advantage of this subduction to test if SOM properties shape microbial community composition, and to identify controls of both on enzyme activities. We found that microbial community composition (estimated by phospholipid fatty acid analysis), was similar in cryoturbated material and in surrounding subsoil, although carbon and nitrogen contents were similar in cryoturbated material and topsoils. This suggests that the microbial community in cryoturbated material was not well adapted to SOM properties. We also measured three potential enzyme activities (cellobiohydrolase, leucine-amino-peptidase and phenoloxidase) and used structural equation models (SEMs) to identify direct and indirect drivers of the three enzyme activities. The models included microbial community composition, carbon and nitrogen contents, clay content, water content, and pH. Models for regular horizons, excluding cryoturbated material, showed that all enzyme activities were mainly controlled by carbon or nitrogen. Microbial community composition had no effect. In contrast, models for cryoturbated material showed that enzyme activities were also related to microbial community composition. The additional control of microbial community composition could have restrained enzyme activities and furthermore decomposition in general. The functional decoupling of SOM properties and microbial community composition might thus be one of the reasons for low decomposition rates and the persistence of 400 Gt carbon stored in cryoturbated material. PMID:24705618
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VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A
2016-12-01
The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao
2014-07-01
Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.
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Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao
2014-07-01
Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.
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Wang, Bing; Liu, Huiling; Cai, Chen; Thabit, Mohamed; Wang, Pu; Li, Guomin; Duan, Ziheng
2016-10-01
The dry mycelium fertilizer (DMF) was produced from penicillin fermentation fungi mycelium (PFFM) following an acid-heating pretreatment to degrade the residual penicillin. In this study, it was applied into soil as fertilizer to investigate its effects on soil properties, phytotoxicity, microbial community composition, enzyme activities, and growth of snap bean in greenhouse. As the results show, pH, total nitrogen, total phosphorus, total potassium, and organic matter of soil with DMF treatments were generally higher than CON treatment. In addition, the applied DMF did not cause heavy metal and residual drug pollution of the modified soil. The lowest GI values (<0.3) were recorded at DMF8 (36 kg DMF/plat) on the first days after applying the fertilizer, indicating that severe phytotoxicity appeared in the DMF8-modified soil. Results of microbial population and enzyme activities illustrated that DMF was rapidly decomposed and the decomposition process significantly affected microbial growth and enzyme activities. The DMF-modified soil phytotoxicity decreased at the late fertilization time. DMF1 was considered as the optimum amount of DMF dose based on principal component analysis scores. Plant height and plant yield of snap bean were remarkably enhanced with the optimum DMF dose.
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Kitagishi, Hiroaki; Kurosawa, Shun; Kano, Koji
2016-11-22
The intramolecular oxidation of ROCH 3 to ROCH 2 OH, where the latter compound spontaneously decomposed to ROH and HCHO, was observed during the reaction of the supramolecular complex (met-hemoCD3) with cumene hydroperoxide in aqueous solution. Met-hemoCD3 is composed of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(III) (Fe III TPPS) and a per-O-methylated β-cyclodextrin dimer having an -OCH 2 PyCH 2 O- linker (Py=pyridine-3,5-diyl). The O=Fe IV TPPS complex was formed by the reaction of met-hemoCD3 with cumene hydroperoxide, and isolated by gel-filtration chromatography. Although the isolated O=Fe IV TPPS complex in the cyclodextrin cage was stable in aqueous solution at 25 °C, it was gradually converted to Fe II TPPS (t 1/2 =7.6 h). This conversion was accompanied by oxidative O-demethylation of an OCH 3 group in the cyclodextrin dimer. The results indicated that hydrogen abstraction by O=Fe IV TPPS from ROCH 3 yields HO-Fe III TPPS and ROCH 2 . . This was followed by radical coupling to afford Fe II TPPS and ROCH 2 OH. The hemiacetal (ROCH 2 OH) immediately decomposed to ROH and HCHO. This study revealed the ability of oxoferryl porphyrin to induce two-electron oxidation. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi
2016-07-01
We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.
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Enhanced cellulose degradation using cellulase-nanosphere complexes.
Blanchette, Craig; Lacayo, Catherine I; Fischer, Nicholas O; Hwang, Mona; Thelen, Michael P
2012-01-01
Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.
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Enhanced Cellulose Degradation Using Cellulase-Nanosphere Complexes
Blanchette, Craig; Lacayo, Catherine I.; Fischer, Nicholas O.; Hwang, Mona; Thelen, Michael P.
2012-01-01
Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production. PMID:22870287
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Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol▿†
Trinh, Cong T.; Li, Johnny; Blanch, Harvey W.; Clark, Douglas S.
2011-01-01
Fermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design an Escherichia coli strain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism of E. coli was decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growth E. coli cannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesigned E. coli strain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producing E. coli strain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production. PMID:21642415
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.
In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less
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Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; ...
2016-06-08
In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less
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Lloyd, Julie C.; Raines, Christine A.
2011-01-01
In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632
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Howard, Thomas P; Lloyd, Julie C; Raines, Christine A
2011-07-01
In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.
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Temporal Fine Structure and Applications to Cochlear Implants
ERIC Educational Resources Information Center
Li, Xing
2013-01-01
Complex broadband sounds are decomposed by the auditory filters into a series of relatively narrowband signals, each of which conveys information about the sound by time-varying features. The slow changes in the overall amplitude constitute envelope, while the more rapid events, such as zero crossings, constitute temporal fine structure (TFS).…
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Insect-mediated nitrogen dynamics in decomposing wood
Michael D. Ulyshen
2015-01-01
1.Wood decomposition is characterised by complex and poorly understood nitrogen (N) dynamics with unclear implications for forest nutrient cycling and productivity.Wood-dwelling microbes have developed unique strategies for coping with the N limitations imposed by their substrate, including the translocation of N into wood by cord-forming fungi and the fixation of...
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Prospects and challenges for fungal metatranscriptomics of complex communities
Cheryl R. Kuske; Cedar N. Hesse; Jean F. Challacombe; Daniel Cullen; Joshua R. Herr; Rebecca C. Mueller; Adrian Tsang; Rytas Vilgalys
2015-01-01
The ability to extract and purify messenger RNA directly from plants, decomposing organic matter and soil, followed by highthroughput sequencing of the pool of expressed genes, has spawned the emerging research area of metatranscriptomics. Each metatranscriptome provides a snapshot of the composition and relative abundance of actively transcribed genes, and thus...
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Effects of Temperature on Microstructure and Wear of Salt Bath Nitrided 17-4PH Stainless Steel
NASA Astrophysics Data System (ADS)
Wang, Jun; Lin, Yuanhua; Fan, Hongyuan; Zeng, Dezhi; Peng, Qian; Shen, Baoluo
2012-08-01
Salt bath nitriding of 17-4 PH martensitic precipitation hardening stainless steels was conducted at 610, 630, and 650 °C for 2 h using a complex salt bath heat-treatment, and the properties of the nitrided surface were systematically evaluated. Experimental results revealed that the microstructure and phase constituents of the nitrided surface alloy are highly process condition dependent. When 17-4PH stainless steel was subjected to complex salt bathing nitriding, the main phase of the nitrided layer was expanded martensite (α'), expanded austenite (γN), CrN, Fe4N, and (Fe,Cr) x O y . In the sample nitrided above 610 °C, the expanded martensite transformed into expanded austenite. But in the sample nitrided at 650 °C, the expanded austenite decomposed into αN and CrN. The decomposed αN then disassembled into CrN and alpha again. The nitrided layer depth thickened intensively with the increasing nitriding temperature. The activation energy of nitriding in this salt bath was 125 ± 5 kJ/mol.
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Electrical, structural and thermal studies of carbon nanotubes from natural legume seeds: kala chana
NASA Astrophysics Data System (ADS)
Ranu, Rachana; Chauhan, Yatishwar; Singh, Pramod K.; Bhattacharya, B.; Tomar, S. K.
2016-12-01
Carbon nanotubes (CNTs) are the carbon materials measured at nanoscale level and they are defined in two types according to the number of concentric layers, i.e. single-layer tube is single-walled nanotubes, while multi-layer tube structure is called multi-walled nanotubes. The green method synthesis for the preparation of CNTs begins with the smashing of legume seeds kala chana, and then they form complex with cobalt salt. Desiccation of the complex compound forms cobalt salt and seed protein. The complex is then decomposed at 625 °C in muffle furnace for 20 min. Purification of the decomposed sample is done through acid wash treatment and dried in vacuum oven. The confirmations of CNTs are done by nuclear magnetic resonance and Fourier transform infrared, which analyzes the denatured protein, reacted to the metal salt. X-Ray diffraction determines the MWNTs with transmission electron microscope (TEM) reports the network structure of CNTs. thermal gravimetric analysis (TGA)-differential thermal analysis (DTA)-thermogravimetric analysis (DTG) tests the amount of sample under thermal treatment. Vibrating sample magnetometer determines the paramagnetic nature of CNTs. CNTs thus prepared can be used in mechanical fields, in solar cells, in electronics fields, etc. because of their multidisciplinary properties. The synthesized CNTs are eco-friendly in nature, prepared by the legume seed natural precursor.
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Taniguchi, Hironori; Sungwallek, Sathidaphorn; Chotchuang, Phatcharin; Okano, Kenji
2017-01-01
ABSTRACT NAD (NAD+) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD+ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD+ de novo synthesis pathway. Herein, we report the importance of the NAD+ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD+ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (Km of 17 μM, kcat of 50 s−1, kcat/Km of 3.0 × 103 s−1 · mM−1). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD+ salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD+ and NADH. IMPORTANCE NAD+ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD+ decomposition is, in general, more rapid. This study emphasizes that NAD+ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures. PMID:28630126
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hesse, Cedar N.; Mueller, Rebecca C.; Vuyisich, Momchilo
Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes)more » in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.« less
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Taniguchi, Hironori; Sungwallek, Sathidaphorn; Chotchuang, Phatcharin; Okano, Kenji; Honda, Kohsuke
2017-09-01
NAD (NAD + ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD + from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD + de novo synthesis pathway. Herein, we report the importance of the NAD + salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD + salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide ( K m of 17 μM, k cat of 50 s -1 , k cat / K m of 3.0 × 10 3 s -1 · mM -1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD + salvage pathway in T. thermophilus Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD + and NADH. IMPORTANCE NAD + and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD + decomposition is, in general, more rapid. This study emphasizes that NAD + instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures. Copyright © 2017 American Society for Microbiology.
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Assessment of derelict soil quality: Abiotic, biotic and functional approaches.
Vincent, Quentin; Auclerc, Apolline; Beguiristain, Thierry; Leyval, Corinne
2018-02-01
The intensification and subsequent closing down of industrial activities during the last century has left behind large surfaces of derelict lands. Derelict soils have low fertility, can be contaminated, and many of them remain unused. However, with the increasing demand of soil surfaces, they might be considered as a resource, for example for non-food biomass production. The study of their physico-chemical properties and of their biodiversity and biological activity may provide indications for their potential re-use. The objective of our study was to investigate the quality of six derelict soils, considering abiotic, biotic, and functional parameters. We studied (i) the soil bacteria, fungi, meso- and macro-fauna and plant communities of six different derelict soils (two from coking plants, one from a settling pond, two constructed ones made from different substrates and remediated soil, and an inert waste storage one), and (ii) their decomposition function based on the decomposer trophic network, enzyme activities, mineralization activity, and organic pollutant degradation. Biodiversity levels in these soils were high, but all biotic parameters, except the mycorrhizal colonization level, discriminated them. Multivariate analysis showed that biotic parameters co-varied more with fertility proxies than with soil contamination parameters. Similarly, functional parameters significantly co-varied with abiotic parameters. Among functional parameters, macro-decomposer proportion, enzyme activity, average mineralization capacity, and microbial polycyclic aromatic hydrocarbon degraders were useful to discriminate the soils. We assessed their quality by combining abiotic, biotic, and functional parameters: the compost-amended constructed soil displayed the highest quality, while the settling pond soil and the contaminated constructed soil displayed the lowest. Although differences among the soils were highlighted, this study shows that derelict soils may provide a biodiversity ecosystem service and are functional for decomposition. Copyright © 2017 Elsevier B.V. All rights reserved.
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Microbial decomposition is highly sensitive to leaf litter emersion in a permanent temperate stream.
Mora-Gómez, Juanita; Duarte, Sofia; Cássio, Fernanda; Pascoal, Cláudia; Romaní, Anna M
2018-04-15
Drought frequency and intensity in some temperate regions are forecasted to increase under the ongoing global change, which might expose permanent streams to intermittence and have severe repercussions on stream communities and ecosystem processes. In this study, we investigated the effect of drought duration on microbial decomposition of Populus nigra leaf litter in a temperate permanent stream (Oliveira, NW Portugal). Specifically, we measured the response of the structural (assemblage composition, bacterial and fungal biomass) and functional (leaf litter decomposition, extracellular enzyme activities (EEA), and fungal sporulation) parameters of fungal and bacterial communities on leaf litter exposed to emersion during different time periods (7, 14 and 21d). Emersion time affected microbial assemblages and litter decomposition, but the response differed among variables. Leaf decomposition rates and the activity of β-glucosidase, cellobiohydrolase and phosphatase were gradually reduced with increasing emersion time, while β-xylosidase reduction was similar when emersion last for 7 or more days, and the phenol oxidase reduction was similar at 14 and 21days of leaf emersion. Microbial biomass and fungal sporulation were reduced after 21days of emersion. The structure of microbial assemblages was affected by the duration of the emersion period. The shifts in fungal assemblages were correlated with a decreased microbial capacity to degrade lignin and hemicellulose in leaf litter exposed to emersion. Additionally, some resilience was observed in leaf litter mass loss, bacterial biomass, some enzyme activities and structure of fungal assemblages. Our study shows that drought can strongly alter structural and functional aspects of microbial decomposers. Therefore, the exposure of leaf litter to increasing emersion periods in temperate streams is expected to affect decomposer communities and overall decomposition of plant material by decelerating carbon cycling in streams. Copyright © 2017 Elsevier B.V. All rights reserved.
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Moparthi, Vamsi K; Kumar, Brijesh; Al-Eryani, Yusra; Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia
2014-01-01
NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na(+)/H(+) antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity. © 2013. Published by Elsevier B.V. All rights reserved.
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Compson, Zacchaeus G; Adams, Kenneth J; Edwards, Joeseph A; Maestas, Jesse M; Whitham, Thomas G; Marks, Jane C
2013-10-01
Reciprocal subsidies between rivers and terrestrial habitats are common where terrestrial leaf litter provides energy to aquatic invertebrates while emerging aquatic insects provide energy to terrestrial predators (e.g., birds, lizards, spiders). We examined how aquatic insect emergence changed seasonally with litter from two foundation riparian trees, whose litter often dominates riparian streams of the southwestern United States: Fremont (Populus fremontii) and narrowleaf (Populus angustifolia) cottonwood. P. fremontii litter is fast-decomposing and lower in defensive phytochemicals (i.e., condensed tannins, lignin) relative to P. angustifolia. We experimentally manipulated leaf litter from these two species by placing them in leaf enclosures with emergence traps attached in order to determine how leaf type influenced insect emergence. Contrary to our initial predictions, we found that packs with slow-decomposing leaves tended to support more emergent insects relative to packs with fast-decomposing leaves. Three findings emerged. Firstly, abundance (number of emerging insects m(-2) day(-1)) was 25% higher on narrowleaf compared to Fremont leaves for the spring but did not differ in the fall, demonstrating that leaf quality from two dominant trees of the same genus yielded different emergence patterns and that these patterns changed seasonally. Secondly, functional feeding groups of emerging insects differed between treatments and seasons. Specifically, in the spring collector-gatherer abundance and biomass were higher on narrowleaf leaves, whereas collector-filterer abundance and biomass were higher on Fremont leaves. Shredder abundance and biomass were higher on narrowleaf leaves in the fall. Thirdly, diversity (Shannon's H') was higher on Fremont leaves in the spring, but no differences were found in the fall, showing that fast-decomposing leaves can support a more diverse, complex emergent insect assemblage during certain times of the year. Collectively, these results challenge the notion that leaf quality is a simple function of decomposition, suggesting instead that aquatic insects benefit differentially from different leaf types, such that some use slow-decomposing litter for habitat and its temporal longevity and others utilize fast-decomposing litter with more immediate nutrient release.
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Koroiva, R; Souza, C W O; Toyama, D; Henrique-Silva, F; Fonseca-Gessner, A A
2013-04-02
We analyzed the digestive activity of the enzymes that digest cellulose and hemicellulose and the bacterial community that is capable of hydrolyzing wood compounds in the digestive tracts of Stenochironomus (Diptera: Chironomidae) larvae, which are miners of decomposing submerged tree and bush branches. Based on quantification of reducing sugars, these larvae have a limited capacity for cellulose degradation but a good capacity for xylan hydrolysis. We isolated 31 types of colonies from two larval morphotypes, of which 19 tested positive for the capacity to hydrolyze at least one of the four substrates that were used as the main carbon source in the culture media. Their woody compound degradation capacity was assessed using colorimetric tests. The bacteria were identified by the analysis of the 16S rRNA gene. None of the bacteria were capable of degrading lignin. The genus Pseudomonas had the greatest species richness; Bacillus spp exhibited the greatest capacity for degrading the different substrates, and Sphingobium was found in both morphotypes. Microorganisms participate in the degradation of wood consumed by Stenochironomus larvae. This is the first report of lignocellulolytic bacteria and enzymes in the digestive tracts of mining chironomids.
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Han, Xu; Cheng, Zhihui; Meng, Huanwen
2012-01-01
The garlic stalk is a byproduct of garlic production and normally abandoned or burned, both of which cause environmental pollution. It is therefore appropriate to determine the conditions of efficient decomposition, and equally appropriate to determine the impact of this decomposition on soil properties. In this study, the soil properties, enzyme activities and nutrient dynamics associated with the decomposition of garlic stalk at different temperatures, concentrations and durations were investigated. Stalk decomposition significantly increased the values of soil pH and electrical conductivity. In addition, total nitrogen and organic carbon concentration were significantly increased by decomposing stalks at 40°C, with a 5∶100 ratio and for 10 or 60 days. The highest activities of sucrase, urease and alkaline phosphatase in soil were detected when stalk decomposition was performed at the lowest temperature (10°C), highest concentration (5∶100), and shortest duration (10 or 20 days). The evidence presented here suggests that garlic stalk decomposition improves the quality of soil by altering the value of soil pH and electrical conductivity and by changing nutrient dynamics and soil enzyme activity, compared to the soil decomposition without garlic stalks. PMID:23226411
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A novel xylan degrading β-D-xylosidase: purification and biochemical characterization.
Michelin, Michele; Peixoto-Nogueira, Simone C; Silva, Tony M; Jorge, João A; Terenzi, Héctor F; Teixeira, José A; Polizeli, Maria de Lourdes T M
2012-11-01
Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. β-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl₂ (28 %) and MgCl₂ (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⁻¹ respectively, and to a lesser extent p-nitrophenyl-β-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.
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Stone, S R; Morrison, J F
1983-06-29
Binding theory has been developed for the reaction of an ionizing enzyme with an ionizing ligand. Consideration has been given to the most general scheme in which all possible reactions and interconversions occur as well as to schemes in which certain interactions do not take place. Equations have been derived in terms of the variation of the apparent dissociation constant (Kiapp) as a function of pH. These equations indicate that plots of pKiapp against pH can be wave-, half-bell- or bell-shaped according to the reactions involved. A wave is obtained whenever there is formation of the enzyme-ligand complexes, ionized enzyme . ionized ligand and protonated enzyme . protonated ligand. The additional formation of singly protonated enzyme-ligand complexes does not affect the wave form of the plot, but can influence the shape of the overall curve. The formation of either ionized enzyme . ionized ligand or protonated enzyme . protonated ligand, with or without singly protonated enzyme-ligand species, gives rise to a half-bell-shaped plot. If only singly protonated enzyme-ligand complexes are formed the plots are bell-shaped, but it is not possible to deduce the ionic forms of the reactants that participate in complex formation. Depending on the reaction pathways, true values for the ionization and dissociation constants may or may not be determined.
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Actinomycetes benefaction role in soil and plant health.
Bhatti, Asma Absar; Haq, Shamsul; Bhat, Rouf Ahmad
2017-10-01
Actinomycetes are aerobic, spore forming gram-positive bacteria, belonging to the order actinomycetales characterized with substrate and aerial mycelium growth. They are the most abundant organisms that form thread-like filaments in the soil and are responsible for characteristically "earthy" smell of freshly turned healthy soil. They play major roles in the cycling of organic matter; inhibit the growth of several plant pathogens in the rhizosphere and decompose complex mixtures of polymer in dead plant, animal and fungal material results in production of many extracellular enzymes which are conductive to crop production. The major contribution in biological buffering of soils, biological control of soil environments by nitrogen fixation and degradation of high molecular weight compounds like hydrocarbons in the polluted soils are remarkable characteristics of actinomycetes. Besides this, they are known to improve the availability of nutrients, minerals, enhance the production of metabolites and promote plant growth regulators. Furthermore, actinobacteria do not contaminate the environment instead, they help sustainably in improving soil health by formation and stabilization of compost piles, formation of stable humus and combine with other soil microorganisms in breaking down the tough plant residues such as cellulose and animal residues to maintain the biotic equilibrium of soil by cooperating with nutrient cycling. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Tian, Baoyu; Wang, Chunxiang; Lv, Ruirui; Zhou, Junxiong; Li, Xin; Zheng, Yi; Jin, Xiangyu; Wang, Mengli; Ye, Yongxia; Huang, Xinyi; Liu, Ping
2014-01-01
The study aims to investigate fungal community structures and dynamic changes in forest soil lignocellulose-degrading process. rRNA gene clone libraries for the samples collected in different stages of lignocellulose degradation process were constructed and analyzed. A total of 26 representative RFLP types were obtained from original soil clone library, including Mucoromycotina (29.5%), unclassified Zygomycetes (33.5%), Ascomycota (32.4%), and Basidiomycota (4.6%). When soil accumulated with natural lignocellulose, 16 RFLP types were identified from 8-day clone library, including Basidiomycota (62.5%), Ascomycota (36.1%), and Fungi incertae sedis (1.4%). After enrichment for 15 days, identified 11 RFLP types were placed in 3 fungal groups: Basidiomycota (86.9%), Ascomycota (11.5%), and Fungi incertae sedis (1.6%). The results showed richer, more diversity and abundance fungal groups in original forest soil. With the degradation of lignocellulose, fungal groups Mucoromycotina and Ascomycota decreased gradually, and wood-rotting fungi Basidiomycota increased and replaced the opportunist fungi to become predominant group. Most of the fungal clones identified in sample were related to the reported lignocellulose-decomposing strains. Understanding of the microbial community structure and dynamic change during natural lignocellulose-degrading process will provide us with an idea and a basis to construct available commercial lignocellulosic enzymes or microbial complex.
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Kimata-Ariga, Yoko; Hase, Toshiharu
2014-01-01
Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.
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Stepanova, Anna; Shurubor, Yevgeniya; Valsecchi, Federica; Manfredi, Giovanni; Galkin, Alexander
2016-09-01
Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10(-8)M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2±6.0% and 56.4±5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
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Kähkönen, Mika A; Lankinen, Pauliina; Hatakka, Annele
2008-06-01
The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron
2013-04-01
The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes showmore » activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.« less
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Taylor, June S.; Mushak, Paul; Coleman, Joseph E.
1970-01-01
Electron spin resonance (esr) spectra of Cu(II) and Co(II) carbonic anhydrase, and a spin-labeled sulfonamide complex of the Zn(II) enzyme, are reported. The coordination geometry of Cu(II) bound in the enzyme appears to have approximately axial symmetry. Esr spectra of enzyme complexes with metal-binding anions also show axial symmetry and greater covalency, in the order ethoxzolamide < SH- < N3- ≤ CN-. Well-resolved superhyperfine structure in the spectrum of the cyanide complex suggests the presence of two, and probably three, equivalent nitrogen ligands from the protein. Esr spectra of the Co(II) enzyme and its complexes show two types of Co(II) environment, one typical of the native enzyme and the 1:1 CN- complex, and one typical of a 2:1 CN- complex. Co(II) in the 2:1 complex appears to be low-spin and probably has a coordination number of 5. Binding of a spin-labeled sulfonamide to the active center immobilizes the free radical. The similarity of the esr spectra of spin-labeled Zn(II) and Co(II) carbonic anhydrases suggests that the conformation at the active center is similar in the two metal derivatives. PMID:4320976
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Hybrid copper complex-derived conductive patterns printed on polyimide substrates
NASA Astrophysics Data System (ADS)
Lee, Byoungyoon; Jeong, Sooncheol; Kim, Yoonhyun; Jeong, Inbum; Woo, Kyoohee; Moon, Jooho
2012-06-01
We synthesized new copper complexes that can be readily converted into highly conductive Cu film. Mechanochemical milling of copper (I) oxide suspended in formic acid resulted in the submicron-sized Cu formate together Cu nanoparticles. The submicrometer-sized Cu formates are reactive toward inter-particle sintering and metallic Cu seeds present in the Cu complexes assist their decomposition and the nucleation of Cu. The hybrid copper complex film printed on polyimide substrate is decomposed into dense and uniform Cu layer after annealing at 250 °C for 30 min under nitrogen atmosphere. The resulting Cu film exhibited a low resistivity of 8.2 μΩ·cm and good adhesion characteristics.
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RECOVERY OF PROTACTINIUM FROM AQUEOUS SOLUTIONS
Elson, R.E.
1959-07-14
The recovery of fluoride complexed protactinium from aqueous acidic solutions by solvent extraction is described. Generally the prccess of the invention com rises mixing an aqueous solution containing protactinium in a complexed form with an organic solvent which is specific for protactinium, such as diisopropyl carbinol, then decomposing the protactinium complex by adjusting the acidity of the aqueous solution to between 0-3 to 0-9 M in hydrogen ion concentration, and introducing a source of aluminum ions in sufficient quantity to establish a concentration of 0.5 to 1.2 M aluminum ion, whereupon decomposition of the protactinium fluoride complex takes place and the protactinium ion is taken up by the organic solvent phase.
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NASA Astrophysics Data System (ADS)
Kosugi, Takahiro; Hayashi, Shigehiko
2011-01-01
Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.
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ERIC Educational Resources Information Center
Torre, Kjerstin; Balasubramaniam, Ramesh
2011-01-01
We address the complex relationship between the stability, variability, and adaptability of psychological systems by decomposing the global variance of serial performance into two independent parts: the local variance (LV) and the serial correlation structure. For two time series with equal LV, the presence of persistent long-range correlations…
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Raymond L. Czaplewski
2010-01-01
Numerous government surveys of natural resources use Post-Stratification to improve statistical efficiency, where strata are defined by full-coverage, remotely sensed data and geopolitical boundaries. Recursive Restriction Estimation, which may be considered a special case of the static Kalman filter, is an attractive alternative. It decomposes a complex estimation...
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Are CORNER and BROTHER Morphologically Complex? Not in the Long Term
ERIC Educational Resources Information Center
Rueckl, Jay G.; Aicher, Karen A.
2008-01-01
Previous studies haves shown that under masked priming conditions, CORNER primes CORN as strongly as TEACHER primes TEACH and more strongly than BROTHEL primes BROTH. This result has been taken as evidence of a purely structural level of representation at which words are decomposed into morphological constituents in a manner that is independent of…
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2012-01-01
COVERED (From - To) 4. TITLE AND SUBTITLE Multi- enzyme complexes in the thermophilic archaea: The effects of temperature on stability, catalysis and... enzyme interactions in a multi- component system 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-07-1-0058 5c. PROGRAM ELEMENT NUMBER 61102F 6...involves cloning of the genes for the relevant lipoylation enzymes , and characterisation of the protein products 15. SUBJECT TERMS 16. SECURITY
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NASA Astrophysics Data System (ADS)
Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger
2017-07-01
How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.
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Insolubilization process increases enzyme stability
NASA Technical Reports Server (NTRS)
Billingham, J.; Lyn, J.
1971-01-01
Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.
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Averill, Colin
2014-10-01
Allocation trade-offs shape ecological and biogeochemical phenomena at local to global scale. Plant allocation strategies drive major changes in ecosystem carbon cycling. Microbial allocation to enzymes that decompose carbon vs. organic nutrients may similarly affect ecosystem carbon cycling. Current solutions to this allocation problem prioritise stoichiometric tradeoffs implemented in plant ecology. These solutions may not maximise microbial growth and fitness under all conditions, because organic nutrients are also a significant carbon resource for microbes. I created multiple allocation frameworks and simulated microbial growth using a microbial explicit biogeochemical model. I demonstrate that prioritising stoichiometric trade-offs does not optimise microbial allocation, while exploiting organic nutrients as carbon resources does. Analysis of continental-scale enzyme data supports the allocation patterns predicted by this framework, and modelling suggests large deviations in soil C loss based on which strategy is implemented. Therefore, understanding microbial allocation strategies will likely improve our understanding of carbon cycling and climate. © 2014 John Wiley & Sons Ltd/CNRS.
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Librando, Vito; Pappalardo, Matteo
2014-08-01
Polycyclic aromatic hydrocarbons are a family of ubiquitous pollutants whose environmental behavior has been widely studied. Different bacterial species are able to decompose hydrocarbons by using them as a food source. One of the best-studied enzymes is naphthalene 1,2-dioxygenase (NDO). A practical way to optimize the degradation process is by mutating the protein involved, increasing both the degradation capacity of the enzyme and its ability to work under extreme environmental conditions of high temperature and low pH. Herein, we describe the study of NDO using molecular dynamics and docking calculations to discover new mutants with high degrading capabilities. We modeled eleven new mutants of NDO. The results indicate that increasing the size of the active site cavity in the mutants allowed for the insertion of high molecular weight PAHs. Additionally, the physicochemical properties of the NDO active sites make the sites well suited to interactions with PAHs, so most amino-acid modifications should not result in significantly altered behavior of NDO.
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Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A
2013-04-01
The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.
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Enzyme reactor design under thermal inactivation.
Illanes, Andrés; Wilson, Lorena
2003-01-01
Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.
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Vanin, A F; Borodulin, R R; Kubrina, L N; Mikoian, V D; Burbaev, D Sh
2013-01-01
Current notions and new experimental data of the authors on physico-chemical features of dinitrosyl iron complexes with natural thiol-containing ligands (glutathione or cysteine), underlying the ability of the complexes to act as NO molecule and nitrosonium ion donors, are considered. This ability determines various biological activities of dinitrosyl iron complexes--inducing long-lasting vasodilation and thereby long-lasting hypotension in human and animals, inhibiting pellet aggregation, increasing red blood cell elasticity, thereby stimulating microcirculation, and reducing necrotic zone in animals with myocardial infarction. Moreover, dinitrosyl iron complexes are capable of accelerating skin wound healing, improving the function of penile cavernous tissue, blocking apoptosis development in cell cultures. When decomposed dinitrosyl iron complexes can exert cytotoxic effect that can be used for curing infectious and carcinogenic pathologies.
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A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.
Araiza-Olivera, Daniela; Chiquete-Felix, Natalia; Rosas-Lemus, Mónica; Sampedro, José G; Peña, Antonio; Mujica, Adela; Uribe-Carvajal, Salvador
2013-08-01
In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO₂ molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. © 2013 FEBS.
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Bounds on Block Error Probability for Multilevel Concatenated Codes
NASA Technical Reports Server (NTRS)
Lin, Shu; Moorthy, Hari T.; Stojanovic, Diana
1996-01-01
Maximum likelihood decoding of long block codes is not feasable due to large complexity. Some classes of codes are shown to be decomposable into multilevel concatenated codes (MLCC). For these codes, multistage decoding provides good trade-off between performance and complexity. In this paper, we derive an upper bound on the probability of block error for MLCC. We use this bound to evaluate difference in performance for different decompositions of some codes. Examples given show that a significant reduction in complexity can be achieved when increasing number of stages of decoding. Resulting performance degradation varies for different decompositions. A guideline is given for finding good m-level decompositions.
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Timofeyeva, Nadezhda A.; Koval, Vladimir V.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Fedorova, Olga S.
2011-01-01
Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5′-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates. PMID:21912662
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Aras, N; Altinel, I K; Oommen, J
2003-01-01
In addition to the classical heuristic algorithms of operations research, there have also been several approaches based on artificial neural networks for solving the traveling salesman problem. Their efficiency, however, decreases as the problem size (number of cities) increases. A technique to reduce the complexity of a large-scale traveling salesman problem (TSP) instance is to decompose or partition it into smaller subproblems. We introduce an all-neural decomposition heuristic that is based on a recent self-organizing map called KNIES, which has been successfully implemented for solving both the Euclidean traveling salesman problem and the Euclidean Hamiltonian path problem. Our solution for the Euclidean TSP proceeds by solving the Euclidean HPP for the subproblems, and then patching these solutions together. No such all-neural solution has ever been reported.
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Liu, Yong; Lou, Jun; Li, Fang-Bai; Xu, Jian-Ming; Yu, Xiong-Sheng; Zhu, Li-An; Wang, Feng
2014-08-01
Green manuring is a common practice in replenishment of soil organic matter and nutrients in rice paddy field. Owing to the complex interplay of multiple factors, the oxidation--reduction (redox) properties of dissolved organic matter (DOM) from green manure crops are presently not fully understood. In this study, a variety of surrogate parameters were used to evaluate the redox capacity and redox state of DOM derived from Chinese milk vetch (CMV, Astragalus sinicus L.) via microbial decomposition under continuously flooded (CF) and non-flooded (NF) conditions. Additionally, the correlation between the surrogate parameters of CMV-DOM and the kinetic parameters of relevant redox reactions was evaluated in a soil-water system containing CMV-DOM. Results showed that the redox properties of CMV-DOM were substantially different between the fresh and decomposed CMV-DOM treatments. Determination of the surrogate parameters via ultraviolet-visible/Fourier transform infrared absorption spectroscopy and gel permeation chromatography generally provided high-quality data for predicting the redox capacity of CMV-DOM, while the surrogate parameters determined by elemental analysis were suitable for predicting the redox state of CMV-DOM. Depending on the redox capacity and redox state of various moieties/components, NF-decomposed CMV-DOM could easily accelerate soil reduction by shuttling electrons to iron oxides, because it contained more reversible redox-active functional groups (e.g. quinone and hydroquinone pairs) than CF-decomposed CMV-DOM. This work demonstrates that a single index cannot interpret complex changes in multiple factors that jointly determine the redox reactivity of CMV-DOM. Thus, a multi-parametric study is needed for providing comprehensive information on the redox properties of green manure DOM.
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Burgova, E N; Tkachev, N A; Vanin, A F
2012-01-01
It has been shown that the administration of 0,5 ml of 5 mM aqueous solution of dinitrosyl-iron complexes (DNIC) with cysteine alleviated the development of experimental endometriosis in rats induced by surgical way: the size of endometriomes decreased 1.85 times when the DNIC was added every day during 10 days. The effect was suggested to be due to cytotoxic action of NO molecules and nitrosonium ions (NO+) released from rapidly decomposed DNIC in animal organism on endometriome tissues.
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Development of a structured approach for decomposition of complex systems on a functional basis
NASA Astrophysics Data System (ADS)
Yildirim, Unal; Felician Campean, I.
2014-07-01
The purpose of this paper is to present the System State Flow Diagram (SSFD) as a structured and coherent methodology to decompose a complex system on a solution- independent functional basis. The paper starts by reviewing common function modelling frameworks in literature and discusses practical requirements of the SSFD in the context of the current literature and current approaches in industry. The proposed methodology is illustrated through the analysis of a case study: design analysis of a generic Bread Toasting System (BTS).
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White-rot fungal response to fresh and photolytically-weathered pyrogenic organic matter
NASA Astrophysics Data System (ADS)
Gibson, C. D.; Berry, T. D.; Wang, R.; Bird, J. A.; Filley, T. R.
2013-12-01
Pyrogenic organic matter (PyOM or biochar) is the product of the incomplete combustion of biomass. A better understanding of the microbial-mediated degradation of PyOM is critical to assess its role in soil C sequestration and to serve as an agricultural amendment. Recent studies have shown that PyOM additions can prime native soil C but results have been inconsistent, with studies reporting no effect, an increase, or decrease in C mineralization. This study investigated the ability of saprotrophic white-rot fungus, Trametes versicolor, to decompose an unaltered 'fresh' PyOM and a photo-oxidized PyOM. In addition, we measured PyOM-induced priming effects on the mineralization of malt extract agar media (MEA). Enriched (13C) Pinus banksiana-derived PyOM, produced at 450oC under N2, was added fresh and after 4 weeks exposure to 254 nm light to MEA. Vials containing the various types of media were then monitored for CO2 evolution and oxidative enzyme activity. We found that MEA C respired was stimulated (positive priming) by photolyzed PyOM and was inhibited with fresh PyOM addition (negative priming) relative to controls. Vetryl alcohol addition, a laccase production stimulant, resulted in less activity in the presence of PyOM compared with a control, indicating PyOM may disrupt enzyme induction processes. Loss of PyOM-13CO2 was 0.2% (× 0.001) for fresh PyOM and 1.2% (×0.001) for photolyzed PyOM C during 10 weeks averaged across media treatments. While MEA C mineralization decreased after fresh PyOM addition, both oxidative (laccase and manganese peroxidase) and hydrolytic (β glucosidase) enzyme production increased with fresh PyOM in the absence of veratryl alcohol. However, there was a decrease in its presence. These results suggest that the physiological response of this common wood decay fungus to PyOM is complex and responsive to enzymatic triggers but that PyOM itself can act to promote or suppress overall litter or soil decay by fungi.
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Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.
NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethylmore » sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex I A and complex I E) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex I A) or NADH oxidation (complex I E). The canonical alphaproteobacterial complex I isozyme (complex I A) was also shown to be important for routing electrons to nitrogenase-mediated H 2 production, while the horizontally acquired enzyme (complex I E) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.« less
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Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes
Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.; ...
2016-02-01
NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethylmore » sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex I A and complex I E) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex I A) or NADH oxidation (complex I E). The canonical alphaproteobacterial complex I isozyme (complex I A) was also shown to be important for routing electrons to nitrogenase-mediated H 2 production, while the horizontally acquired enzyme (complex I E) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.« less
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... enough of the enzyme to break down certain complex molecules, the molecules build up in harmful amounts. ... chromosome, an enzyme that's needed to break down complex sugar molecules is missing or malfunctioning. Without this ...
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ERIC Educational Resources Information Center
Wang, Ze
2015-01-01
Using data from the Trends in International Mathematics and Science Study (TIMSS) 2007, this study examined the big-fish-little-pond-effects (BFLPEs) in 49 countries. In this study, the effect of math ability on math self-concept was decomposed into a within- and a between-level components using implicit mean centring and the complex data…
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Acid sorption regeneration process using carbon dioxide
King, C. Judson; Husson, Scott M.
2001-01-01
Carboxylic acids are sorbed from aqueous feedstocks onto a solid adsorbent in the presence of carbon dioxide under pressure. The acids are freed from the sorbent phase by a suitable regeneration method, one of which is treating them with an organic alkylamine solution thus forming an alkylamine-carboxylic acid complex which thermally decomposes to the desired carboxylic acid and the alkylamine.
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Improving engineering system design by formal decomposition, sensitivity analysis, and optimization
NASA Technical Reports Server (NTRS)
Sobieski, J.; Barthelemy, J. F. M.
1985-01-01
A method for use in the design of a complex engineering system by decomposing the problem into a set of smaller subproblems is presented. Coupling of the subproblems is preserved by means of the sensitivity derivatives of the subproblem solution to the inputs received from the system. The method allows for the division of work among many people and computers.
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Hoskovcová, Monika; Halámek, Emil; Kobliha, Zbynĕk
2009-01-01
Reactivation with bis quaternary aldoxime HI-6, chemical formula 1-(2-hydroxyamino-methylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride of immobilized enzyme acetylcholinesterase inhibited by nerve agent type "G" was studied. This aldoxime is effective in reactivation of sarin-inhibited acetylcholinesterase. Substantially lower reactivation potency was observed with cyclosarin-inhibited enzyme and almost no effect was found for that acetylcholinesterase is the enzyme complex. HI 6 is completely ineffective towards the soman-inhibited enzyme: After a 2-minute inhibition of the enzyme with soman no ability to define reactivator the inhibited enzymes and complexes.
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Study on the technology of compound enzymatic hydrolysis of whole passion fruit
NASA Astrophysics Data System (ADS)
Yang, Yu-xia; Duan, Zhen-hua; Kang, Chao; Zhu, Xiang-hao; Li, Ding-jin
2017-12-01
Fresh Whole Passion Fruit was used as raw material, The enzymatic hydrolysis technology of Passion Fruit by Complex enzyme were studied, The effects of enzyme dosage, Enzyme ratio(cellulose: pectinase), pH, temperature and time on the hydrolysis were investigated by single-tests and orthogonal tests, the hydrolysis indicators of single-factor tests and orthogonal tests were juice yield. The optimal hydrolysis conditions of Passion Fruit by Complex enzyme were enzyme dosage 0.12%, Enzyme ratio 5:1, hydrolysis temperature 50°C, pH4.0 and time 3.5 h. Under such conditions, juice yield of Passion Fruit was 92.91%.
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Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...
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Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.
2013-01-01
The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases. PMID:23519665
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Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L; Lata, James P; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M; Bergkvist, Magnus; Sherwood, Robert W; Zhang, Sheng; Travis, Alexander J
2017-01-02
For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Mukai, Chinatsu; Gao, Lizeng; Nelson, Jacquelyn L.; Lata, James P.; Cohen, Roy; Wu, Lauren; Hinchman, Meleana M.; Bergkvist, Magnus; Sherwood, Robert W.; Zhang, Sheng; Travis, Alexander J.
2016-01-01
For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo. PMID:27901298
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The structure and function of Alzheimer's gamma secretase enzyme complex.
Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N
2009-01-01
The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity.
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Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.
Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi
2015-01-01
Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.
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USDA-ARS?s Scientific Manuscript database
CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions t...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Pazhanisamy, S.; Pratt, R.F.
The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-(((phenylacetyl)glycyl)oxy)benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presencemore » of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.« less
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Tchong, Shih-I; Xu, Huimin; White, Robert H
2005-02-08
A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the alpha-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an alpha,beta-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed.
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Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.
Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M
2013-01-01
With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V
1997-07-20
Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.
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Acetylcholinesterase Inhibitors: Pharmacology and Toxicology
Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M
2013-01-01
Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466
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Luan, Qian; Gan, Ning; Cao, Yuting; Li, Tianhua
2017-07-19
A mimicking-enzyme-based colorimetric aptasensor was developed for the detection of kanamycin (KANA) in milk using magnetic loop-DNA-NMOF-Pt (m-L-DNA) probes and catalytic hairpin assembly (CHA)-assisted target recycling for signal amplification. The m-L-DNA probes were constructed via hybridization of hairpin DNA H1 (containing aptamer sequence) immobilized magnetic beads (m-H1) and signal DNA (sDNA, partial hybridization with H1) labeled nano Fe-MIL-88NH 2 -Pt (NMOF-Pt-sDNA). In the presence of KANA and complementary hairpin DNA H2, the m-L-DNA probes decomposed and formed an m-H1/KANA intermediate, which triggered the CHA reaction to form a stable duplex strand (m-H1-H2) while releasing KANA again for recycling. Consequently, numerous NMOF-Pt-sDNA as mimicking enzymes can synergistically catalyze 3,3',5,5'-tetramethylbenzidine (TMB) for color development. The aptasensor exhibited high selectivity and sensitivity for KANA in milk with a detection limit of 0.2 pg mL -1 within 30 min. The assay can be conveniently extended for on-site screening of other antibiotics in foods by simply changing the base sequence of the probes.
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A Proteomic Study of Pectin Degrading Enzymes Secreted by Botrytis cinerea Grown in Liquid Culture
Shah, Punit; Gutierrez-Sanchez, Gerardo; Orlando, Ron; Bergmann, Carl
2009-01-01
Botrytis cinerea is a pathogenic filamentous fungus which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty-seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a Signal P motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues. PMID:19526562
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X-ray diffraction study of Penicillium Vitale catalase in the complex with aminotriazole
DOE Office of Scientific and Technical Information (OSTI.GOV)
Borovik, A. A.; Grebenko, A. I.; Melik-Adamyan, V. R., E-mail: mawr@ns.crys.ras.ru
2011-07-15
The three-dimensional structure of the enzyme catalase from Penicillium vitale in a complex with the inhibitor aminotriazole was solved and refined by protein X-ray crystallography methods. An analysis of the three-dimensional structure of the complex showed that the inhibition of the enzyme occurs as a result of the covalent binding of aminotriazole to the amino-acid residue His64 in the active site of the enzyme. An investigation of the three-dimensional structure of the complex resulted in the amino-acid residues being more precisely identified. The binding sites of saccharide residues and calcium ions in the protein molecule were found.
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Electrochemical alkaline Fe(VI) water purification and remediation.
Licht, Stuart; Yu, Xingwen
2005-10-15
Fe(VI) is an unusual and strongly oxidizing form of iron, which provides a potentially less hazardous water-purifying agent than chlorine. A novel on-line electrochemical Fe(VI) water purification methodology is introduced. Fe(VI) addition had been a barrier to its effective use in water remediation, because solid Fe(VI) salts require complex (costly) syntheses steps and solutions of Fe(VI) decompose. Online electrochemical Fe(VI) water purification avoids these limitations, in which Fe(VI) is directly prepared in solution from an iron anode as the FeO42- ion, and is added to the contaminant stream. Added FeO42- decomposes, by oxidizing a wide range of water contaminants including sulfides (demonstrated in this study) and other sulfur-containing compounds, cyanides (demonstrated in this study), arsenic (demonstrated in this study), ammonia and other nitrogen-containing compounds (previously demonstrated), a wide range of organics (phenol demonstrated in this study), algae, and viruses (each previously demonstrated).
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[Spectral characteristics of decomposition of incorporated straw in compound polluted arid loess].
Fan, Chun-Hui; Zhang, Ying-Chao; Xu, Ji-Ting; Wang, Jia-Hong
2014-04-01
The original loess from western China was used as soil sample, the spectral methods of scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDS), elemental analysis, Fourier transform infrared spectroscopy (FT-IR) and 13C nuclear magnetic resonance (13C NMR) were used to investigate the characteristics of decomposed straw and formed humic acids in compound polluted arid loess. The SEM micrographs show the variation from dense to decomposed surface, and finally to damaged structure, and the EDS data reveal the phenomenon of element transfer. The newly-formed humic acids are of low aromaticity, helpful for increasing the activity of organic matters in loess. The FTIR spectra in the whole process are similar, indicating the complexity of transformation dynamics of humic acids. The molecular structure of humic acids becomes simpler, shown from 13C NMR spectra. The spectral methods are useful for humic acids identification in loess region in straw incorporation process.
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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
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Crofts, Naoko; Nakamura, Yasunori; Fujita, Naoko
2017-09-01
Starch accounts for the majority of edible carbohydrate resources generated through photosynthesis. Amylopectin is the major component of starch and is one of highest-molecular-weight biopolymers. Rapid and systematic synthesis of frequently branched hydro-insoluble amylopectin and efficient accumulation into amyloplasts of cereal endosperm is crucial. The functions of multiple starch biosynthetic enzymes, including elongation, branching, and debranching enzymes, must be temporally and spatially coordinated. Accordingly, direct evidence of protein-protein interactions of starch biosynthetic enzymes were first discovered in developing wheat endosperm in 2004, and they have since been shown in the developing seeds of other cereals. This review article describes structural characteristics of starches as well as similarities and differences in protein complex formation among different plant species and among mutant plants that are deficient in specific starch biosynthetic enzymes. In addition, evidence for protein complexes that are involved in the initiation stages of starch biosynthesis is summarized. Finally, we discuss the significance of protein complexes and describe new methods that may elucidate the mechanisms and roles of starch biosynthetic enzyme complexes. Copyright © 2017 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Polášek, Miroslav; Makrlík, Emanuel; Kvíčala, Jaroslav; Křížová, Věra; Vaňura, Petr
2018-02-01
By using electrospray ionization mass spectrometry (ESI-MS), it was proven experimentally that the univalent thallium cation (Tl+) forms with meso-octamethylcalix[4]pyrrole (1) the cationic complex species 1 Tl+. When this kinetically stable cation-π complex 1 Tl+ is collisionally activated, it decomposes by elimination of the whole ligand 1 or small meso-octamethylcalix[4]pyrrole fragments. Further, applying quantum chemical DFT calculations, four different conformations of the resulting complex 1 Tl+ were derived. It means that under the present experimental conditions, this ligand 1 can be considered as a very effective macrocyclic receptor for the thallium cation.
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Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro
2015-10-01
Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.
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Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E
2015-05-15
The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.
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Liu, Sidong; Charlesworth, Thomas J.; Bason, John V.; Montgomery, Martin G.; Harbour, Michael E.; Fearnley, Ian M.; Walker, John E.
2015-01-01
The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex. PMID:25759169
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Pootakham, Wirulda; Sonthirod, Chutima; Naktang, Chaiwat; Jomchai, Nukoon; Sangsrakru, Duangjai; Tangphatsornruang, Sithichoke
2016-01-01
Advances in next generation sequencing have facilitated a large-scale single nucleotide polymorphism (SNP) discovery in many crop species. Genotyping-by-sequencing (GBS) approach couples next generation sequencing with genome complexity reduction techniques to simultaneously identify and genotype SNPs. Choice of enzymes used in GBS library preparation depends on several factors including the number of markers required, the desired level of multiplexing, and whether the enrichment of genic SNP is preferred. We evaluated various combinations of methylation-sensitive ( Aat II, Pst I, Msp I) and methylation-insensitive ( Sph I, Mse I) enzymes for their effectiveness in genome complexity reduction and enrichment of genic SNPs. We discovered that the use of two methylation-sensitive enzymes effectively reduced genome complexity and did not require a size selection step. On the contrary, the genome coverage of libraries constructed with methylation-insensitive enzymes was quite high, and the additional size selection step may be required to increase the overall read depth. We also demonstrated the effectiveness of methylation-sensitive enzymes in enriching for SNPs located in genic regions. When two methylation-insensitive enzymes were used, only 16% of SNPs identified were located in genes and 18% in the vicinity (± 5 kb) of the genic regions, while most SNPs resided in the intergenic regions. In contrast, a remarkable degree of enrichment was observed when two methylation-sensitive enzymes were employed. Almost two thirds of the SNPs were located either inside (32-36%) or in the vicinity (28-31%) of the genic regions. These results provide useful information to help researchers choose appropriate GBS enzymes in oil palm and other crop species.
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Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto
2013-01-01
In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected.
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Martínez-Núñez, Mario Alberto; Poot-Hernandez, Augusto Cesar; Rodríguez-Vázquez, Katya; Perez-Rueda, Ernesto
2013-01-01
In this work, the content of enzymes and DNA-binding transcription factors (TFs) in 794 non-redundant prokaryotic genomes was evaluated. The identification of enzymes was based on annotations deposited in the KEGG database as well as in databases of functional domains (COG and PFAM) and structural domains (Superfamily). For identifications of the TFs, hidden Markov profiles were constructed based on well-known transcriptional regulatory families. From these analyses, we obtained diverse and interesting results, such as the negative rate of incremental changes in the number of detected enzymes with respect to the genome size. On the contrary, for TFs the rate incremented as the complexity of genome increased. This inverse related performance shapes the diversity of metabolic and regulatory networks and impacts the availability of enzymes and TFs. Furthermore, the intersection of the derivatives between enzymes and TFs was identified at 9,659 genes, after this point, the regulatory complexity grows faster than metabolic complexity. In addition, TFs have a low number of duplications, in contrast to the apparent high number of duplications associated with enzymes. Despite the greater number of duplicated enzymes versus TFs, the increment by which duplicates appear is higher in TFs. A lower proportion of enzymes among archaeal genomes (22%) than in the bacterial ones (27%) was also found. This low proportion might be compensated by the interconnection between the metabolic pathways in Archaea. A similar proportion was also found for the archaeal TFs, for which the formation of regulatory complexes has been proposed. Finally, an enrichment of multifunctional enzymes in Bacteria, as a mechanism of ecological adaptation, was detected. PMID:23922780
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Modeling of substrate and inhibitor binding to phospholipase A2.
Sessions, R B; Dauber-Osguthorpe, P; Campbell, M M; Osguthorpe, D J
1992-09-01
Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.
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Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin
2016-01-01
The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates. PMID:27271847
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DOE Office of Scientific and Technical Information (OSTI.GOV)
DeAngelis, Kristen M.; Sharma, Deepak; Varney, Rebecca
2013-08-29
The anaerobic isolate Enterobacter lignolyticus SCF1 was initially cultivated based on anaerobic growth on lignin as sole carbon source. The source of the isolated bacteria was from tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, making it likely that bacteria using oxygen-independent enzymes play an important role in decomposition. We have examined differential expression of the anaerobic isolate Enterobacter lignolyticus SCF1 during growth on lignin. After 48 hours of growth, we used transcriptomics and proteomics to define the enzymes and other regulatory machinery that these organisms use to degrade lignin, as well as metabolomics tomore » measure lignin degradation and monitor the use of lignin and iron as terminal electron acceptors that facilitate more efficient use of carbon. Proteomics revealed accelerated xylose uptake and metabolism under lignin-amended growth, and lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. Our data shows the advantages of a multi-omics approach, where incomplete pathways identified by genomics were completed, and new observations made on coping with poor carbon availability. The fast growth, high efficiency and specificity of enzymes employed in bacterial anaerobic litter deconstruction makes these soils useful templates for improving biofuel production.« less
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Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin
2016-06-08
The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.
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Amy Ross-Davis; Jane E. Stewart; Matt Settles; John W. Hanna; John D. Shaw; Andrew T. Hudak; Deborah S. Page-Dumroese; Ned B. Klopfenstein
2016-01-01
Forests are home to some of the most complex microbial communities (Fierer et al. 2012) which drive biogeochemical cycles (Clemmensen et al. 2013; van der Heijden et al. 2008) and account for substantial terrestrial biomass (Nielsen et al. 2011). Fungi, through their ecological roles as decomposers, mutualists, or pathogens, are particularly important in...
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Program Helps Decompose Complex Design Systems
NASA Technical Reports Server (NTRS)
Rogers, James L., Jr.; Hall, Laura E.
1994-01-01
DeMAID (A Design Manager's Aid for Intelligent Decomposition) computer program is knowledge-based software system for ordering sequence of modules and identifying possible multilevel structure for design problem. Groups modular subsystems on basis of interactions among them. Saves considerable money and time in total design process, particularly in new design problem in which order of modules has not been defined. Available in two machine versions: Macintosh and Sun.
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Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme
Vallières, Cindy; Fisher, Nicholas; Meunier, Brigitte
2013-01-01
The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials. PMID:23951230
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Smith, Steven P; Bayer, Edward A
2013-10-01
Cellulosomes are multi-enzyme complexes produced by anaerobic bacteria for the efficient deconstruction of plant cell wall polysaccharides. The assembly of enzymatic subunits onto a central non-catalytic scaffoldin subunit is mediated by a highly specific interaction between the enzyme-bearing dockerin modules and the resident cohesin modules of the scaffoldin, which affords their catalytic activities to work synergistically. The scaffoldin also imparts substrate-binding and bacterial-anchoring properties, the latter of which involves a second cohesin-dockerin interaction. Recent structure-function studies reveal an ever-growing array of unique and increasingly complex cohesin-dockerin complexes and cellulosomal enzymes with novel activities. A 'build' approach involving multimodular cellulosomal segments has provided a structural model of an organized yet conformationally dynamic supramolecular assembly with the potential to form higher order structures. Copyright © 2013. Published by Elsevier Ltd.
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Siswoyo, Tri Agus; Morita, Naofumi
2003-05-07
Complexes of starch and monoacyl-sn-glycerophosphatidylcholine (GPC) containing various acyl (myristoyl, palmitoyl, and stearoyl) chains were subjected to hydrolysis with glucoamylase (EC 3.2.1.3). The enzyme hydrolyzed approximately 40% of starch control and 20-28% of starch-GPC complexes. Among the GPCs examined, 1- and 2-monomyristoyl-sn-GPC showed the highest resistance to enzyme hydrolysis, and the hydrolysis rate of starch-GPCs was greater with longer chains. Enzymatic hydrolysis strongly affected the thermal properties of the starch. After enzymatic hydrolysis of starch-GPC complexes for 24 h, their thermograms had broader peaks with lower enthalpies than the corresponding starch without enzyme; however, the starch-GPC complexes showed little change. The surface of starch-GPC granules was less eroded. These results showed that the increasing amount of starch-GPC complexes could be more resistant to hydrolysis.
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Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro
2013-03-08
Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Carlsson, Mats; Johansson, Mikael; Larson, Jeffrey
Previous approaches for scheduling a league with round-robin and divisional tournaments involved decomposing the problem into easier subproblems. This approach, used to schedule the top Swedish handball league Elitserien, reduces the problem complexity but can result in suboptimal schedules. This paper presents an integrated constraint programming model that allows to perform the scheduling in a single step. Particular attention is given to identifying implied and symmetry-breaking constraints that reduce the computational complexity significantly. The experimental evaluation of the integrated approach takes considerably less computational effort than the previous approach.
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Mas-Marzá, Elena; Poyatos, Macarena; Sanaú, Mercedes; Peris, Eduardo
2004-03-22
Two tripodal trisimidazolium ligand precursors have been tested in the synthesis of new N-heterocyclic carbene rhodium and iridium complexes. [Tris(3-methylbenzimidazolium-1-yl)]methane sulfate gave products with coordination of the decomposed precursor. [1,1,1-Tris(3-butylimidazolium-1-yl)methyl]ethane trichloride (TIMEH(3)(Bu)) coordinated to the metal in a chelate and bridged-chelate form, depending on the reaction conditions. The crystal structures of two of the products are described. The compounds resulting from the coordination with TIME(Bu) were tested in the catalytic hydrosilylation of terminal alkynes.
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The History of Maltose-active Disaccharidases.
Lentze, Michael J
2018-06-01
The history of maltose-active disaccharidases is closely related to the history of the sugar and starch industry. It began in the 19th century, when a shortage of cane sugar occurred in continental Europe, because Napoleon Bonaparte decreed that no goods could be imported from England to the countries he occupied. Other sugar sources had to be found, and it led to the identification of sugar beets as alternative source of sugar by Marggraf in 1774, to the detection of starch hydrolysis by diluted sulfuric acid by Kirchhoff in 1812, and to the starch digestion enzyme, α-amylase, by Payen in 1833. In the 20th century, Borkström's group in Sweden investigated the absorption of nutrients in human adults by transintubation techniques and found that the luminal concentration of invertase was small compared to that of α-amylase. They speculated that the major locus of this enzyme activity must be in the intestinal cells. Borkström's coworker, Dahlqvist, investigated the maltose-active enzymes in pig intestine, and a second group around Semenza studied the maltase-active enzymes in rabbit intestine. After the first descriptions of congenital sucrase-isomaltase deficiency in 1960 and 1961, the research on disaccharidases increased. Dahlqvist published the first quantitative method to measure these enzymes. Consecutive research led to the discovery of 4 maltases, which were later identified as 2 complex enzymes: the sucrase-isomaltase complex and the maltase-glucoamylase complex. The homology of the 2 enzyme complexes was later determined when the cDNA sequences of the 2 complexes in human intestine were identified.
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An optical Fourier transform coprocessor with direct phase determination.
Macfaden, Alexander J; Gordon, George S D; Wilkinson, Timothy D
2017-10-20
The Fourier transform is a ubiquitous mathematical operation which arises naturally in optics. We propose and demonstrate a practical method to optically evaluate a complex-to-complex discrete Fourier transform. By implementing the Fourier transform optically we can overcome the limiting O(nlogn) complexity of fast Fourier transform algorithms. Efficiently extracting the phase from the well-known optical Fourier transform is challenging. By appropriately decomposing the input and exploiting symmetries of the Fourier transform we are able to determine the phase directly from straightforward intensity measurements, creating an optical Fourier transform with O(n) apparent complexity. Performing larger optical Fourier transforms requires higher resolution spatial light modulators, but the execution time remains unchanged. This method could unlock the potential of the optical Fourier transform to permit 2D complex-to-complex discrete Fourier transforms with a performance that is currently untenable, with applications across information processing and computational physics.
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Yu, Qilin; Jin, Xiaochen; Zhang, Yaobin
2018-05-15
Sludge cell disruption was generally considered as the rate-limiting step for the anaerobic digestion of waste activated sludge (WAS). Advanced oxidation processes and bio-electro-chemical systems were recently reported to enhance the hydrolysis of WAS and sludge cell disruption, while the cell-breaking processes of these systems remain unclear yet. In this study, an innovative Bio-electro-Fenton system was developed to pretreat the WAS sequentially with cathode Fenton process and anode anaerobic digestion. Significant cell disruption and dissolution intracellular organics were founded after the treatment. X-ray photoelectron spectroscopy (XPS) analysis and fourier transform infrared spectroscopy (FT-IR) spectra indicated that Gram-negative bacteria were more sensitive to free radicals yielded in cathode to induce a chain reaction that destroyed the lipid-contained outer membrane, while Gram-positive bacteria with thick peptidoglycan layer were liable to be biologically decomposed in the anode. Compared with the oxidation of organic matters in the cathode Fenton, the secretion of enzyme increased in the anode which was beneficial to break down the complex matters (peptidoglycans) into simples that were available for anode oxidation by exoelectrogens. The results also showed a possible prospect for the application of this sequential pretreatment in bio-electro-Fenton systems to disrupt sludge cells and enhance the anaerobic digestion. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Campbell, I D; Jones, R B; Kiener, P A; Waley, S G
1979-01-01
The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix. PMID:38777
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Artificial enzymes based on supramolecular scaffolds.
Dong, Zeyuan; Luo, Quan; Liu, Junqiu
2012-12-07
Enzymes are nanometer-sized molecules with three-dimensional structures created by the folding and self-assembly of polymeric chain-like components through supramolecular interactions. They are capable of performing catalytic functions usually accompanied by a variety of conformational states. The conformational diversities and complexities of natural enzymes exerted in catalysis seriously restrict the detailed understanding of enzymatic mechanisms in molecular terms. A supramolecular viewpoint is undoubtedly helpful in understanding the principle of enzyme catalysis. The emergence of supramolecular artificial enzymes therefore provides an alternative way to approach the structural complexity and thus to unravel the mystery of enzyme catalysis. This critical review covers the recent development of artificial enzymes designed based on supramolecular scaffolds ranging from the synthetic macrocycles to self-assembled nanometer-sized objects. Such findings are anticipated to facilitate the design of supramolecular artificial enzymes as well as their potential uses in important fields, such as manufacturing and food industries, environmental biosensors, pharmaceutics and so on.
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Pazhanisamy, S; Pratt, R F
1989-08-22
The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-[[(phenylacetyl)glycyl]oxy]benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first [Pazhanisamy, S., Govardhan, C. P., & Pratt, R. F. (1989) Biochemistry (first of three papers in this issue)]. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.(ABSTRACT TRUNCATED AT 250 WORDS)
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Choosing the Best Enzyme Complex Structure Made Easy.
Das, Sayoni; Orengo, Christine
2018-04-03
In this issue of Structure, Tyzack et al. (2018) present a study of enzyme-ligand complexes in the PDB and show that the molecular similarity of bound and cognate ligands can be used to choose the most biologically appropriate complex structure for analysis when multiple structures are available. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Nitrogen Alters Fungal Communities in Boreal Forest Soil: Implications for Carbon Cycling
NASA Astrophysics Data System (ADS)
Allison, S. D.; Treseder, K. K.
2005-12-01
One potential effect of climate change in high latitude ecosystems is to increase soil nutrient availability. In particular, greater nitrogen availability could impact decomposer communities and lead to altered rates of soil carbon cycling. Since fungi are the primary decomposers in many high-latitude ecosystems, we used molecular techniques and field surveys to test whether fungal communities and abundances differed in response to nitrogen fertilization in a boreal forest ecosystem. We predicted that fungi that degrade recalcitrant carbon would decline under nitrogen fertilization, while fungi that degrade labile carbon would increase, leading to no net change in rates of soil carbon mineralization. The molecular data showed that basidiomycete fungi dominate the active fungal community in both fertilized and unfertilized soils. However, we found that fertilization reduced peak mushroom biomass by 79%, although most of the responsive fungi were ectomycorrhizal and therefore their capacity to degrade soil carbon is uncertain. Fertilization increased the activity of the cellulose-degrading enzyme beta-glucosidase by 78%, while protease activity declined by 39% and polyphenol oxidase, a lignin-degrading enzyme, did not respond. Rates of soil respiration did not change in response to fertilization. These results suggest that increased nitrogen availability does alter the composition of the fungal community, and its potential to degrade different carbon compounds. However, these differences do not affect the total flux of CO2 from the soil, even though the contribution to CO2 respiration from different carbon pools may vary with fertilization. We conclude that in the short term, increased nitrogen availability due to climate warming or nitrogen deposition is more likely to alter the turnover of individual carbon pools rather than total carbon fluxes from the soil. Future work should determine if changes in fungal community structure and associated differences in substrate utilization will also affect total carbon fluxes over longer time scales.
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de Vries, Ronald P.; Timonen, Sari; Hildén, Kristiina
2014-01-01
Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes. PMID:24505339
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Hesse, Cedar N.; Mueller, Rebecca C.; Vuyisich, Momchilo; ...
2015-04-23
Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes)more » in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.« less
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Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.
Hersh, L B; Jorns, M S
1975-11-25
The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
University of California, Berkeley; Lawrence Berkeley National Laboratory; Raymond, Kenneth
2007-09-27
Nature uses enzymes to activate otherwise unreactive compounds in remarkable ways. For example, DNases are capable of hydrolyzing phosphate diester bonds in DNA within seconds,[1-3]--a reaction with an estimated half-life of 200 million years without an enzyme.[4] The fundamental features of enzyme catalysis have been much discussed over the last sixty years in an effort to explain the dramatic rate increases and high selectivities of enzymes. As early as 1946, Linus Pauling suggested that enzymes must preferentially recognize and stabilize the transition state over the ground state of a substrate.[5] Despite the intense study of enzymatic selectivity and ability tomore » catalyze chemical reactions, the entire nature of enzyme-based catalysis is still poorly understood. For example, Houk and co-workers recently reported a survey of binding affinities in a wide variety of enzyme-ligand, enzyme-transition-state, and synthetic host-guest complexes and found that the average binding affinities were insufficient to generate many of the rate accelerations observed in biological systems.[6] Therefore, transition-state stabilization cannot be the sole contributor to the high reactivity and selectivity of enzymes, but rather, other forces must contribute to the activation of substrate molecules. Inspired by the efficiency and selectivity of Nature, synthetic chemists have admired the ability of enzymes to activate otherwise unreactive molecules in the confines of an active site. Although much less complex than the evolved active sites of enzymes, synthetic host molecules have been developed that can carry out complex reactions with their cavities. While progress has been made toward highly efficient and selective reactivity inside of synthetic hosts, the lofty goal of duplicating enzymes specificity remains.[7-9] Pioneered by Lehn, Cram, Pedersen, and Breslow, supramolecular chemistry has evolved well beyond the crown ethers and cryptands originally studied.[10-12] Despite the increased complexity of synthetic host molecules, most assembly conditions utilize self-assembly to form complex highly-symmetric structures from relatively simple subunits. For supramolecular assemblies able to encapsulate guest molecules, the chemical environment in each assembly--defined by the size, shape, charge, and functional group availability--greatly influences the guest-binding characteristics.[6, 13-17]« less
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Sun, Jian; Cui, Wen-Hui; Du, Kun; Gao, Qian; Du, Mengmeng; Ji, Peijun; Feng, Wei
2017-03-10
R-ɷ-transaminases transfer an amino group from an amino donor (e.g. (R)-1-phenylethylamine) onto an amino acceptor (e.g. pyruvate), resulting a co-product (e.g. d-alanine). This work intends to immobilize R-ɷ-Transaminase on MnO 2 nanorods to achieve multienzyme catalysis. R-ɷ-Transaminase (RTA) and d-amino acid oxidase (DAAO) have been fused to an elastin-like polypeptide (ELP) separately through genetic engineering of the enzymes. ELP-RTA and ELP-DAAO have been separately immobilized on polydopamine-coated MnO 2 nanorods. When the two immobilized enzymes were used together in one pot, the transformation of (R)-1-phenylethylamine was catalyzed by the immobilized ELP-RTA, and the co-product d-alanine was converted back to pyruvate under the catalysis of the immobilized ELP-DAAO, achieving the recycling of pyruvate in situ. Thus pyruvate was maintained at a low concentration in order to reduce its negative effect. On the other hand, the generated H 2 O 2 of ELP-DAAO was decomposed by the MnO 2 nanorods, and the evolved oxygen oxidized the reduced cofactors of ELP-DAAO. Forming the circles of hydrogen peroxide→oxygen→hydrogen peroxide accelerated the deamination reaction. The highly efficient conversion of the co-product d-alanine back to pyruvate accelerated the forming of the pyruvate→d-alanine→pyruvate cycle between the two immobilized enzymes. The coordination of the pyruvate→d-alanine→pyruvate and hydrogen peroxide→oxygen→hydrogen peroxide cycles accelerated the transformation of (R)-1-phenylethylamine. As a result, As a result, the immobilized enzymes achieved a conversion of 98±1.8% in comparison to 69.6±1.2% by free enzymes. Copyright © 2017 Elsevier B.V. All rights reserved.
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Yuan, Jiao-Jiao; Wang, Cheng-Zhang; Ye, Jian-Zhong; Tao, Ran; Zhang, Yu-Si
2015-02-11
Oleuropein (OE), the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT) and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity) optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE) were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL) was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.
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NASA Astrophysics Data System (ADS)
Amatangelo, K. L.; Cordova, T. P.; Vitousek, P. M.
2007-12-01
Microbial growth and enzyme production during decomposition is controlled by the availability of carbon substrates, essential elements, and the ratios of these (such as lignin:N). We manipulated carbon:nutrient stoichiometry during decomposition using a natural fertility gradient in Hawaii and litter of varying initial biochemistry. We collected freshly senesced litter of seven biochemically distinct species from three sites offering differing levels of N, P, cations, and 15N , but similar yearly rainfall and temperature patterns. Litter types were decomposed at both the sites they were collected, and at the other site(s) that species was found. Litter was collected at multiple time points, and after one year of decomposition, calculated K constants varied an order of magnitude, from 0.276 to 2.76. Decomposition rates varied significantly with both litter site of origin and deployment, except at the oldest, P-limited site, where litter site of origin was not significantly correlated with decomposition within species. As microbial exocellular enzymes provide the catalyst for the breakdown of organic molecules including phenols, cellulose, and cutin, we assayed polyphenol oxidase, cellobiohydrolase, cutinase, chitinase, and lignin peroxidase to evaluate the breakdown sequence of different litter types. To measure the fungal biomass accumulating during decomposition, we extracted (22E)-Ergosta-5,7,22-trien-3beta- ol (ergosterol) on a subset of samples. The production of particular exocellular enzymes on litter species responded distinctly to origin and decomposition sites: after six months, chitinase and cellobiohydrolase were significantly affected by origin site, whereas polyphenol oxidase activity was controlled by deployment site. We conclude that site characteristics can alter the interaction between litter carbon:nutrient ratios and decomposition rate, mediated through microbial biomass and enzyme production.
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Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour.
Mathy, Nathalie; Hébert, Agnès; Mervelet, Peggy; Bénard, Lionel; Dorléans, Audrey; Li de la Sierra-Gallay, Inés; Noirot, Philippe; Putzer, Harald; Condon, Ciarán
2010-01-01
Ribonucleases J1 and J2 are recently discovered enzymes with dual 5'-to-3' exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5'-to-3' exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.
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Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes
Rytioja, Johanna; Hildén, Kristiina; Yuzon, Jennifer; Hatakka, Annele; de Vries, Ronald P.
2014-01-01
SUMMARY Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation. PMID:25428937
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Group Decision Support System to Aid the Process of Design and Maintenance of Large Scale Systems
1992-03-23
from a fuzzy set of user requirements. The overall objective of the project is to develop a system combining the characteristics of a compact computer... AHP ) for hierarchical prioritization. 4) Individual Evaluation and Selection of Alternatives - Allows the decision maker to individually evaluate...its concept of outranking relations. The AHP method supports complex decision problems by successively decomposing and synthesizing various elements
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Removal of methylmercury and tributyltin (TBT) using marine microorganisms.
Lee, Seong Eon; Chung, Jin Wook; Won, Ho Shik; Lee, Dong Sup; Lee, Yong-Woo
2012-02-01
Two marine species of bacteria were isolated that are capable of degrading organometallic contaminants: Pseudomonas balearica, which decomposes methylmercury; and Shewanella putrefaciens, which decomposes tributyltin. P. balearica decomposed 97% of methylmercury (20.0 μg/L) into inorganic mercury after 3 h, while S. putrefaciens decomposed 88% of tributyltin (55.3 μg Sn/L) in real wastewater after 36 h. These data indicate that the two bacteria efficiently decomposed the targeted substances and may be applied to real wastewater.
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Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.
Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M
2016-02-23
Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at
2012-01-20
Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less
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Constructing the wonders of the bacterial world: biosynthesis of complex enzymes.
Sargent, Frank
2007-03-01
The prokaryotic cytoplasmic membrane not only maintains cell integrity and forms a barrier between the cell and its outside environment, but is also the location for essential biochemical processes. Microbial model systems provide excellent bases for the study of fundamental problems in membrane biology including signal transduction, chemotaxis, solute transport and, as will be the topic of this review, energy metabolism. Bacterial respiration requires a diverse array of complex, multi-subunit, cofactor-containing redox enzymes, many of which are embedded within, or located on the extracellular side of, the membrane. The biosynthesis of these enzymes therefore requires carefully controlled expression, assembly, targeting and transport processes. Here, focusing on the molybdenum-containing respiratory enzymes central to anaerobic respiration in Escherichia coli, recent descriptions of a chaperone-mediated 'proofreading' system involved in coordinating assembly and export of complex extracellular enzymes will be discussed. The paradigm proofreading chaperones are members of a large group of proteins known as the TorD family, and recent research in this area highlights common principles that underpin biosynthesis of both exported and non-exported respiratory enzymes.
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Thermal stability of bioactive enzymatic papers.
Khan, Mohidus Samad; Li, Xu; Shen, Wei; Garnier, Gil
2010-01-01
The thermal stability of two enzymes adsorbed on paper, alkaline phosphatase (ALP) and horseradish peroxidase (HRP), was measured using a colorimetric technique quantifying the intensity of the product complex. The enzymes adsorbed on paper retained their functionality and selectivity. Adsorption on paper increased the enzyme thermal stability by 2-3 orders of magnitude compared to the same enzyme in solution. ALP and HRP enzymatic papers had half-lives of 533 h and 239 h at 23 degrees C, respectively. The thermal degradation of adsorbed enzyme was found to follow two sequential first-order reactions, indication of a reaction system. A complex pattern of enzyme was printed on paper using a thermal inkjet printer. Paper and inkjet printing are ideal material and process to manufacture low-cost-high volume bioactive surfaces.
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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.
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Nanoparticle bioconjugates as "bottom-up" assemblies of artifical multienzyme complexes
NASA Astrophysics Data System (ADS)
Keighron, Jacqueline D.
2010-11-01
The sequential enzymes of several metabolic pathways have been shown to exist in close proximity with each other in the living cell. Although not proven in all cases, colocalization may have several implications for the rate of metabolite formation. Proximity between the sequential enzymes of a metabolic pathway has been proposed to have several benefits for the overall rate of metabolite formation. These include reduced diffusion distance for intermediates, sequestering of intermediates from competing pathways and the cytoplasm. Restricted diffusion in the vicinity of an enzyme can also cause the pooling of metabolites, which can alter reaction equilibria to control the rate of reaction through inhibition. Associations of metabolic enzymes are difficult to isolate ex vivo due to the weak interactions believed to colocalize sequential enzymes within the cell. Therefore model systems in which the proximity and diffusion of intermediates within the experiment system are controlled are attractive alternatives to explore the effects of colocalization of sequential enzymes. To this end three model systems for multienzyme complexes have been constructed. Direct adsorption enzyme:gold nanoparticle bioconjugates functionalized with malate dehydrogenase (MDH) and citrate synthase (CS) allow for proximity between to the enzymes to be controlled from the nanometer to micron range. Results show that while the enzymes present in the colocalized and non-colocalized systems compared here behaved differently overall the sequential activity of the pathway was improved by (1) decreasing the diffusion distance between active sites, (2) decreasing the diffusion coefficient of the reaction intermediate to prevent escape into the bulk solution, and (3) decreasing the overall amount of bioconjugate in the solution to prevent the pathway from being inhibited by the buildup of metabolite over time. Layer-by-layer (LBL) assemblies of MDH and CS were used to examine the layering effect of sequential enzymes found in multienzyme complexes such as the pyruvate dehydrogenase complex (PDC). By controlling the orientation of enzymes in the complex (i.e. how deeply embedded each enzyme is) it was hypothesized that differences in sequential activity would determine an optimal orientation for a multienzyme complex. It was determined during the course of these experiments that the polyelectrolyte (PE) assembly itself served to slow diffusion of intermediates, leading to a buildup of oxaloacetate within the PE layers to form a pool of metabolite that equalized the rate of sequential reaction between the different orientations tested. Hexahistidine tag -- Ni(II) nitriliotriacetic acid (NTA) chemistry is an attractive method to control the proximity between sequential enzymes because each enzyme can be bound in a specific orientation, with minimal loss of activity, and the interaction is reversible. Modifying gold nanoparticles or large unilamellar vesicles with this functionality allows for another class of model to be constructed in which proximity between enzymes is dynamic. Some metabolic pathways (such as the de novo purine biosynthetic pathway), have demonstrated dynamic proximity of sequential enzymes in response to specific cellular stimuli. Results indicate that Ni(II)NTA scaffolds immobilize histidine-tagged enzymes non-destructively, with a near 100% reversibility. This model can be used to demonstrate the possible implications of dynamic proximity such as pathway regulation. Insight into the benefits and mechanisms of sequential enzyme colocalization can enhance the general understanding of cellular processes, as well as allow for the development of new and innovative ways to modulate pathway activity. This may provide new designs for treatments of metabolic diseases and cancer, where metabolic pathways are altered.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Castro, C.; Gratson, A.A.; Evans, J.C.
2010-03-05
Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind andmore » Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.« less
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2010-01-01
Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763
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Soil Carbon Inputs and Ecosystem Respiration: a Field Priming Experiment in Arctic Coastal Tundra
NASA Astrophysics Data System (ADS)
Vaughn, L. S.; Zhu, B.; Bimueller, C.; Curtis, J. B.; Chafe, O.; Bill, M.; Abramoff, R. Z.; Torn, M. S.
2016-12-01
In Arctic ecosystems, climate change is expected to influence soil carbon stocks through changes in both plant carbon inputs and organic matter decomposition. This study addresses the potential for a priming effect, an interaction between these changes in which root-derived carbon inputs alter SOM decomposition rates via microbial biomass increases, co-metabolism of substrates, induced nitrogen limitation, or other possible mechanisms. The priming effect has been observed in numerous laboratory and greenhouse experiments, and is increasingly included in ecosystem models. Few studies, however, have evaluated the priming effect with in situ field manipulations. In a two-year field experiment in Barrow, Alaska, we tested for a priming effect under natural environmental variability. In September 2014 and August 2015, we added 6.1g of 13C-labeled glucose to 25cm diameter mesocosms, 15cm below the soil surface in the mineral soil layer. Over the following month, we quantified effects on the rate and temperature sensitivity of native (non-glucose) ecosystem respiration and GPP. Following the 2014 treatment, soil samples were collected at 1 and 3 weeks for microbial biomass carbon and 13C/12C analysis, and ion exchange membranes were buried for one week to assess nitrate and ammonium availability. In contrast with many laboratory incubation studies using soils from a broad range of ecosystems, we observed no significant priming effect. In spite of a clear signal of 13C-glucose decomposition in respired CO2 and microbial biomass, we detected no treatment effect on background ecosystem respiration or total microbial biomass carbon. Our findings suggest that glucose taken up by microbes was not used for production of additional SOM-decomposing enzymes, possibly due to stoichiometric limitations on enzyme production. To best inform models representing complex and dynamic ecosystems, this study calls for further research relating theory, laboratory findings, and field experimentation.
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Multi-segmental movement patterns reflect juggling complexity and skill level.
Zago, Matteo; Pacifici, Ilaria; Lovecchio, Nicola; Galli, Manuela; Federolf, Peter Andreas; Sforza, Chiarella
2017-08-01
The juggling action of six experts and six intermediates jugglers was recorded with a motion capture system and decomposed into its fundamental components through Principal Component Analysis. The aim was to quantify trends in movement dimensionality, multi-segmental patterns and rhythmicity as a function of proficiency level and task complexity. Dimensionality was quantified in terms of Residual Variance, while the Relative Amplitude was introduced to account for individual differences in movement components. We observed that: experience-related modifications in multi-segmental actions exist, such as the progressive reduction of error-correction movements, especially in complex task condition. The systematic identification of motor patterns sensitive to the acquisition of specific experience could accelerate the learning process. Copyright © 2017 Elsevier B.V. All rights reserved.
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Thermal regeneration of an electrochemical concentration cell
Krumpelt, Michael; Bates, John K.
1981-01-01
A system and method for thermally regenerating an electrochemical concentration cell having first and second aluminum electrodes respectively positioned in contact with first and second electrolytes separated by an ion exchange member, the first and second electrolytes being composed of different concentrations of an ionic solvent and a salt, preferably an aluminum halide. The ionic solvent may be either organic or inorganic with a relatively low melting point, the ionic solvent and the salt form a complex wherein the free energy of formation of said complex is less than about -5 Kcal/mole. A distillation column using solar heat or low grade industrial waste heat receives the first and second electrolytes and thermally decomposes the salt-solvent complex to provide feed material for the two half cells.
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Early evolution of efficient enzymes and genome organization
2012-01-01
Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight. PMID:23114029
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Multienzyme kinetics and sequential metabolism.
Wienkers, Larry C; Rock, Brooke
2014-01-01
Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYP) enzymes are catalytically promiscuous, and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appears to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein is a discussion of two distinct types of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.
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USDA-ARS?s Scientific Manuscript database
Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...
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A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.
Mashimo, Yasumasa; Mie, Masayasu; Kobatake, Eiry
2018-04-01
We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.
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Temperature Sensitivities of Extracellular Enzyme Vmax and Km Across Thermal Environments
NASA Astrophysics Data System (ADS)
Allison, S. D.; Romero-Olivares, A.; Lu, Y.; Taylor, J.; Treseder, K. K.
2017-12-01
The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivity of microbial processes in soil. Enzymatic rates could increase at higher temperatures, but this response is determined by multiple parameters that may change over time if soil microbes adapt to warming. We used the Michaelis-Menten relationship, the Arrhenius relationship, and biochemical transition state theory to construct hypotheses about the responses of extracellular enzyme Vmax and Km to temperature. Based on the Arrhenius relationship, we hypothesized that Vmax and Km would show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower Vmax, Km, and Km temperature sensitivity but higher Vmax temperature sensitivity. We tested these hypotheses with enzymes from isolates of the filamentous fungus Neurospora discreta collected around the globe and from decomposing leaf litter in a warming experiment in Alaskan boreal forest. Vmax and Km of most Neurospora extracellular enzymes were temperature sensitive with average Vmax Q10 ranging from 1.48 to 2.25 and Km Q10 ranging from 0.71 to 2.80. For both Vmax and Km, there was a tendency for the parameter to correlate negatively with its temperature sensitivity, a pattern predicted by biochemical theory. Also in agreement with theory, Vmax and Km were positively correlated for some enzymes. In contrast, there was little support for biochemical theory when comparing Vmax and Km across thermal environments. There was no relationship between temperature sensitivity of Vmax or Km and mean annual temperature of the isolation site for Neurospora strains. There was some evidence for greater Vmax under experimental warming in Alaskan litter, but the temperature sensitivities of Vmax and Km did not vary with warming as expected. We conclude that relationships among Vmax, Km, and temperature are largely consistent with biochemical theory, and our enzyme data should be useful for parameterizing trait-based models of microbial processes. However, theoretical predictions about adaptation to thermal environment were not supported by our data, suggesting that covarying edaphic and ecological factors may play a dominant role in soil enzyme responses to climate warming.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi
The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P{submore » R} and P{sub S} stereoisomers at the P-chiral center. The tabun complex displayed only the P{sub R} stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.« less
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Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J
2009-08-15
The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.
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Decomposed bodies--still an unrewarding autopsy?
Ambade, Vipul Namdeorao; Keoliya, Ajay Narmadaprasad; Deokar, Ravindra Baliram; Dixit, Pradip Gangadhar
2011-04-01
One of the classic mistakes in forensic pathology is to regard the autopsy of decomposed body as unrewarding. The present study was undertaken with a view to debunk this myth and to determine the characteristic pattern in decomposed bodies brought for medicolegal autopsy. From a total of 4997 medicolegal deaths reported at an Apex Medical Centre, Yeotmal, a rural district of Maharashtra over seven year study period, only 180 cases were decomposed, representing 3.6% of the total medicolegal autopsies with the rate of 1.5 decomposed body/100,000 population per year. Male (79.4%) predominance was seen in decomposed bodies with male female ratio of 3.9:1. Most of the victims were between the ages of 31 and 60 years with peak at 31-40 years (26.7%) followed by 41-50 years (19.4%). Older age above 60 years was found in 8.6% cases. Married (64.4%) outnumbered unmarried ones in decomposition. Most of the decomposed bodies were complete (83.9%) and identified (75%). But when the body was incomplete/mutilated or skeletonised then 57.7% of the deceased remains unidentified. The cause and manner of death was ascertained in 85.6% and 81.1% cases respectively. Drowning (35.6%) was the commonest cause of death in decomposed bodies with suicide (52.8%) as the commonest manner of death. Decomposed bodies were commonly recovered from open places (43.9%), followed by water sources (43.3%) and enclosed place (12.2%). Most of the decomposed bodies were retrieved from well (49 cases) followed by barren land (27 cases) and forest (17 cases). 83.8% of the decomposed bodies were recovered before 72 h and only in 16.2% cases the time since death was more than 72 h, mostly recovered from barren land, forest and river. Most of the decomposed bodies were found in summer season (42.8%) with peak in the month of May. Despite technical difficulties in handling the body and artefactual alteration of the tissue, the decomposed body may still reveal cause and manner of death in significant number of cases. Copyright © 2011 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
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Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger
2017-01-01
How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.
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Refojo, Patrícia N; Teixeira, Miguel; Pereira, Manuela M
2012-10-01
Alternative complexes III (ACIII) are recently identified membrane-bound enzymes that replace functionally the cytochrome bc(1/)b(6)f complexes. In general, ACIII are composed of four transmembrane proteins and three peripheral subunits that contain iron-sulfur centers and C-type hemes. ACIII are built by a combination of modules present in different enzyme families, namely the complex iron-sulfur molybdenum containing enzymes. In this article a historical perspective on the investigation of ACIII is presented, followed by an overview of the present knowledge on these enzymes. Electron transfer pathways within the protein are discussed taking into account possible different locations (cytoplasmatic or periplasmatic) of the iron-sulfur containing protein and their contribution to energy conservation. In this way several hypotheses for energy conservation modes are raised including linear and bifurcating electron transfer pathways. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.
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Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes
Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat
2015-01-01
Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736
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Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.
Liu, Lei; Wen, Wei; Zhang, Ruifen; Wei, Zhencheng; Deng, Yuanyuan; Xiao, Juan; Zhang, Mingwei
2017-01-01
In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Kim, Kwang-Wook; Lee, Keun-Young; Chung, Dong-Yong; Lee, Eil-Hee; Moon, Jei-Kwon; Shin, Dong-Woo
2012-09-30
This work studied the stability of peroxide in uranyl peroxo carbonato complex ions in a carbonate solution with hydrogen peroxide using absorption and Raman spectroscopies, and evaluated the temperature dependence of the decomposition characteristics of uranyl peroxo carbonato complex ions in the solution. The uranyl peroxo carbonato complex ions self-decomposed more rapidly into uranyl tris-carbonato complex ions in higher temperature carbonate solutions. The concentration of peroxide in the solution without free hydrogen peroxide represents the concentration of uranyl peroxo carbonato complex ions in a mixture of uranyl peroxo carbonato complex and uranyl tris-carbonato complex ions. The self-decomposition of the uranyl peroxo carbonato complex ions was a first order reaction, and its activation energy was evaluated to be 7.144×10(3) J mol(-1). The precipitation of sodium uranium oxide hydroxide occurred when the amount of uranyl tris-carbonato complex ions generated from the decomposition of the uranyl peroxo carbonato complex ions exceeded the solubility of uranyl tris-carbonato ions in the solution at the solution temperature. Copyright © 2012 Elsevier B.V. All rights reserved.
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Microbial Activity and Silica Degradation in Rice Straw
NASA Astrophysics Data System (ADS)
Kim, Esther Jin-kyung
Abundantly available agricultural residues like rice straw have the potential to be feedstocks for bioethanol production. Developing optimized conditions for rice straw deconstruction is a key step toward utilizing the biomass to its full potential. One challenge associated with conversion of rice straw to bioenergy is its high silica content as high silica erodes machinery. Another obstacle is the availability of enzymes that hydrolyze polymers in rice straw under industrially relevant conditions. Microbial communities that colonize compost may be a source of enzymes for bioconversion of lignocellulose to products because composting systems operate under thermophilic and high solids conditions that have been shown to be commercially relevant. Compost microbial communities enriched on rice straw could provide insight into a more targeted source of enzymes for the breakdown of rice straw polysaccharides and silica. Because rice straw is low in nitrogen it is important to understand the impact of nitrogen concentrations on the production of enzyme activity by the microbial community. This study aims to address this issue by developing a method to measure microbial silica-degrading activity and measure the effect of nitrogen amendment to rice straw on microbial activity and extracted enzyme activity during a high-solids, thermophilic incubation. An assay was developed to measure silica-degrading enzyme or silicase activity. This process included identifying methods of enzyme extraction from rice straw, identifying a model substrate for the assay, and optimizing measurement techniques. Rice straw incubations were conducted with five different levels of nitrogen added to the biomass. Microbial activity was measured by respiration and enzyme activity. A microbial community analysis was performed to understand the shift in community structure with different treatments. With increased levels of nitrogen, respiration and cellulose and hemicellulose degrading activity increased. Silicase activity did not change across nitrogen treatments despite a shift in microbial community with varied nitrogen concentration. Samples treated with different nitrogen concentrations had similar levels of diversity, however the microbial community composition differed with added nitrogen. The results demonstrated that adding nitrogen to rice straw during thermophilic decomposition nurtured a more active microbial community and promoted enzyme secretion thus improving the ability to discover enzymes for rice straw deconstruction. These results can inform future experiments for cultivating a unique, thriving compost-derived microbial community that can successfully decompose rice straw. Understanding the silicase activity of microorganisms may alleviate the challenges associated with silica in various feedstocks.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Volkov, Oleg A.; Kinch, Lisa; Ariagno, Carson
Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures ofTrypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomericTbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving acis-to-transproline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanismmore » was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.« less
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Cell endogenous activities of fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5.
Nishihara, Ryuta; Kobayashi, Kazuhiro; Imae, Rieko; Tsumoto, Hiroki; Manya, Hiroshi; Mizuno, Mamoru; Kanagawa, Motoi; Endo, Tamao; Toda, Tatsushi
2018-03-18
Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function. Copyright © 2018 Elsevier Inc. All rights reserved.
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Fungal-to-bacterial dominance of soil detrital food-webs: Consequences for biogeochemistry
NASA Astrophysics Data System (ADS)
Rousk, Johannes; Frey, Serita
2015-04-01
Resolving fungal and bacterial groups within the microbial decomposer community is thought to capture disparate microbial life strategies, associating bacteria with an r-selected strategy for carbon (C) and nutrient use, and fungi with a K-selected strategy. Additionally, food-web models have established a widely held belief that the bacterial decomposer pathway in soil supports high turnover rates of easily available substrates, while the slower fungal pathway supports the decomposition of more complex organic material, thus characterising the biogeochemistry of the ecosystem. Three field-experiments to generate gradients of SOC-quality were assessed. (1) the Detritus Input, Removal, and Trenching - DIRT - experiment in a temperate forest in mixed hardwood stands at Harvard Forest LTER, US. There, experimentally adjusted litter input and root input had affected the SOC quality during 23 years. (2) field-application of 14-C labelled glucose to grassland soils, sampled over the course of 13 months to generate an age-gradient of SOM (1 day - 13 months). (3) The Park Grass Experiment at Rothamsted, UK, where 150-years continuous N-fertilisation (0, 50, 100, 150 kg N ha-1 y-1) has affected the quality of SOM in grassland soils. A combination of carbon stable and radio isotope studies, fungal and bacterial growth and biomass measurements, and C and N mineralisation (15N pool dilution) assays were used to investigate how SOC-quality influenced fungal and bacterial food-web pathways and the implications this had for C and nutrient turnover. There was no support that decomposer food-webs dominated by bacteria support high turnover rates of easily available substrates, while slower fungal-dominated decomposition pathways support the decomposition of more complex organic material. Rather, an association between high quality SOC and fungi emerges from the results. This suggests that we need to revise our basic understanding for soil microbial communities and the processes they regulate in soil.
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Luminescence of europium (III) complexes for visualization
NASA Astrophysics Data System (ADS)
Kolontaeva, Olga A.; Pozharov, Mikhail V.; Korolovich, Vladimir F.; Khokhlova, Anastasia R.; Kirdyanova, Anna N.; Burmistrova, Natalia A.; Zakharova, Tamara V.; Goryacheva, Irina Y.
2016-04-01
With the purpose to develop bright non-toxic luminescent label for theranostic application we have studied complexation of lanthanide dipicolinates (2,6-pyridinedicarboxylates) by sodium alginate and effect of thermal exposure of synthesized micro-capsules on their luminescent properties. Synthesized micro-capsules are stable in acidic medium but dissolve at pH ~ 4 due to transformation of cationic europium dipicolinate complex to anionic. Luminescence studies have shown that emission spectra of europium(III)-alginate complexes (both chloride and dipicolinate) contain two intensive bands characteristic to Eu3+ ion (5D0 --> 7F1 (590 nm) and 5D0 --> 7F1 (612 nm)). We have also found that at 160ºC europium(III)- alginate micro-capsules decompose to black, soot-like substance, therefore, their thermal treatment must be performed in closed environment (i.e., sealed ampoules).
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Ecology of coarse wood decomposition by the saprotrophic fungus Fomes fomentarius.
Větrovský, Tomáš; Voříšková, Jana; Snajdr, Jaroslav; Gabriel, Jiří; Baldrian, Petr
2011-07-01
Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.
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NASA Astrophysics Data System (ADS)
Ostrovtsova, Svetlana A.; Volodenkov, Alexander P.; Maskevich, Alexander A.; Artsukevich, Irina M.; Anufrik, Slavomir S.; Makarchikov, Alexander F.; Chernikevich, Ivan P.; Stepuro, Vitali I.
1998-05-01
Three enzymes differing in their structural composition were irradiated by UV lasers to study the effect of temperature, protein concentration and addition of small molecules on their sensitivity to radiation exposure. The laser-induced effects were due to the structural complexity of the protein molecules and depended on the dose applied, the wavelength and the density of irradiation. The multi-enzyme 2- oxoglutarate dehydrogenase complex was subjected to pronounced irradiation-induced changes whereas the response of the two other enzymes was less significant. Reduction of the protein levels in irradiated samples was important under the XeCl laser coercion and the effects depended on the doses applied. The laser irradiation effects are suggested to be realized by means of conformational changes in the protein molecules and intermolecular association- dissociation processes.
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USDA-ARS?s Scientific Manuscript database
Two experiments were conducted to determine the effects of treating sorghum WDG with solubles (SWDG) with an enzyme, or enzyme-buffer combination on diet digestibility and feedlot performance. Experimental treatments are; 1) untreated SWDG (control), 2) addition of an enzyme complex to SWDG (enzyme...
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Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass
dos Santos, Hevila Brognaro; Bezerra, Thais Milena Souza; Pradella, Jose G. C.; ...
2016-11-02
Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitativemore » protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. Furthermore, the participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.« less
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Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass
DOE Office of Scientific and Technical Information (OSTI.GOV)
dos Santos, Hevila Brognaro; Bezerra, Thais Milena Souza; Pradella, Jose G. C.
Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitativemore » protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. Furthermore, the participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.« less
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Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio
2011-04-01
Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.
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Grosse, R; Eckert, K; Malur, J; Repke, K R
1978-01-01
The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.
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Szymula, M
2004-01-01
The antioxidant efficiency of two hydrophilic species, ascorbic acid (AA) and propyl gallate (PG), in an anionic surfactant system are studied. Ascorbic acid and propyl gallate are dissolved/solubilized in a microemulsion formed by water, pentanol, and sodium dodecyl sulfate. The determination of propyl gallate decomposition/oxidation kinetics shows enhanced oxidation of PG with increasing pentanol concentration in the system. When ascorbic acid and propyl gallate are both present in water, in surfactant aqueous solution, and in the studied microemulsion systems, the molecular complex AAPG is formed. After some time the complex decomposes.
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Localized motion in random matrix decomposition of complex financial systems
NASA Astrophysics Data System (ADS)
Jiang, Xiong-Fei; Zheng, Bo; Ren, Fei; Qiu, Tian
2017-04-01
With the random matrix theory, we decompose the multi-dimensional time series of complex financial systems into a set of orthogonal eigenmode functions, which are classified into the market mode, sector mode, and random mode. In particular, the localized motion generated by the business sectors, plays an important role in financial systems. Both the business sectors and their impact on the stock market are identified from the localized motion. We clarify that the localized motion induces different characteristics of the time correlations for the stock-market index and individual stocks. With a variation of a two-factor model, we reproduce the return-volatility correlations of the eigenmodes.
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Polymethacrylic acid as a new precursor of CuO nanoparticles
NASA Astrophysics Data System (ADS)
Hosny, Nasser Mohammed; Zoromba, Mohamed Shafick
2012-11-01
Polymethacrylic acid and its copper complexes have been synthesized and characterized. These complexes have been used as precursors to produce CuO nanoparticles by thermal decomposition in air. The stages of decompositions and the calcination temperature of the precursors have been determined from thermal analyses (TGA). The obtained CuO nanoparticles have been characterized by X-ray diffraction (XRD), scanning tunneling microscopy (STM) and transmission electron microscopy (TEM). XRD showed a monoclinic structure with particle size 8-20 nm for the synthesized copper oxide nanoparticles. These nanoparticles are catalytically active in decomposing hydrogen peroxide and a mechanism of decomposition has been suggested.
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Chutani, Preeti; Sharma, Krishna Kant
2016-05-01
Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil having decomposed leaf litter and compost samples collected from different regions of India. Fifteen fungal cultures were selected qualitatively for the production of xylanase and cellulases and were identified employing ITS, NS and MNS primers. The enzyme cocktail consisting of 3811 IU g(-1) of xylanase and 9.9 IU g(-1) of cellulase from Trichoderma longibrachiatum MDU-6 was selected quantitatively for the deinking of diverse paper wastes. The enzyme production increased two fold when produced at tray level in comparison with flasks. The enzyme cocktail was effective in the deinking of old newspaper samples with significant removal of chromophores, phenolics and hydrophobic compounds and less sugar loss. While in case of examination papers and laser printed papers, ink removal was not very significant. Moreover, the sugar loss was significantly high in case of examination papers. The deinking results were further confirmed with FTIR analysis. Deinked newspaper pulp sample shows brightness of 52%, which was 9.6% high than its control sample. The ERIC value for deinked newspaper pulp was found to be 655.9 ppm. Thereafter, the deinked newspaper pulp was examined under light microscope after differential staining with safranin and malachite green and also examined under scanning and transmission electron microscope, which revealed fibrillation and perforation.
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2017-12-01
inefficiencies of a more complex system. Additional time may also be due to the longer distances traveled . The fulfillment time for a requisition to...Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time ...advanced manufacturing methods with additive manufacturing. This work decomposes the additive manufacturing processes into 11 primary functions. The time
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Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata
2017-03-01
The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.
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Preuveneers, M. J.; Peacock, D.; Crook, E. M.; Clark, J. B.; Brocklehurst, K.
1973-01-01
1. The reversible NAD+-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0°C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate–NAD+ oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2μmol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme–nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme–nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme. PMID:4352835
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Uznanski, Pawel; Zakrzewska, Joanna; Favier, Frederic; Kazmierski, Slawomir; Bryszewska, Ewa
2017-01-01
A comparative study of amine and silver carboxylate adducts [R 1 COOAg-2(R 2 NH 2 )] (R 1 = 1, 7, 11; R 2 = 8, 12) as a key intermediate in NPs synthesis is carried out via differential scanning calorimetry, solid-state FT-infrared spectroscopy, 13 C CP MAS NMR, powder X-ray diffraction and X-ray photoelectron spectroscopy, and various solution NMR spectroscopies ( 1 H and 13 C NMR, pulsed field gradient spin-echo NMR, and ROESY). It is proposed that carboxyl moieties in the presence of amine ligands are bound to silver ions via chelating bidentate type of coordination as opposed to bridging bidentate coordination of pure silver carboxylates resulting from the formation of dimeric units. All complexes are packed as lamellar bilayer structures. Silver carboxylate/amine complexes show one first-order melting transition. The evidence presented in this study shows that phase behavior of monovalent metal carboxylates are controlled, mainly, by head group bonding. In solution, insoluble silver salt is stabilized by amine molecules which exist in dynamic equilibrium. Using (bis)amine-silver carboxylate complex as precursor, silver nanoparticles were fabricated. During high-temperature thermolysis, the (bis)amine-carboxylate adduct decomposes to produce silver nanoparticles of small size. NPs are stabilized by strongly interacting carboxylate and trace amounts of amine derived from the silver precursor interacting with carboxylic acid. A corresponding aliphatic amide obtained from silver precursor at high-temperature reaction conditions is not taking part in the stabilization. Combining NMR techniques with FTIR, it was possible to follow an original stabilization mechanism. Graphical abstractThe synthesis of a series (bis)alkylamine silver(I) carboxylate complexes in nonpolar solvents were carried out and fully characterized both in the solid and solution. Carboxyl moieties in the presence of amine ligands are bound to silver ions via chelating bidentate type of coordination. The complexes form layered structures which thermally decompose forming nanoparticles stabilized only by aliphatic carboxylates.
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Optimal reconstruction of the states in qutrit systems
NASA Astrophysics Data System (ADS)
Yan, Fei; Yang, Ming; Cao, Zhuo-Liang
2010-10-01
Based on mutually unbiased measurements, an optimal tomographic scheme for the multiqutrit states is presented explicitly. Because the reconstruction process of states based on mutually unbiased states is free of information waste, we refer to our scheme as the optimal scheme. By optimal we mean that the number of the required conditional operations reaches the minimum in this tomographic scheme for the states of qutrit systems. Special attention will be paid to how those different mutually unbiased measurements are realized; that is, how to decompose each transformation that connects each mutually unbiased basis with the standard computational basis. It is found that all those transformations can be decomposed into several basic implementable single- and two-qutrit unitary operations. For the three-qutrit system, there exist five different mutually unbiased-bases structures with different entanglement properties, so we introduce the concept of physical complexity to minimize the number of nonlocal operations needed over the five different structures. This scheme is helpful for experimental scientists to realize the most economical reconstruction of quantum states in qutrit systems.
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An integrated condition-monitoring method for a milling process using reduced decomposition features
NASA Astrophysics Data System (ADS)
Liu, Jie; Wu, Bo; Wang, Yan; Hu, Youmin
2017-08-01
Complex and non-stationary cutting chatter affects productivity and quality in the milling process. Developing an effective condition-monitoring approach is critical to accurately identify cutting chatter. In this paper, an integrated condition-monitoring method is proposed, where reduced features are used to efficiently recognize and classify machine states in the milling process. In the proposed method, vibration signals are decomposed into multiple modes with variational mode decomposition, and Shannon power spectral entropy is calculated to extract features from the decomposed signals. Principal component analysis is adopted to reduce feature size and computational cost. With the extracted feature information, the probabilistic neural network model is used to recognize and classify the machine states, including stable, transition, and chatter states. Experimental studies are conducted, and results show that the proposed method can effectively detect cutting chatter during different milling operation conditions. This monitoring method is also efficient enough to satisfy fast machine state recognition and classification.
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Augmenting the decomposition of EMG signals using supervised feature extraction techniques.
Parsaei, Hossein; Gangeh, Mehrdad J; Stashuk, Daniel W; Kamel, Mohamed S
2012-01-01
Electromyographic (EMG) signal decomposition is the process of resolving an EMG signal into its constituent motor unit potential trains (MUPTs). In this work, the possibility of improving the decomposing results using two supervised feature extraction methods, i.e., Fisher discriminant analysis (FDA) and supervised principal component analysis (SPCA), is explored. Using the MUP labels provided by a decomposition-based quantitative EMG system as a training data for FDA and SPCA, the MUPs are transformed into a new feature space such that the MUPs of a single MU become as close as possible to each other while those created by different MUs become as far as possible. The MUPs are then reclassified using a certainty-based classification algorithm. Evaluation results using 10 simulated EMG signals comprised of 3-11 MUPTs demonstrate that FDA and SPCA on average improve the decomposition accuracy by 6%. The improvement for the most difficult-to-decompose signal is about 12%, which shows the proposed approach is most beneficial in the decomposition of more complex signals.
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Using "big data" to optimally model hydrology and water quality across expansive regions
Roehl, E.A.; Cook, J.B.; Conrads, P.A.
2009-01-01
This paper describes a new divide and conquer approach that leverages big environmental data, utilizing all available categorical and time-series data without subjectivity, to empirically model hydrologic and water-quality behaviors across expansive regions. The approach decomposes large, intractable problems into smaller ones that are optimally solved; decomposes complex signals into behavioral components that are easier to model with "sub- models"; and employs a sequence of numerically optimizing algorithms that include time-series clustering, nonlinear, multivariate sensitivity analysis and predictive modeling using multi-layer perceptron artificial neural networks, and classification for selecting the best sub-models to make predictions at new sites. This approach has many advantages over traditional modeling approaches, including being faster and less expensive, more comprehensive in its use of available data, and more accurate in representing a system's physical processes. This paper describes the application of the approach to model groundwater levels in Florida, stream temperatures across Western Oregon and Wisconsin, and water depths in the Florida Everglades. ?? 2009 ASCE.
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Multiscale structure of time series revealed by the monotony spectrum.
Vamoş, Călin
2017-03-01
Observation of complex systems produces time series with specific dynamics at different time scales. The majority of the existing numerical methods for multiscale analysis first decompose the time series into several simpler components and the multiscale structure is given by the properties of their components. We present a numerical method which describes the multiscale structure of arbitrary time series without decomposing them. It is based on the monotony spectrum defined as the variation of the mean amplitude of the monotonic segments with respect to the mean local time scale during successive averagings of the time series, the local time scales being the durations of the monotonic segments. The maxima of the monotony spectrum indicate the time scales which dominate the variations of the time series. We show that the monotony spectrum can correctly analyze a diversity of artificial time series and can discriminate the existence of deterministic variations at large time scales from the random fluctuations. As an application we analyze the multifractal structure of some hydrological time series.
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NASA Astrophysics Data System (ADS)
Korotkova, T. I.; Popova, V. I.
2017-11-01
The generalized mathematical model of decision-making in the problem of planning and mode selection providing required heat loads in a large heat supply system is considered. The system is multilevel, decomposed into levels of main and distribution heating networks with intermediate control stages. Evaluation of the effectiveness, reliability and safety of such a complex system is carried out immediately according to several indicators, in particular pressure, flow, temperature. This global multicriteria optimization problem with constraints is decomposed into a number of local optimization problems and the coordination problem. An agreed solution of local problems provides a solution to the global multicriterion problem of decision making in a complex system. The choice of the optimum operational mode of operation of a complex heat supply system is made on the basis of the iterative coordination process, which converges to the coordinated solution of local optimization tasks. The interactive principle of multicriteria task decision-making includes, in particular, periodic adjustment adjustments, if necessary, guaranteeing optimal safety, reliability and efficiency of the system as a whole in the process of operation. The degree of accuracy of the solution, for example, the degree of deviation of the internal air temperature from the required value, can also be changed interactively. This allows to carry out adjustment activities in the best way and to improve the quality of heat supply to consumers. At the same time, an energy-saving task is being solved to determine the minimum required values of heads at sources and pumping stations.
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The crystallogenesis of a human estradiol dehydrogenase-substrate complex
NASA Astrophysics Data System (ADS)
Zhu, Dao-Wei; Azzi, Arezki; Rehse, Peter; Lin, Sheng-Xiang
1996-10-01
Human 17β-hydroxysteroid dehydrogenase type 1 is an important steroidogenic enzyme catalyzing the synthesis of the most active estrogen: estradiol. The enzyme is formed by two identical subunits (34.5 kDa). In this paper, we report the preparation of a stoichiometric 17β-HSD1-estradiol complex sample at a much higher concentration than the solubility of the free substrate, using a gradual concentration of the enzyme-substrate mixture starting at low concentration. The complex is successfully crystallized by vapor diffusion at pH 7.5 with polyethyleneglycol 4000 as the precipitating agent. The space group is C2 with a = 123.56 Å, b = 45.21 Å, c = 61.30 Å and β = 99.06°. There is one monomer in the asymmetric unit and two molecules of the enzyme in a unit cell. A diffraction data set to 2.5 Å has been collected to 86% completeness on native crystals. The high quality of the electronic density map of estradiol supports the full occupancy of the binding site, thus confirming the efficiency of the complex preparation. This method will also be useful in crystallizing other steroid-dehydrogenase complexes.
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Reed, James R.; Backes, Wayne L.
2017-01-01
Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450–P450 interactions. PMID:28194112
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Computational multiscale modeling in protein--ligand docking.
Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles
2009-01-01
In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].
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Hoeprich, Paul D.; Whalen, Maureen
2016-04-05
Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.
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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.
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Forward design of a complex enzyme cascade reaction
Hold, Christoph; Billerbeck, Sonja; Panke, Sven
2016-01-01
Enzymatic reaction networks are unique in that one can operate a large number of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biological processes such as glycolysis. For this, we adapt the characterization of the biochemical system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply standard system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chemical production purposes. PMID:27677244
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INTERRELATION BETWEEN ACTIVATION AND POLYMERIZATION IN GRAMICIDIN S BIOSYNTHESIS*
Kleinkauf, Horst; Gevers, Wieland; Lipmann, Fritz
1969-01-01
The nucleic acid-independent biosynthesis of the peptide antibiotic gramicidin S results from the interaction of an enzyme bearing phenylalanine in activated form with a polyenzyme system charged with the other four component amino acids. After reaction with ATP, magnesium, and any or all of its amino acid substrates, the polyenzyme system (mol wt 280,000) yields complexes containing AMP and the respective amino acids in the proportion of 1 to 2. Similar complexes are formed by another enzyme (mol wt 100,000) on incubation with ATP, magnesium, and L- or D-phenylalanine. The amino acids are probably bound as aminoacyl adenylates and then transferred to another function on the enzyme. Initiation of polymerization is achieved by combination of the two complexes. No ATP is needed for completion of synthesis, and free intermediates are not released. Enzyme organization and specificity are responsible for the ordering of the amino acid sequence. PMID:5253659
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Reduced Toxicity Fuel Satellite Propulsion System
NASA Technical Reports Server (NTRS)
Schneider, Steven J. (Inventor)
2001-01-01
A reduced toxicity fuel satellite propulsion system including a reduced toxicity propellant supply for consumption in an axial class thruster and an ACS class thruster. The system includes suitable valves and conduits for supplying the reduced toxicity propellant to the ACS decomposing element of an ACS thruster. The ACS decomposing element is operative to decompose the reduced toxicity propellant into hot propulsive gases. In addition the system includes suitable valves and conduits for supplying the reduced toxicity propellant to an axial decomposing element of the axial thruster. The axial decomposing element is operative to decompose the reduced toxicity propellant into hot gases. The system further includes suitable valves and conduits for supplying a second propellant to a combustion chamber of the axial thruster, whereby the hot gases and the second propellant auto-ignite and begin the combustion process for producing thrust.
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Reduced Toxicity Fuel Satellite Propulsion System Including Plasmatron
NASA Technical Reports Server (NTRS)
Schneider, Steven J. (Inventor)
2003-01-01
A reduced toxicity fuel satellite propulsion system including a reduced toxicity propellant supply for consumption in an axial class thruster and an ACS class thruster. The system includes suitable valves and conduits for supplying the reduced toxicity propellant to the ACS decomposing element of an ACS thruster. The ACS decomposing element is operative to decompose the reduced toxicity propellant into hot propulsive gases. In addition the system includes suitable valves and conduits for supplying the reduced toxicity propellant to an axial decomposing element of the axial thruster. The axial decomposing element is operative to decompose the reduced toxicity propellant into hot gases. The system further includes suitable valves and conduits for supplying a second propellant to a combustion chamber of the axial thruster. whereby the hot gases and the second propellant auto-ignite and begin the combustion process for producing thrust.
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Pan, Xu; Cornelissen, Johannes H C; Zhao, Wei-Wei; Liu, Guo-Fang; Hu, Yu-Kun; Prinzing, Andreas; Dong, Ming; Cornwell, William K
2014-09-01
Leaf litter decomposability is an important effect trait for ecosystem functioning. However, it is unknown how this effect trait evolved through plant history as a leaf 'afterlife' integrator of the evolution of multiple underlying traits upon which adaptive selection must have acted. Did decomposability evolve in a Brownian fashion without any constraints? Was evolution rapid at first and then slowed? Or was there an underlying mean-reverting process that makes the evolution of extreme trait values unlikely? Here, we test the hypothesis that the evolution of decomposability has undergone certain mean-reverting forces due to strong constraints and trade-offs in the leaf traits that have afterlife effects on litter quality to decomposers. In order to test this, we examined the leaf litter decomposability and seven key leaf traits of 48 tree species in the temperate area of China and fitted them to three evolutionary models: Brownian motion model (BM), Early burst model (EB), and Ornstein-Uhlenbeck model (OU). The OU model, which does not allow unlimited trait divergence through time, was the best fit model for leaf litter decomposability and all seven leaf traits. These results support the hypothesis that neither decomposability nor the underlying traits has been able to diverge toward progressively extreme values through evolutionary time. These results have reinforced our understanding of the relationships between leaf litter decomposability and leaf traits in an evolutionary perspective and may be a helpful step toward reconstructing deep-time carbon cycling based on taxonomic composition with more confidence.
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A bacterial hydrogen-dependent CO2 reductase forms filamentous structures.
Schuchmann, Kai; Vonck, Janet; Müller, Volker
2016-04-01
Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 μm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state. © 2016 Federation of European Biochemical Societies.
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Thermal regeneration of an electrochemical concentration cell
Krumpelt, M.; Bates, J.K.
1980-05-09
A system and method are described for thermally regenerating an electrochemical concentration cell having first and second aluminum electrodes respectively positioned in contact with first and second electrolytes separated by an ion exchange member, the first and second electrolytes being composed of different concentrations of an ionic solvent and a salt, preferably an aluminum halide. The ionic solvent may be either organic or inorganic with a relatively low melting point, the ionic solvent and the salt form a complex wherein the free energy of formation of said complex is less than about -5 kcal/mole. A distillation column using solar heat or low grade industrial waste heat receives the first and second electrolytes and thermally decomposes the salt-solvent complex to provide feed material for the two half cells.
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Overset grid applications on distributed memory MIMD computers
NASA Technical Reports Server (NTRS)
Chawla, Kalpana; Weeratunga, Sisira
1994-01-01
Analysis of modern aerospace vehicles requires the computation of flowfields about complex three dimensional geometries composed of regions with varying spatial resolution requirements. Overset grid methods allow the use of proven structured grid flow solvers to address the twin issues of geometrical complexity and the resolution variation by decomposing the complex physical domain into a collection of overlapping subdomains. This flexibility is accompanied by the need for irregular intergrid boundary communication among the overlapping component grids. This study investigates a strategy for implementing such a static overset grid implicit flow solver on distributed memory, MIMD computers; i.e., the 128 node Intel iPSC/860 and the 208 node Intel Paragon. Performance data for two composite grid configurations characteristic of those encountered in present day aerodynamic analysis are also presented.
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A Global Approach to the Optimal Trajectory Based on an Improved Ant Colony Algorithm for Cold Spray
NASA Astrophysics Data System (ADS)
Cai, Zhenhua; Chen, Tingyang; Zeng, Chunnian; Guo, Xueping; Lian, Huijuan; Zheng, You; Wei, Xiaoxu
2016-12-01
This paper is concerned with finding a global approach to obtain the shortest complete coverage trajectory on complex surfaces for cold spray applications. A slicing algorithm is employed to decompose the free-form complex surface into several small pieces of simple topological type. The problem of finding the optimal arrangement of the pieces is translated into a generalized traveling salesman problem (GTSP). Owing to its high searching capability and convergence performance, an improved ant colony algorithm is then used to solve the GTSP. Through off-line simulation, a robot trajectory is generated based on the optimized result. The approach is applied to coat real components with a complex surface by using the cold spray system with copper as the spraying material.
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Kudryashova, Elena V; Artemova, Tatiana M; Vinogradov, Alexei A; Gladilin, Alexander K; Mozhaev, Vadim V; Levashov, Andrey V
2003-04-01
Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.
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Pick, Frances M.; Bray, R. C.
1969-01-01
The origin of the Rapid molybdenum electron-paramagnetic-resonance signals, which are obtained on reducing xanthine oxidase with purine or with xanthine, and whose parameters were measured by Bray & Vänngård (1969), was studied. It is concluded that these signals represent complexes of reduced enzyme with substrate molecules. Xanthine forms one complex at high concentrations and a different one at low concentrations. Purine forms a complex indistinguishable from the low-concentration xanthine complex. There are indications that some other substrates also form complexes, but uric acid, a reaction product, does not appear to do so. The possible significance of the complexes in the catalytic cycle of the enzyme is discussed and it is suggested that they represent substrate molecules bound at the reduced active site, waiting their turn to react there, when the enzyme has been reoxidized. Support for this role for the complexes was deduced from experiments in which frozen samples of enzyme–xanthine mixtures, prepared by the rapid-freezing method, were warmed until the signals began to change. Under these conditions an increase in amplitude of the Very Rapid signal took place. Data bearing on the origin of the Slow molybdenum signal are also discussed. This signal disappears only slowly in the presence of oxygen, and its appearance rate is unaffected by change in the concentration of dithionite. It is concluded that, like other signals from the enzyme, it is due to Mov but that a slow change of ligand takes place before it is seen. The Slow species, like the Rapid, seems capable of forming complexes with purines. PMID:4310056
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Wuxiuer, Yimingjiang; Morgunova, Ekaterina; Cols, Neus; Popov, Alexander; Karshikoff, Andrey; Sylte, Ingebrigt; Gonzàlez-Duarte, Roser; Ladenstein, Rudolf; Winberg, Jan-Olof
2012-08-01
All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis. © 2012 The Authors Journal compilation © 2012 FEBS.
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The nature and function of microbial enzymes in subsurface marine sediments
NASA Astrophysics Data System (ADS)
Steen, A. D.; Schmidt, J.
2016-02-01
Isotopic and genomic evidence indicates that marine sediments contain populations of active heterotrophic microorganisms which appear to metabolize old, detrital, apparently recalcitrant organic matter. In surface communities, heterotrophs use extracellular enzymes to access complex organic matter. In subsurface sediments, in which microbial doubling times can be on the order of hundreds or thousands of years, it is not clear whether extracellular enzymes could remain stable and active long enough to constitute a 'profitable' stragtegy for accessing complex organic carbon. Here we present evidence that a wide range of extracellular enzyme are active in subsurface sediments from two different environments: the White Oak River, NC, and deep (up to 80 m) sediments of the Baltic Sea Basin recovered from IODP Expedition 347. In the White Oak River, enzymes from deeper sediments appear to be better-adapted to highly-degraded organic matter than enzymes from surface sediments. In the Baltic Sea, preliminary data suggest that enzymes related to nitrogen acquisition are preferentially expressed. By characterizing the extracellular enzymes present in marine sediments, we hope to achieve a better understanding of the mechanisms that control sedimentary organic matter remineralization and preservation.
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Mishra, Arjun K; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J Venkatesh
2015-01-09
Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. Copyright © 2014 Elsevier Inc. All rights reserved.
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Detection of damaged DNA bases by DNA glycosylase enzymes.
Friedman, Joshua I; Stivers, James T
2010-06-22
A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.
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Liu, Lei; Zhang, Ruifen; Deng, Yuanyuan; Zhang, Yan; Xiao, Juan; Huang, Fei; Wen, Wei; Zhang, Mingwei
2017-04-15
In this study, rice bran was successively steamed with α-amylase, fermented with lactic acid bacteria, and hydrolyzed with complex enzymes. The changes in phenolic profiles and antioxidant activities of the corresponding aqueous solutions from three stages were investigated. Compared to the first stage, fermentation and complex enzyme hydrolysis significantly increased the total phenolics, total flavonoids, total FRAP and ORAC values by 59.2%, 56.6%, 73.6% and 45.4%, respectively. Twelve individual phenolics present in free or soluble conjugate forms were also analyzed during the processing. Ferulic acid was released in the highest amount among different phenolics followed by protocatechuic acid. Moreover, a major proportion of phenolics existed as soluble conjugates. The results showed that fermentation and complex enzyme hydrolysis enhanced total phenolics and antioxidant activities of aqueous solution from rice bran pretreated by steaming with α-amylase. This research could provide basis for the processing of rice bran beverage rich in phenolics. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Narayan, Srinivas B.; Master, Stephen R.; Sireci, Anthony N.; Bierl, Charlene; Stanley, Paige E.; Li, Changhong; Stanley, Charles A.; Bennett, Michael J.
2012-01-01
Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein. PMID:22496890
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de Vasconcellos, Adriano; Miller, Alex Henrique; Aranda, Donato A G; Nery, José Geraldo
2018-05-01
Nanozeolites with different crystallographic structures (Nano/TS1, Nano/GIS, Nano/LTA, Nano/BEA, Nano/X, and Nano-X/Ni), functionalized with (3-aminopropyl)trimethoxysilane (APTMS) and crosslinked with glutaraldehyde (GA), were studied as solid supports for Thermomyces lanuginosus lipase (TLL) immobilization. Physicochemical characterizations of the surface-functionalized nanozeolites and nanozeolite-enzyme complexes were performed using XRD, SEM, AFM, ATR-FTIR, and zeta potential measurements. The experimental enzymatic activity results indicated that the nanozeolitic supports functionalized with APTMS and GA immobilized larger amounts of enzymes and provided higher enzymatic activities, compared to unfunctionalized supports. Correlations were observed among the nanozeolite surface charges, the enzyme immobilization efficiencies, and the biocatalyst activities. The catalytic performance and reusability of these enzyme-nanozeolite complexes were evaluated in the ethanolysis transesterification of microalgae oil to fatty acid ethyl esters (FAEEs). TLL immobilized on the nanozeolite supports functionalized with APTMS and GA provided the most efficient biocatalysis, with FAEEs yields above 93% and stability during five reaction cycles. Lower FAEEs yields and poorer catalytic stability were found for nanozeolite-enzyme complexes prepared only by physical adsorption. The findings indicated the viability of designing highly efficient biocatalysts for biofuel production by means of chemical modulation of nanozeolite surfaces. The high biocatalyst catalytic efficiency observed in ethanolysis reactions using a lipid feedstock that does not compete with food production is an advantage that should encourage the industrial application of these biocatalysts. Copyright © 2018 Elsevier B.V. All rights reserved.
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Extracellular enzyme kinetics scale with resource availability
Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimi...
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Salwiński, Aleksander; Da Silva, David; Delépée, Raphaël; Maunit, Benoît
2014-04-01
In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: 'ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and 'ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.
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Megavoltage cargo radiography with dual energy material decomposition
NASA Astrophysics Data System (ADS)
Shikhaliev, Polad M.
2018-02-01
Megavoltage (MV) radiography has important applications in imaging large cargos for detecting illicit materials. A useful feature of MV radiography is the possibility of decomposing and quantifying materials with different atomic numbers. This can be achieved by imaging cargo at two different X-ray energies, or dual energy (DE) radiography. The performance of both single energy and DE radiography depends on beam energy, beam filtration, radiation dose, object size, and object content. The purpose of this work was to perform comprehensive qualitative and quantitative investigations of the image quality in MV radiography depending on the above parameters. A digital phantom was designed including Fe background with thicknesses of 2cm, 6cm, and 18cm, and materials samples of Polyethylene, Fe, Pb, and U. The single energy images were generated at x-ray beam energies 3.5MV, 6MV, and 9MV. The DE material decomposed images were generated using interlaced low and high energy beams 3.5/6MV and 6/9MV. The X-ray beams were filtered by low-Z (Polyethylene) and high-Z (Pb) filters with variable thicknesses. The radiation output of the accelerator was kept constant for all beam energies. The image quality metrics was signal-to-noise ratio (SNR) of the particular sample over a particular background. It was found that the SNR depends on the above parameters in a complex way, but can be optimized by selecting a particular set of parameters. For some imaging setups increased filter thicknesses, while strongly absorbing the beams, increased the SNR of material decomposed images. Beam hardening due to polyenergetic x-ray spectra resulted in material decomposition errors, but this could be addressed using region of interest decomposition. It was shown that it is not feasible to separate the materials with close atomic numbers using the DE method. Particularly, Pb and U were difficult to decompose, at least at the dose levels allowed by radiation source and safety requirements.
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Zhong, Hai-Jing; Wang, Wanhe; Kang, Tian-Shu; Yan, Hui; Yang, Yali; Xu, Lipeng; Wang, Yuqiang; Ma, Dik-Lung; Leung, Chung-Hang
2017-01-12
We report herein the identification of the rhodium(III) complex [Rh(phq) 2 (MOPIP)] + (1) as a potent and selective ATP-competitive neural precursor cell expressed, developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) inhibitor. Structure-activity relationship analysis indicated that the overall organometallic design of complex 1 was important for anti-inflammatory activity. Complex 1 showed promising anti-inflammatory activity in vivo for the potential treatment of inflammatory bowel disease.
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A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions
Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.
2013-01-01
SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014
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The topological requirements for robust perfect adaptation in networks of any size.
Araujo, Robyn P; Liotta, Lance A
2018-05-01
Robustness, and the ability to function and thrive amid changing and unfavorable environments, is a fundamental requirement for living systems. Until now it has been an open question how large and complex biological networks can exhibit robust behaviors, such as perfect adaptation to a variable stimulus, since complexity is generally associated with fragility. Here we report that all networks that exhibit robust perfect adaptation (RPA) to a persistent change in stimulus are decomposable into well-defined modules, of which there exist two distinct classes. These two modular classes represent a topological basis for all RPA-capable networks, and generate the full set of topological realizations of the internal model principle for RPA in complex, self-organizing, evolvable bionetworks. This unexpected result supports the notion that evolutionary processes are empowered by simple and scalable modular design principles that promote robust performance no matter how large or complex the underlying networks become.
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King, C. Judson; Husson, Scott M.
1999-01-01
Carboxylic acids are sorbed from aqueous feedstocks onto a solid adsorbent. The acids are freed from the sorbent phase by treating it with an organic solution of alkylamine thus forming an alkylamine/carboxylic acid complex which is decomposed with improved efficiency to the desired carboxylic acid and the alkylamine. Carbon dioxide addition can be used to improve the adsorption or the carboxylic acids by the solid phase sorbent.
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Mission planning for autonomous systems
NASA Technical Reports Server (NTRS)
Pearson, G.
1987-01-01
Planning is a necessary task for intelligent, adaptive systems operating independently of human controllers. A mission planning system that performs task planning by decomposing a high-level mission objective into subtasks and synthesizing a plan for those tasks at varying levels of abstraction is discussed. Researchers use a blackboard architecture to partition the search space and direct the focus of attention of the planner. Using advanced planning techniques, they can control plan synthesis for the complex planning tasks involved in mission planning.
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The Design, Synthesis and Screening of Potential Pyridinium Oxime Prodrugs.
1985-07-31
copper sulfate pentahydrate , and 15 g (87 mol) of the mixture of bromo- picolines 13c and 131. The combined reactions produced 27 g (96%) of a brown...extracted with ethyl ether. The ether extracts were washed with brine, dried with sodium sulfate , filtered and flashed. The residue was then purified by...stirring to the reaction mix. The addition was exothermic as the copper complexes decomposed. The cooled mixture was extracted with several 20 ml
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Kivi, Rait; Solovjova, Karina; Haljasorg, Tõiv; Arukuusk, Piret; Järv, Jaak
2016-12-01
The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.
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NASA Technical Reports Server (NTRS)
Schneider, Steven J. (Inventor)
2001-01-01
A reduced toxicity fuel satellite propulsion system including a reduced toxicity propellant supply for consumption in an axial class thruster and an ACS class thruster. The system includes suitable valves and conduits for supplying the reduced toxicity propellant to the ACS decomposing element of an ACS thruster. The ACS decomposing element is operative to decompose the reduced toxicity propellant into hot propulsive gases. In addition the system includes suitable valves and conduits for supplying the reduced toxicity propellant to an axial decomposing element of the axial thruster. The axial decomposing element is operative to decompose the reduced toxicity propellant into hot gases. The system further includes suitable valves and conduits for supplying a second propellant to a combustion chamber of the axial thruster, whereby the hot gases and the second propellant auto-ignite and begin the combustion process for producing thrust.
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Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey
2010-02-01
Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.
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Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion
Lawton, Thomas J.; Rosenzweig, Amy C.
2017-01-01
Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16–13 s−1, these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock. PMID:27366961
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Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion.
Lawton, Thomas J; Rosenzweig, Amy C
2016-08-03
Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16-13 s(-1), these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock.
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Simulated nitrogen deposition affects wood decomposition by cord-forming fungi.
Bebber, Daniel P; Watkinson, Sarah C; Boddy, Lynne; Darrah, Peter R
2011-12-01
Anthropogenic nitrogen (N) deposition affects many natural processes, including forest litter decomposition. Saprotrophic fungi are the only organisms capable of completely decomposing lignocellulosic (woody) litter in temperate ecosystems, and therefore the responses of fungi to N deposition are critical in understanding the effects of global change on the forest carbon cycle. Plant litter decomposition under elevated N has been intensively studied, with varying results. The complexity of forest floor biota and variability in litter quality have obscured N-elevation effects on decomposers. Field experiments often utilize standardized substrates and N-levels, but few studies have controlled the decay organisms. Decomposition of beech (Fagus sylvatica) blocks inoculated with two cord-forming basidiomycete fungi, Hypholoma fasciculare and Phanerochaete velutina, was compared experimentally under realistic levels of simulated N deposition at Wytham Wood, Oxfordshire, UK. Mass loss was greater with P. velutina than with H. fasciculare, and with N treatment than in the control. Decomposition was accompanied by growth of the fungal mycelium and increasing N concentration in the remaining wood. We attribute the N effect on wood decay to the response of cord-forming wood decay fungi to N availability. Previous studies demonstrated the capacity of these fungi to scavenge and import N to decaying wood via a translocating network of mycelium. This study shows that small increases in N availability can increase wood decomposition by these organisms. Dead wood is an important carbon store and habitat. The responses of wood decomposers to anthropogenic N deposition should be considered in models of forest carbon dynamics.
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Multi-enzyme logic network architectures for assessing injuries: digital processing of biomarkers.
Halámek, Jan; Bocharova, Vera; Chinnapareddy, Soujanya; Windmiller, Joshua Ray; Strack, Guinevere; Chuang, Min-Chieh; Zhou, Jian; Santhosh, Padmanabhan; Ramirez, Gabriela V; Arugula, Mary A; Wang, Joseph; Katz, Evgeny
2010-12-01
A multi-enzyme biocatalytic cascade processing simultaneously five biomarkers characteristic of traumatic brain injury (TBI) and soft tissue injury (STI) was developed. The system operates as a digital biosensor based on concerted function of 8 Boolean AND logic gates, resulting in the decision about the physiological conditions based on the logic analysis of complex patterns of the biomarkers. The system represents the first example of a multi-step/multi-enzyme biosensor with the built-in logic for the analysis of complex combinations of biochemical inputs. The approach is based on recent advances in enzyme-based biocomputing systems and the present paper demonstrates the potential applicability of biocomputing for developing novel digital biosensor networks.
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Jackson, R H; Cole, J A; Cornish-Bowden, A
1981-01-01
The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions. PMID:6279095
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Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William
2004-03-09
Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.
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Trinh, Cong T.; Wlaschin, Aaron; Srienc, Friedrich
2010-01-01
Elementary Mode Analysis is a useful Metabolic Pathway Analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering. PMID:19015845
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Pan, Xu; Cornelissen, Johannes H C; Zhao, Wei-Wei; Liu, Guo-Fang; Hu, Yu-Kun; Prinzing, Andreas; Dong, Ming; Cornwell, William K
2014-01-01
Leaf litter decomposability is an important effect trait for ecosystem functioning. However, it is unknown how this effect trait evolved through plant history as a leaf ‘afterlife’ integrator of the evolution of multiple underlying traits upon which adaptive selection must have acted. Did decomposability evolve in a Brownian fashion without any constraints? Was evolution rapid at first and then slowed? Or was there an underlying mean-reverting process that makes the evolution of extreme trait values unlikely? Here, we test the hypothesis that the evolution of decomposability has undergone certain mean-reverting forces due to strong constraints and trade-offs in the leaf traits that have afterlife effects on litter quality to decomposers. In order to test this, we examined the leaf litter decomposability and seven key leaf traits of 48 tree species in the temperate area of China and fitted them to three evolutionary models: Brownian motion model (BM), Early burst model (EB), and Ornstein-Uhlenbeck model (OU). The OU model, which does not allow unlimited trait divergence through time, was the best fit model for leaf litter decomposability and all seven leaf traits. These results support the hypothesis that neither decomposability nor the underlying traits has been able to diverge toward progressively extreme values through evolutionary time. These results have reinforced our understanding of the relationships between leaf litter decomposability and leaf traits in an evolutionary perspective and may be a helpful step toward reconstructing deep-time carbon cycling based on taxonomic composition with more confidence. PMID:25535551
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nielsen, Tine Kragh; Hildmann, Christian; Riester, Daniel
2007-04-01
The crystal structure of HDAH FB188 in complex with a trifluoromethylketone at 2.2 Å resolution is reported and compared to a previously determined inhibitor complex. Histone deacetylases (HDACs) have emerged as attractive targets in anticancer drug development. To date, a number of HDAC inhibitors have been developed and most of them are hydroxamic acid derivatives, typified by suberoylanilide hydroxamic acid (SAHA). Not surprisingly, structural information that can greatly enhance the design of novel HDAC inhibitors is so far only available for hydroxamic acids in complex with HDAC or HDAC-like enzymes. Here, the first structure of an enzyme complex with amore » nonhydroxamate HDAC inhibitor is presented. The structure of the trifluoromethyl ketone inhibitor 9,9,9-trifluoro-8-oxo-N-phenylnonanamide in complex with bacterial FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188) has been determined. HDAH reveals high sequential and functional homology to human class 2 HDACs and a high structural homology to human class 1 HDACs. Comparison with the structure of HDAH in complex with SAHA reveals that the two inhibitors superimpose well. However, significant differences in binding to the active site of HDAH were observed. In the presented structure the O atom of the trifluoromethyl ketone moiety is within binding distance of the Zn atom of the enzyme and the F atoms participate in interactions with the enzyme, thereby involving more amino acids in enzyme–inhibitor binding.« less
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Ahmed, Zaheer; Tetlow, Ian J; Ahmed, Regina; Morell, Matthew K; Emes, Michael J
2015-04-01
The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Decursin and decursinol angelate selectively inhibit NADH-fumarate reductase of Ascaris suum.
Shiomi, Kazuro; Hatano, Hiroko; Morimoto, Hiromi; Ui, Hideaki; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tomoda, Hiroshi; Lee, Eun Woo; Heo, Tae Ryeon; Kawagishi, Hirokazu; Omura, Satoshi
2007-11-01
NADH-fumarate reductase (NFRD) is a key enzyme in many anaerobic helminths. Decursin and decursinol angelate have been isolated from the roots of ANGELICA GIGAS Nakai (Apiaceae) as NFRD inhibitors. They inhibited ASCARIS SUUM NFRD with IC (50) values of 1.1 and 2.7 microM, respectively. Their target is the electron transport enzyme complex I. Since the inhibitory activities of decursin against bovine heart complexes are weak, it is a selective inhibitor of the nematode complex I. In contrast, decursinol angelate moderately inhibits bovine heart complexes II and III. Decursinol inhibits A. SUUM NFRD to a similar extent, but its target is complex II. It also inhibits bovine heart complexes II and III.
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Flexibility, Diversity, and Cooperativity: Pillars of Enzyme Catalysis
Hammes, Gordon G.; Benkovic, Stephen J.; Hammes-Schiffer, Sharon
2011-01-01
This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, NMR, and single molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model. PMID:22029278
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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.
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Liposome encapsulation of chelating agents
Rahman, Yueh Erh
1976-01-13
A method for transferring a chelating agent across a cellular membrane by encapsulating the charged chelating agent within liposomes and carrying the liposome-encapsulated chelating agent to the cellular membrane where the liposomes containing the chelating agent will be taken up by the cells, thereby transferring the chelating agent across the cellular membrane. A chelating agent can be introduced into the interior of a cell of a living organism wherein the liposomes will be decomposed, releasing the chelating agent to the interior of the cell. The released chelating agent will complex intracellularly deposited toxic heavy metals, permitting the more soluble metal complex to transfer across the cellular membrane from the cell and subsequently be removed from the living organism.
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NASA Astrophysics Data System (ADS)
Dong, Lieqian; Wang, Deying; Zhang, Yimeng; Zhou, Datong
2017-09-01
Signal enhancement is a necessary step in seismic data processing. In this paper we utilize the complementary ensemble empirical mode decomposition (CEEMD) and complex curvelet transform (CCT) methods to separate signal from random noise further to improve the signal to noise (S/N) ratio. Firstly, the original data with noise is decomposed into a series of intrinsic mode function (IMF) profiles with the aid of CEEMD. Then the IMFs with noise are transformed into CCT domain. By choosing different thresholds which are based on the noise level difference of each IMF profile, the noise in original data can be suppressed. Finally, we illustrate the effectiveness of the approach by simulated and field datasets.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gladysz, J. A.
1980-11-10
Mechanistic studies of anionic formyl complexes were made. The rate of decomposition of the formyl (CO)/sub 4/Mn(COC/sub 6/H/sub 5/)(CHO)/sup -/ anion as a function of counter-ion and trialkylborane present was examined. The deuterated formyl (CO)/sub 4/Mn(COC/sub 6/H/sub 5/)(CDO)/sup -/ was prepared and its decomposition was monitored by H/sup 2/ NMR. The data obtained indicate that the formyl decomposes by a disproportionative pathway not by initial CO loss, as commonly noted for formyl complexes.
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Three-dimensional scene encryption and display based on computer-generated holograms.
Kong, Dezhao; Cao, Liangcai; Jin, Guofan; Javidi, Bahram
2016-10-10
An optical encryption and display method for a three-dimensional (3D) scene is proposed based on computer-generated holograms (CGHs) using a single phase-only spatial light modulator. The 3D scene is encoded as one complex Fourier CGH. The Fourier CGH is then decomposed into two phase-only CGHs with random distributions by the vector stochastic decomposition algorithm. Two CGHs are interleaved as one final phase-only CGH for optical encryption and reconstruction. The proposed method can support high-level nonlinear optical 3D scene security and complex amplitude modulation of the optical field. The exclusive phase key offers strong resistances of decryption attacks. Experimental results demonstrate the validity of the novel method.
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Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20.
Crocker, Fiona H; Indest, Karl J; Fredrickson, Herbert L
2006-11-01
Cyclic nitramine explosives are synthesized globally mainly as military munitions, and their use has resulted in environmental contamination. Several biodegradation pathways have been proposed, and these are based mainly on end-product characterization because many of the metabolic intermediates are hypothetical and unstable in water. Biodegradation mechanisms for cyclic nitramines include (a) formation of a nitramine free radical and loss of nitro functional groups, (b) reduction of nitro functional groups, (c) direct enzymatic cleavage, (d) alpha-hydroxylation, or (e) hydride ion transfer. Pathway intermediates spontaneously decompose in water producing nitrite, nitrous oxide, formaldehyde, or formic acid as common end-products. In vitro enzyme and functional gene expression studies have implicated a limited number of enzymes/genes involved in cyclic nitramine catabolism. Advances in molecular biology methods such as high-throughput DNA sequencing, microarray analysis, and nucleic acid sample preparation are providing access to biochemical and genetic information on cultivable and uncultivable microorganisms. This information can provide the knowledge base for rational engineering of bioremediation strategies, biosensor development, environmental monitoring, and green biosynthesis of explosives. This paper reviews recent developments on the biodegradation of cyclic nitramines and the potential of genomics to identify novel functional genes of explosive metabolism.
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Low dose X -ray effects on catalase activity in animal tissue
NASA Astrophysics Data System (ADS)
Focea, R.; Nadejde, C.; Creanga, D.; Luchian, T.
2012-12-01
This study was intended to investigate the effect of low-dose X ray-irradiation upon the activity of catalase (CAT) in freshly excised chicken tissues (liver, kidney, brain, muscle). The tissue samples were irradiated with 0.5Gy and 2Gy respectively, in a 6 MV photon beam produced by a clinical linear accelerator (VARIAN CLINAC 2100SC). The dose rate was of 260.88cGy/min. at 100 cm source to sample distance. The catalase level was assayed spectrophotometrically, based on reaction kinetics, using a catalase UV assay kit (SIGMA). Catalase increased activity in various tissue samples exposed to the studied X ray doses (for example with 24 % in the liver cells, p<0.05) suggested the stimulation of the antioxidant enzyme biosynthesis within several hours after exposure at doses of 0.5 Gy and 2 Gy; the putative enzyme inactivation could also occur (due to the injuries on the hydrogen bonds that ensure the specificity of CAT active site) but the resulted balance of the two concurrent processes indicates the cell ability of decomposing the hydrogen peroxide-with benefits for the cell physiology restoration for the chosen low dose radiation.
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Polyelectrolyte Complex Optimization for Macrophage Delivery of Redox Enzyme Nanoparticles
Zhao, Yuling; Haney, Matthew J.; Klyachko, Natalia L.; Li, Shu; Booth, Stephanie L.; Higginbotham, Sheila M.; Jones, Jocelyn; Zimmerman, Matthew C.; Mosley, R. Lee; Kabanov, Alexander V.; Gendelman, Howard E.; Batrakova, Elena V.
2011-01-01
Background We posit that cell-mediated drug delivery can improve transport of therapeutic enzymes to the brain and decrease inflammation and neurodegeneration induced during Parkinson’s disease. Our prior work demonstrated that macrophages loaded with nanoformulated catalase (“nanozyme”) protect the nigrostriatum in a murine model of Parkinson’s disease. Packaging of catalase into block ionomer complex with a synthetic polyelectrolyte block copolymers protects the enzyme degradation in macrophages. Methods We examined relationships between the composition and structure of block ionomer complexes, their physicochemical characteristics, and loadings, release rates, and catalase activity in bone marrow-derived macrophages. Results Formation of block-ionomer complexes resulted in improved aggregation stability. Block ionomer complexes with ε-polylisine, and poly-L-glutamic acid -poly(ethylene glycol) demonstrated the least cytotoxicity and high loading and release rates, however, did not efficiently protect catalase inside macrophages. Conclusion nanozymes with polyethyleneimine- and poly(L-lysine)10-poly(ethylene glycol) provided the best protection of enzymatic activity for cell-mediated drug delivery. PMID:21182416
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Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute
2009-01-01
Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively. PMID:19851019
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Ji, Mingshan; Liang, Yaping; Gu, Zumin; Li, Xiuwei
2015-01-01
Previous studies in our laboratory found that the extract from seeds of Amorpha fruticosa in the Leguminosae family had lethal effects against mosquito larvae, and an insecticidal compound amorphigenin was isolated. In this study, the inhibitory effects of amorphigenin against the mitochondrial complex I of Culex pipiens pallens (Diptera: Culicidae) were investigated and compared with that of rotenone. The results showed that amorphigenin and rotenone can decrease the mitochondrial complex I activity both in vivo and in vitro as the in vivo IC50 values (the inhibitor concentrations leading to 50% of the enzyme activity lost) were determined to be 2.4329 and 2.5232 μmol/L, respectively, while the in vitro IC50 values were 2.8592 and 3.1375 μmol/L, respectively. Both amorphigenin and rotenone were shown to be reversible and mixed-I type inhibitors of the mitochondrial complex I of Cx. pipiens pallens, indicating that amorphigenin and rotenone inhibited the enzyme activity not only by binding with the free enzyme but also with the enzyme-substrate complex, and the values of KI and KIS for amorphigenin were determined to be 20.58 and 87.55 μM, respectively, while the values for rotenone were 14.04 and 69.23 μM, respectively. PMID:26307964
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NASA Astrophysics Data System (ADS)
Khandeparker, Rakhee; Verma, Preeti; Meena, Ram M.; Deobagkar, Deepti D.
2011-12-01
Coastal and estuarine waters are highly productive and dynamic ecosystems. The complex carbohydrate composition of the ecosystem would lead to colonisation of microbial communities with abilities to produce an array of complex carbohydrate degrading enzymes. We have examined the abundance and phylogenetic diversity of culturable bacteria with abilities to produce complex carbohydrate degrading enzymes in the Mondovi and Zuari eustauri. It was interesting to note that 65% of isolated bacteria could produce complex carbohydrate degrading enzymes. A majority of these bacteria belonged to Bacillus genera followed by Vibrio, Marinobacter, Exiquinobacterium, Alteromonas, Enterobacter and Aeromonas. Most abundant bacterial genus to degrade hemicellulose and cellulose were Bacillus and Vibrio respectively. Most abundant bacterial genus to degrade hemicellulose and cellulose were Bacillus and Vibrio respectively. It was seen that 46% of Bacillus had ability to degrade both the substrate while only 14% of Vibrio had bifunctionality.
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DeAngelis, Kristen M.; Sharma, Deepak; Varney, Rebecca; Simmons, Blake; Isern, Nancy G.; Markilllie, Lye Meng; Nicora, Carrie; Norbeck, Angela D.; Taylor, Ronald C.; Aldrich, Joshua T.; Robinson, Errol W.
2013-01-01
Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability. PMID:24065962
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Deangelis, Kristen M; Sharma, Deepak; Varney, Rebecca; Simmons, Blake; Isern, Nancy G; Markilllie, Lye Meng; Nicora, Carrie; Norbeck, Angela D; Taylor, Ronald C; Aldrich, Joshua T; Robinson, Errol W
2013-01-01
Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Suzuki, Hitoshi; MacDonald, Jacqueline; Syed, Khajamohiddin
Background Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reportedmore » P. chrysosporium genome. Results P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. Conclusions The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.« less
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2012-01-01
Background Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. Results P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. Conclusions The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species. PMID:22937793
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Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi
2016-01-01
The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169
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Baker, Perrin; Hillis, Colleen; Carere, Jason; Seah, Stephen Y K
2012-03-06
Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.
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Soil actinomycetes in the National Forest Park in northeastern China
NASA Astrophysics Data System (ADS)
Shirokikh, I. G.; Shirokikh, A. A.
2017-01-01
The taxonomic and functional structure of actinomycete complexes in the litters and upper horizons of the soils under an artificial coniferous-broad-leaved forest located around the town of Chanchun (Tszilin province, PRC). The complex of actinomycetes included representatives of the Streptomyces, Micromonospora, Streptosporangium, and Streptoverticillium genera and oligosporous forms. In the actinomycete complexes, streptomycetes prevailed in the abundance (61-95%) and frequency of occurrence (100%). In the parcels of Korean pine ( Pinus koraiensis) and Mongolian oak ( Quercus mongolica), streptomycetes of 19 species from 8 series and 4 sections were isolated. The most representative, as in European forest biomes, was the Cinereus Achromogenes series. A distinguishing feature of the streptomycete complex in the biomes studied was the high participation of species from the Imperfectus series. The verification of the functional activity of natural isolates made it possible to reveal strains with high antagonistic and cellulolytic abilities. A high similarity of actinomycete complexes was found in Eurasian forest ecosystems remote from each other, probably due to the similarity of plant polymers decomposable by actinomycetes.
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Visser, Franziska; Müller, Boje; Rose, Judith; Prüfer, Dirk; Noll, Gundula A
2016-08-09
The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.
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Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok
2015-06-25
An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.
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Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem
2015-01-01
Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.
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Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B
2016-06-01
Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.
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Gonzalez-Vogel, Alvaro; Eyzaguirre, Jaime; Oleas, Gabriela; Callegari, Eduardo; Navarrete, Mario
2011-01-01
Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein-protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.
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Degli Esposti, Mauro
2016-01-01
Respiratory complex I (NADH:ubiquinone oxidoreductase) is a ubiquitous bioenergetic enzyme formed by over 40 subunits in eukaryotes and a minimum of 11 subunits in bacteria. Recently, crystal structures have greatly advanced our knowledge of complex I but have not clarified the details of its reaction with ubiquinone (Q). This reaction is essential for bioenergy production and takes place in a large cavity embedded within a conserved module that is homologous to the catalytic core of Ni–Fe hydrogenases. However, how a hydrogenase core has evolved into the protonmotive Q reductase module of complex I has remained unclear. This work has exploited the abundant genomic information that is currently available to deduce structure–function relationships in complex I that indicate the evolutionary steps of Q reactivity and its adaptation to natural Q substrates. The results provide answers to fundamental questions regarding various aspects of complex I reaction with Q and help re-defining the old concept that this reaction may involve two Q or inhibitor sites. The re-definition leads to a simplified classification of the plethora of complex I inhibitors while throwing a new light on the evolution of the enzyme function. PMID:26615219
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Enzyme catalysis: C-H activation is a Reiske business
NASA Astrophysics Data System (ADS)
Bruner, Steven D.
2011-05-01
Enzymes that selectively oxidize unactivated C-H bonds are capable of constructing complex molecules with high efficiency. A new member of this enzyme family is RedG, a Reiske-type oxygenase that catalyses chemically challenging cyclizations in the biosynthesis of prodiginine natural products.
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Lignocellulose Degradation Mechanisms Across the Tree of Life
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cragg, Simon M.; Beckham, Gregg T.; Bruce, Neil C.
Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. We found that the Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however,more » house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. Moreover, the omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.« less
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Lignocellulose Degradation Mechanisms Across the Tree of Life
Cragg, Simon M.; Beckham, Gregg T.; Bruce, Neil C.; ...
2015-11-14
Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. We found that the Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however,more » house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. Moreover, the omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.« less
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1987-12-01
Normally, the system is decomposed into manageable parts with accurately defined interfaces. By rigidly controlling this process, aerospace companies have...Reference A CHANGE IN SYSTEM DESIGN EMPHASIS: FROM MACHINE TO MAN by M.L.Metersky and J.L.Ryder 16 SESSION I1 - MANAGING THE FUl URE SYSTEM DESIGN...PROCESS MANAGING ADVANCED AVIONIC SYSTEM DESIGN by P.Simons 17 ERGONOMIE PSYCHOSENSORIELLE DES COCKPITS, INTERET DES SYSTEMES INFORMATIQUES INTELLIGENTS
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Super-resolution with an SLM and two intensity images
NASA Astrophysics Data System (ADS)
Alcalá Ochoa, Noé; de León, Y. Ponce
2018-06-01
It is reported a method which may simplify the optical setups used to achieve super-resolution through the amplitude multiplication of two waves. For this end we decompose a super-resolving pupil into two complex masks and with the aid of a Spatial Light Modulator (LCoS) we obtain two intensity images that are subtracted. With this proposal, the traditional experimental optical setups are considerably simplified, with the additional benefit that different masks can be utilized without needing to perform the setup alignment each time.
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Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.
Tan, Zaigao; Chen, Jing; Zhang, Xueli
2016-01-01
Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.
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Michelin, Michele; Polizeli, Maria de Lourdes T M; Ruzene, Denise S; Silva, Daniel P; Ruiz, Héctor A; Vicente, António A; Jorge, João A; Terenzi, Héctor F; Teixeira, José A
2012-09-01
Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.
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Heneghan, Mary N.; Burns, Claire; Costa, Ana M. S. B.; Burton, Kerry S.; Challen, Michael P.
2016-01-01
Summary Agaricus bisporus is a secondary decomposer fungus and an excellent model for the adaptation, persistence and growth of fungi in humic‐rich environments such as soils of temperate woodland and pastures. The A. bisporus serine proteinase SPR1 is induced by humic acids and is highly expressed during growth on compost. Three Spr1 gene silencing cassettes were constructed around sense, antisense and non‐translatable‐stop strategies (pGRsensehph, pGRantihph and pGRstophph). Transformation of A. bisporus with these cassettes generated cultures showing a reduction in extracellular proteinase activity as demonstrated by the reduction, or abolition, of a clearing zone on plate‐based bioassays. These lines were then assessed by detailed enzyme assay, RT‐qPCR and fruiting. Serine proteinase activity in liquid cultures was reduced in 83% of transformants. RT‐qPCR showed reduced Spr1 mRNA levels in all transformants analysed, and these correlated with reduced enzyme activity. When fruiting was induced, highly‐silenced transformant AS5 failed to colonize the compost, whilst for those that did colonize the compost, 60% gave a reduction in mushroom yield. Transcriptional, biochemical and developmental observations, demonstrate that SPR1 has an important role in nutrient acquisition in compost and that SPR1 is a key enzyme in the adaptation of Agaricus to the humic‐rich ecological niche formed during biomass degradation. PMID:27113919
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The complex between a four-way DNA junction and T7 endonuclease I
Déclais, Anne-Cécile; Fogg, Jonathan M.; Freeman, Alasdair D.J.; Coste, Franck; Hadden, Jonathan M.; Phillips, Simon E.V.; Lilley, David M.J.
2003-01-01
The junction-resolving enzyme endonuclease I is selective for the structure of the DNA four-way (Holliday) junction. The enzyme binds to a four-way junction in two possible orientations, with a 4:1 ratio, opening the DNA structure at the centre and changing the global structure into a 90° cross of approximately coaxial helices. The nuclease cleaves the continuous strands of the junction in each orientation. Binding leads to pronounced regions of protection of the DNA against hydroxyl radical attack. Using all this information together with the known structure of the enzyme and the structure of the BglI–DNA complex, we have constructed a model of the complex of endonuclease I and a DNA junction. This shows how the enzyme is selective for the structure of a four-way junction, such that both continuous strands can be accommodated into the two active sites so that a productive resolution event is possible. PMID:12628932
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Israyilova, Aygun; Buroni, Silvia; Forneris, Federico; Scoffone, Viola Camilla; Shixaliyev, Namiq Q; Riccardi, Giovanna; Chiarelli, Laurent Roberto
2016-01-01
The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 μM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.
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NASA Technical Reports Server (NTRS)
Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.
2003-01-01
The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.
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Forensic entomology of decomposing humans and their decomposing pets.
Sanford, Michelle R
2015-02-01
Domestic pets are commonly found in the homes of decedents whose deaths are investigated by a medical examiner or coroner. When these pets become trapped with a decomposing decedent they may resort to feeding on the body or succumb to starvation and/or dehydration and begin to decompose as well. In this case report photographic documentation of cases involving pets and decedents were examined from 2009 through the beginning of 2014. This photo review indicated that in many cases the pets were cats and dogs that were trapped with the decedent, died and were discovered in a moderate (bloat to active decay) state of decomposition. In addition three cases involving decomposing humans and their decomposing pets are described as they were processed for time of insect colonization by forensic entomological approach. Differences in timing and species colonizing the human and animal bodies were noted as was the potential for the human or animal derived specimens to contaminate one another at the scene. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Yang, Xiu-Yan; Xue, Zhi-Yuan; Yang, Ya-Fei; Fang, Yao-Yao; Zhou, Xiang-Lin; Zhao, Liang-Gong; Feng, Shi-Lan
2018-06-01
In this study, complex enzymes combined with ultrasonic extraction technology（MC） were used, to select optimal extraction combinations by single factor and orthogonal test, with Hedysarum polysaccharides yield and content as the comprehensive indexes. The components, physicochemical properties and antioxidant activity of Hedysarum polysaccharides from complex enzyme combined with ultrasonic extraction（HPS-MC）and the Hedysarum polysaccharides from hot water extraction（HPS-R）were analyzed. The results showed that：complex enzymes had significant effect on the yield and content of Hedysarum polysaccharides, and the ultrasonic power could significantly improve the content of Hedysarum polysaccharides. The optimum technological parameters were as follows: complex enzyme ratio 1:1, ultrasonic power 105 W, ultrasonic time 60 min, and enzymatic hydrolysis pH 5, achieving （14.01±0.64）% and （92.45±1.47）% respectively for the yield and content of Polysaccharides. As compared with HPS-R, the molecular weight, absolute viscosity and protein content of HPS-MC were decreased, while the content of uronic acid was increased. In the antioxidant system, the concentration of polysaccharide was within the range of 1-7 g·L⁻¹; the antioxidant activity of HPS-MC was higher than that of HPS-R, and HPS-MC (80%) with the lowest molecular weight showed a significant dose effect relationship with the increase of the experimental concentration. In conclusion, MC is a simple, convenient, economical and environmentally friendly extraction technology, and the Hedysarum polysaccharides extracted by this method have obvious antioxidant activity. Copyright© by the Chinese Pharmaceutical Association.
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Attention theory and training research
NASA Technical Reports Server (NTRS)
Connelly, James G., Jr.; Wickens, Christopher D.; Lintern, Gavan; Harwood, Kelly
1987-01-01
This study used elements of attention theory as a methodological basis to decompose a complex training task in order to improve training efficiency. The complex task was a microcomputer flight simulation where subjects were required to control the stability of their own helicopter while acquiring and engaging enemy helicopers in a threat enviroment. Subjects were divided into whole-task, part-task, and part/open loop adaptive task groups in a transfer of training paradigm. The effect of reducing mental workload at the early stages of learning was examined with respect to the degree that subordinate elements of the complex task could be automated through practice of consistent, learnable stimulus-response relationships. Results revealed trends suggesting the benefit of isolating consistently mapped sub-tasks for part-task training and the presence of a time-sharing skill over and above the skill required for the separate subtasks.
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Cross linking in the radiolysis of some enzymes and related proteins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lynn, K.R.
1977-01-01
In non-covalently bound complexes of several serine proteases and of ribonuclease with DNA the enzymes were protected against the effects of ionizing radiation. No scavenging by the nucleic acids was observed. Similarly, complexing trypsin with silica protected the enzyme from radiolytic destruction. Irradiation of solutions of serine proteases required about twice the D37 dose to produce about 10% polymerization: significantly lower relative doses were effective in causing polymerization in both lima bean protease inhibitor and in the octapeptidal hormone oxytocin. Several sulfhydryl enzymes which have been examined were very efficiently inactivated by ionizing radiation. There was, at the same time,more » apparent formation of novel intra-molecular -S-S- bonds.« less
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Philpott, Timothy J; Barker, Jason S; Prescott, Cindy E; Grayston, Sue J
2018-02-01
Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on β-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia. IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.
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Microbial Activity in Peat Soil Treated With Ordinary Portland Cement (OPC) and Coal Ashes
NASA Astrophysics Data System (ADS)
Rahman, J. A.; Mohamed, R. M. S. R.; Al-Gheethi, A. A.
2018-04-01
Peat soil is a cumulative of decayed plant fragment which developed as a result of microbial activity. The microbes degrade the organic matter in the peat soils by the production of hydrolysis enzyme. The least decomposed peat, known as fibric peat has big particles and retain lots of water. This made peat having high moisture content, up to 1500 %. The most decomposed peat known as sapric peat having fines particles and less void ratio. The present study aimed to understand the effects of solidification process on the bacterial growth and cellulase (CMCase) enzyme activity. Two types of mixing were designed for fibric, hemic and sapric peats; (i) Ordinary Portland cement (OPC) at an equal amount of dry peat, with 25 % of fly ash (FA) and total of coarse particle, a combination of bottom ash and fibre of 22 – 34 %, (ii) fibric peat was using water-to-binder ratio (w/b) = 1, 50% OPC, 25 % bottom ash (BA) and 25 % FA. For hemic and sapric peat, w/b=3 with 50 % OPC and 50 % BA were used. All samples were prepared triplicates, and were cured for 7, 14, 28 and 56 days in a closed container at room temperature. The results revealed that the first mix design giving a continuous strength development. However, the second mix design shows a decreased in strength pattern after day 28. The influence of the environment factors such as alkaline pH, reduction of the water content and peat temperature has no significant on the reduction amount of native microbes in the peat. The microbes survived in the solidified peat but the amount of microbes were found reduced for all types of mixing Fibric Mixed 1 (FM1), Hemic Mixed 1(HM1) and Sapric Mixed 1 (SM1) were having good strength increment for about 330 – 1427 % with enzymatic activity recorded even after D56. Nevertheless, with increase in the strength development through curing days, the enzymatic activities were reduced. For the time being, it can be concluded that the microbes have the ability to adapt with new environment. The reactivity of the microbes relates with the strength of solidified peat.
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On the Temperature Dependence of Enzyme-Catalyzed Rates.
Arcus, Vickery L; Prentice, Erica J; Hobbs, Joanne K; Mulholland, Adrian J; Van der Kamp, Marc W; Pudney, Christopher R; Parker, Emily J; Schipper, Louis A
2016-03-29
One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems.
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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.
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A DNA enzyme with N-glycosylase activity
NASA Technical Reports Server (NTRS)
Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.
2000-01-01
In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.
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A Quantitative Measure of Conformational Changes in Apo, Holo and Ligand-Bound Forms of Enzymes.
Singh, Satendra; Singh, Atul Kumar; Wadhwa, Gulshan; Singh, Dev Bukhsh; Dwivedi, Seema; Gautam, Budhayash; Ramteke, Pramod W
2016-06-01
Determination of the native geometry of the enzymes and ligand complexes is a key step in the process of structure-based drug designing. Enzymes and ligands show flexibility in structural behavior as they come in contact with each other. When ligand binds with active site of the enzyme, in the presence of cofactor some structural changes are expected to occur in the active site. Motivation behind this study is to determine the nature of conformational changes as well as regions where such changes are more pronounced. To measure the structural changes due to cofactor and ligand complex, enzyme in apo, holo and ligand-bound forms is selected. Enzyme data set was retrieved from protein data bank. Fifteen triplet groups were selected for the analysis of structural changes based on selection criteria. Structural features for selected enzymes were compared at the global as well as local region. Accessible surface area for the enzymes in entire triplet set was calculated, which describes the change in accessible surface area upon binding of cofactor and ligand with the enzyme. It was observed that some structural changes take place during binding of ligand in the presence of cofactor. This study will helps in understanding the level of flexibility in protein-ligand interaction for computer-aided drug designing.
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Ligninolytic Activity at 0 °C of Fungi on Oak Leaves Under Snow Cover in a Mixed Forest in Japan.
Miyamoto, Toshizumi; Koda, Keiichi; Kawaguchi, Arata; Uraki, Yasumitsu
2017-08-01
Despite the importance of litter decomposition under snow cover in boreal forests and tundra, very little is known regarding the characteristics and functions of litter-decomposing fungi adapted to the cold climate. We investigated the decomposition of oak leaves in a heavy snowfall forest region of Japan. The rate of litter weight loss reached 26.5% during the snow cover period for 7 months and accounted for 64.6% of the annual loss (41.1%). Although no statistically significant lignin loss was detected, decolourization portions of oak leaf litter, which was attributable to the activities of ligninolytic fungi, were observed during snow cover period. This suggests that fungi involved in litter decomposition can produce extracellular enzymes to degrade lignin that remain active at 0 °C. Fungi were isolated from oak leaves collected from the forest floor under the snow layer. One hundred and sixty-six strains were isolated and classified into 33 operational taxonomic units (OTUs) based on culture characteristics and nuclear rDNA internal transcribed spacer (ITS) region sequences. To test the ability to degrade lignin, the production of extracellular phenoloxidases by isolates was quantified at 0 °C. Ten OTUs (9 Ascomycota and 1 Basidiomycota) of fungi exhibited mycelial growth and ligninolytic activity. These results suggested that some litter-decomposing fungi that had the potential to degrade lignin at 0 °C significantly contribute to litter decomposition under snow cover.
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Hennen-Bierwagen, Tracie A.; Lin, Qiaohui; Grimaud, Florent; Planchot, Véronique; Keeling, Peter L.; James, Martha G.; Myers, Alan M.
2009-01-01
Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds. PMID:19168640
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The Cobalt cyclo‐P4 Sandwich Complex and Its Role in the Formation of Polyphosphorus Compounds
Dielmann, Fabian; Timoshkin, Alexey; Piesch, Martin; Balázs, Gábor
2017-01-01
Abstract A synthetic approach to the sandwich complex [Cp′′′Co(η4‐P4)] (2) containing a cyclo‐P4 ligand as an end‐deck was developed. Complex 2 is the missing homologue in the series of first‐row cyclo‐Pn sandwich complexes, and shows a unique tendency to dimerize in solution to form two isomeric P8 complexes [(Cp′′′Co)2(μ,η4:η2:η1‐P8)] (3 and 4). Reactivity studies indicate that 2 and 3 react with further [Cp′′′Co] fragments to give [(Cp′′′Co)2(μ,η2:η2‐P2)2] (5) and [(Cp′′′Co)3P8] (6), respectively. Furthermore, complexes 2, 3, and 4 thermally decompose forming 5, 6, and the P12 complex [(Cp′′′Co)3P12] (7). DFT calculations on the P4 activation process suggest a η3‐P4 Co complex as the key intermediate in the synthesis of 2 as well as in the formation of larger polyphosphorus complexes via a unique oligomerization pathway. PMID:28078794
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Our World without Decomposers: How Scary!
ERIC Educational Resources Information Center
Spring, Patty; Harr, Natalie
2014-01-01
Bugs, slugs, bacteria, and fungi are decomposers at the heart of every ecosystem. Fifth graders at Dodge Intermediate School in Twinsburg, Ohio, ventured outdoors to learn about the necessity of these amazing organisms. With the help of a naturalist, students explored their local park and discovered the wonder of decomposers and their…
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Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si
2017-04-01
The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.
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NASA Astrophysics Data System (ADS)
Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si
2017-04-01
The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.
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Francesch, M; Geraert, P A
2009-09-01
One experiment was conducted to investigate the benefits of a multi-enzyme complex, containing carbohydrases (from Penicillium funiculosum) and phytase (bacterial 6-phytase) activities, on the performance and bone mineralization of broiler chickens fed corn-soybean meal diets. A total of 2,268 male broilers were allocated to 9 treatments, replicated 6 times, in a randomized complete block design from 1 to 43 d. A positive control (PC) diet formulated to be adequate in nutrients and 4 reduced nutrient diets (NC1 to NC4), with gradual decrease on AME, CP, and digestible amino acids (CP-dAA) and available P (avP) and Ca contents, with or without enzyme supplementation, were tested. The nutrient reductions applied were NC1 (-65 kcal/kg, -1.5% CP-dAA) and NC2 (-85 kcal/kg, -3.0% CP-dAA) both with -0.15 percent point avP and -0.12 percent point Ca and NC3 (-65 kcal/kg, -1.5% CP-dAA) and NC4 (-85 kcal/kg, -3.0% CP-dAA) both with -0.20 percent point avP and -0.16 percent point Ca. Supplementation of the NC diets with the enzyme complex increased ADFI (P<0.001), ADG (P<0.001), and reduced feed:gain (P<0.01). The magnitude of the enzyme effect in increasing feed intake and weight gain was greater for the diets with greatest reductions in avP and Ca. Enzyme supplementation increased (P<0.001) feed intake of birds fed on NC diets close to the level of feed consumption of the PC. Enzyme supplementation to NC diets resulted in all cases in lower (P<0.05) feed:gain than the PC. Enzyme supplementation to NC1 and NC3 diets restored bone mineralization to that of the PC, whereas ash and Ca with NC2 and NC4 diets and P with NC4 diet remained lower (P<0.05). These results suggest that the dietary supplementation with a multi-enzyme complex containing nonstarch polysaccharide enzymes and phytase is efficient in reducing the P, energy, protein, and amino acid specifications of corn-soybean meal diets.
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Specific starch digestion of maize alpha-limit dextrins by recombinant mucosal glucosidase enzymes
USDA-ARS?s Scientific Manuscript database
Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose b...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Firestone, Mary
2015-03-31
It is now understood that most plant C is utilized or transformed by soil microorganisms en route to stabilization. Hence the composition of microbial communities that mediate decomposition and transformation of root C is critical, as are the metabolic capabilities of these communities. The change in composition and function of the C-transforming microbial communities over time in effect defines the biological component of soil C stabilization. Our research was designed to test 2 general hypotheses; the first two hypotheses are discussed first; H1: Root-exudate interactions with soil microbial populations results in the expression of enzymatic capacities for macromolecular, complex carbonmore » decomposition; and H2: Microbial communities surrounding roots undergo taxonomic succession linked to functional gene activities as roots grow, mature, and decompose in soil. Over the term of the project we made significant progress in 1) quantifying the temporal pattern of root interactions with the soil decomposing community and 2) characterizing the role of root exudates in mediating these interactions.« less
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Iguchi, Masayuki; Zhong, Heng; Himeda, Yuichiro; Kawanami, Hajime
2017-12-14
The hydroxyl groups of a 2,2'-bipyridine (bpy) ligand near the metal center activated the catalytic performance of the Ir complex for the dehydrogenation of formic acid at high pressure. The position of the hydroxyl groups on the ligand affected the catalytic durability for the high-pressure H 2 generation through the decomposition of formic acid. The Ir complex with a bipyridine ligand functionalized with para-hydroxyl groups shows a good durability with a constant catalytic activity during the reaction even under high-pressure conditions, whereas deactivation was observed for an Ir complex with a bipyridine ligand with ortho-hydroxyl groups (2). In the presence of high-pressure H 2 , complex 2 decomposed into the ligand and an Ir trihydride complex through the isomerization of the bpy ligand. This work provides the development of a durable catalyst for the high-pressure H 2 production from formic acid. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Technetium and rhenium pentacarbonyl complexes with C₂ and C₁₁ ω-isocyanocarboxylic acid esters.
Miroslavov, Alexander E; Polotskii, Yuriy S; Gurzhiy, Vladislav V; Ivanov, Alexander Yu; Lumpov, Alexander A; Tyupina, Margarita Yu; Sidorenko, Georgy V; Tolstoy, Peter M; Maltsev, Daniil A; Suglobov, Dmitry N
2014-08-04
Technetium(I) and rhenium(I) pentacarbonyl complexes with ethyl 2-isocyanoacetate and methyl 11-isocyanoundecanoate, [M(CO)5(CNCH2COOEt)]ClO4 (M = Tc (1) and Re (2)) and [M(CO)5(CN(CH2)10COOMe)]ClO4 (M = Tc (3) and Re (4)), were prepared and characterized by IR, (1)H NMR, and (13)C{(1)H} NMR spectroscopy. The crystal structures of 1 and 2 were determined using single-crystal X-ray diffraction. The kinetics of thermal decarbonylation of technetium complexes 1 and 3 in ethylene glycol was studied by IR spectroscopy. The rate constants and activation parameters of this reaction were determined and compared with those for [Tc(CO)6](+). It was found that rhenium complexes 2 and 4 were stable with respect to thermal decarbonylation. Histidine challenge reaction of complexes 1 and 2 in phosphate buffer was examined by IR spectroscopy. In the presence of histidine, the rhenium pentacarbonyl isocyanide complex partially decomposes to form an unidentified yellow precipitate. Technetium analogue 1 is more stable under these conditions.
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Bumb, Iris; Garnier, Eric; Coq, Sylvain; Nahmani, Johanne; Del Rey Granado, Maria; Gimenez, Olivier; Kazakou, Elena
2018-03-05
Forage quality for herbivores and litter quality for decomposers are two key plant properties affecting ecosystem carbon and nutrient cycling. Although there is a positive relationship between palatability and decomposition, very few studies have focused on larger vertebrate herbivores while considering links between the digestibility of living leaves and stems and the decomposability of litter and associated traits. The hypothesis tested is that some defences of living organs would reduce their digestibility and, as a consequence, their litter decomposability, through 'afterlife' effects. Additionally in high-fertility conditions the presence of intense herbivory would select for communities dominated by fast-growing plants, which are able to compensate for tissue loss by herbivory, producing both highly digestible organs and easily decomposable litter. Relationships between dry matter digestibility and decomposability were quantified in 16 dominant species from Mediterranean rangelands, which are subject to management regimes that differ in grazing intensity and fertilization. The digestibility and decomposability of leaves and stems were estimated at peak standing biomass, in plots that were either fertilized and intensively grazed or unfertilized and moderately grazed. Several traits were measured on living and senesced organs: fibre content, dry matter content and nitrogen, phosphorus and tannin concentrations. Digestibility was positively related to decomposability, both properties being influenced in the same direction by management regime, organ and growth forms. Digestibility of leaves and stems was negatively related to their fibre concentrations, and positively related to their nitrogen concentration. Decomposability was more strongly related to traits measured on living organs than on litter. Digestibility and decomposition were governed by similar structural traits, in particular fibre concentration, affecting both herbivores and micro-organisms through the afterlife effects. This study contributes to a better understanding of the interspecific relationships between forage quality and litter decomposition in leaves and stems and demonstrates the key role these traits play in the link between plant and soil via herbivory and decomposition. Fibre concentration and dry matter content can be considered as good predictors of both digestibility and decomposability. © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Droux, M; Ruffet, M L; Douce, R; Job, D
1998-07-01
The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.
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Ahmedov, Merdin Lyutviev; Kemerdere, Rahsan; Baran, Oguz; Inal, Berrin Bercik; Gumus, Alper; Coskun, Cihan; Yeni, Seher Naz; Eren, Bulent; Uzan, Mustafa; Tanriverdi, Taner
2017-10-01
We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r 2 = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r 2 = 0.7, P = 0.00001 and r 2 = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r 2 = 0.06, P = 0.00001). Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature. Copyright © 2017 Elsevier Inc. All rights reserved.
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Spatial organization of multi-enzyme biocatalytic cascades.
Quin, M B; Wallin, K K; Zhang, G; Schmidt-Dannert, C
2017-05-23
Industrial biocatalysis is an economically attractive option for the production of valuable chemicals. Our repertoire of cheap building blocks and commodity target molecules is vastly enhanced by multi-enzyme biocatalytic cascades. In order to achieve suitable titers in complex novel biocatalytic schemes, spatial organization may become necessary to overcome barriers caused by slow or inhibited enzymes as well as instability of biocatalysts. A number of spatial organization strategies are currently available, which could be integrated in the design of complex cascades. These include fusion proteins, immobilization on solid supports, multi-dimensional scaffolding, and encapsulation within vessels. This review article highlights recent advances in cascade biocatalysis, discusses the role of spatial organization in reaction kinetics, and presents some of the currently employed strategies for spatial organization of multi-enzyme cascades.
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Chung, Ivy Yeuk Wah; Paetzel, Mark
2013-05-03
Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kohler, Annegret; Kuo, Alan; Nagy, Laszlo G.
To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergentmore » evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.« less
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Kohler, Annegret; Kuo, Alan; Nagy, Laszlo G.; ...
2015-02-23
To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergentmore » evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.« less
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Enhanced production and application of acidothermophilic Streptomyces cellulase.
Budihal, Saikumar R; Agsar, Dayanand; Patil, Sarvamangala R
2016-01-01
An efficient cellulolytic and acidothermophilic actinobacterium was isolated from soil, adhered to decomposing tree bark and was identified as Streptomyces DSK59. Screening of synthetic media and the media components identified that, a medium based on starch casein minerals containing carboxy methyl cellulose (CMC) and beef extract (BE) could support enhanced cellulase production by the organism. CMC, BE, NaCl, temperature and pH were accounted as significant for cellulase production and these were optimized using a response surface central composite design (CCD). Optimization of cellulase production resulted in an enhancement of endoglucanase activity to 27IUml(-1). Acidothermophillic Streptomyces cellulase was found to be efficient for hydrolysis of pretreated sorghum stover and liberated 0.413gg(-1) of total reducing sugars which was higher than previously reported sugar yields obtained using fungal enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Denitrification Associated with Periphyton Communities
Triska, Frank J.; Oremland, Ronald S.
1981-01-01
Scrapings of decomposing Cladophora sp. mats (periphyton) covering stream bed rocks produced N2O when incubated under N2 plus 15% C2H2. Denitrification (N2O formation) was enhanced by NO3− and was inhibited by autoclaving, Hg2+, and O2. No N2O was formed in the absence of C2H2 (air or N2 atmosphere). Chloramphenicol did not block N2O formation, indicating that the enzymes were constitutive. In field experiments, incubation of periphyton scrapings in the light inhibited denitrification because of algal photosynthetic O2 production. The diurnal periphyton-associated denitrification rate was estimated to be 45.8 μmol of N2O·m−2·day−1, as determined by averaging light, aerobic plus dark, and anaerobic rates over a 24-h period. PMID:16345874
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Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V
1997-01-01
Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.
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Integrated boiler, superheater, and decomposer for sulfuric acid decomposition
Moore, Robert [Edgewood, NM; Pickard, Paul S [Albuquerque, NM; Parma, Jr., Edward J.; Vernon, Milton E [Albuquerque, NM; Gelbard, Fred [Albuquerque, NM; Lenard, Roger X [Edgewood, NM
2010-01-12
A method and apparatus, constructed of ceramics and other corrosion resistant materials, for decomposing sulfuric acid into sulfur dioxide, oxygen and water using an integrated boiler, superheater, and decomposer unit comprising a bayonet-type, dual-tube, counter-flow heat exchanger with a catalytic insert and a central baffle to increase recuperation efficiency.
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Procedures for Decomposing a Redox Reaction into Half-Reaction
ERIC Educational Resources Information Center
Fishtik, Ilie; Berka, Ladislav H.
2005-01-01
A simple algorithm for a complete enumeration of the possible ways a redox reaction (RR) might be uniquely decomposed into half-reactions (HRs) using the response reactions (RERs) formalism is presented. A complete enumeration of the possible ways a RR may be decomposed into HRs is equivalent to a complete enumeration of stoichiometrically…
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Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays
NASA Astrophysics Data System (ADS)
Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan
2015-03-01
The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications. Electronic supplementary information (ESI) available: Experimental procedures and analytical data are provided. See DOI: 10.1039/c5nr00697j
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On Design Mining: Coevolution and Surrogate Models.
Preen, Richard J; Bull, Larry
2017-01-01
Design mining is the use of computational intelligence techniques to iteratively search and model the attribute space of physical objects evaluated directly through rapid prototyping to meet given objectives. It enables the exploitation of novel materials and processes without formal models or complex simulation. In this article, we focus upon the coevolutionary nature of the design process when it is decomposed into concurrent sub-design-threads due to the overall complexity of the task. Using an abstract, tunable model of coevolution, we consider strategies to sample subthread designs for whole-system testing and how best to construct and use surrogate models within the coevolutionary scenario. Drawing on our findings, we then describe the effective design of an array of six heterogeneous vertical-axis wind turbines.
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Segmental Refinement: A Multigrid Technique for Data Locality
Adams, Mark F.; Brown, Jed; Knepley, Matt; ...
2016-08-04
In this paper, we investigate a domain decomposed multigrid technique, termed segmental refinement, for solving general nonlinear elliptic boundary value problems. We extend the method first proposed in 1994 by analytically and experimentally investigating its complexity. We confirm that communication of traditional parallel multigrid is eliminated on fine grids, with modest amounts of extra work and storage, while maintaining the asymptotic exactness of full multigrid. We observe an accuracy dependence on the segmental refinement subdomain size, which was not considered in the original analysis. Finally, we present a communication complexity analysis that quantifies the communication costs ameliorated by segmental refinementmore » and report performance results with up to 64K cores on a Cray XC30.« less
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[2+2] cycloaddition of 1,3-dienes by visible light photocatalysis.
Hurtley, Anna E; Lu, Zhan; Yoon, Tehshik P
2014-08-18
[2+2] photocycloadditions of 1,3-dienes represent a powerful yet synthetically underutilized class of reactions. We report that visible light absorbing transition metal complexes enable the [2+2] cycloaddition of a diverse range of 1,3-dienes. The ability to use long-wavelength visible light is attractive because these reaction conditions tolerate the presence of sensitive functional groups that might be readily decomposed by the high-energy UVC radiation required for direct photoexcitation of 1,3-dienes. The resulting vinylcyclobutane products are poised for a variety of further diversification reactions, and this method is consequently expected to be powerfully enabling in the synthesis of complex organic targets. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko
β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less
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Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; ...
2017-06-14
β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less
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Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross
2017-08-25
Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .
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Regulation of yeast central metabolism by enzyme phosphorylation
Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe
2012-01-01
As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688
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A multi-step chromatographic strategy to purify three fungal endo-β-glucanases.
McCarthy, Tracey; Tuohy, Maria G
2011-01-01
Fungi and fungal enzymes have traditionally occupied a central role in biotechnology. Understanding the biochemical properties of the variety of enzymes produced by these eukaryotes has been an area of research interest for decades and again more recently due to global interest in greener bio-production technologies. Purification of an individual enzyme allows its unique biochemical and functional properties to be determined, can provide key information as to the role of individual biocatalysts within a complex enzyme system, and can inform both protein engineering and enzyme production strategies in the development of novel green technologies based on fungal biocatalysts. Many enzymes of current biotechnological interest are secreted by fungi into the extracellular culture medium. These crude enzyme mixtures are typically complex, multi-component, and generally also contain other non-enzymatic proteins and secondary metabolites. In this chapter, we describe a multi-step chromatographic strategy required to isolate three new endo-β-glucanases (denoted EG V, EG VI, and EG VII) with activity against cereal mixed-linkage β-glucans from the thermophilic fungus Talaromyces emersonii. This work also illustrates the challenges frequently involved in isolating individual extracellular fungal proteins in general.
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Efremova, Maria V; Veselov, Maxim M; Barulin, Alexander V; Gribanovsky, Sergey L; Le-Deygen, Irina M; Uporov, Igor V; Kudryashova, Elena V; Sokolsky-Papkov, Marina; Majouga, Alexander G; Golovin, Yuri I; Kabanov, Alexander V; Klyachko, Natalia L
2018-04-24
Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.
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Respiratory complex I: 'steam engine' of the cell?
Efremov, Rouslan G; Sazanov, Leonid A
2011-08-01
Complex I is the first enzyme of the respiratory chain and plays a central role in cellular energy production. It has been implicated in many human neurodegenerative diseases, as well as in ageing. One of the biggest membrane protein complexes, it is an L-shaped assembly consisting of hydrophilic and membrane domains. Previously, we have determined structures of the hydrophilic domain in several redox states. Last year was marked by fascinating breakthroughs in the understanding of the complete structure. We described the architecture of the membrane domain and of the entire bacterial complex I. X-ray analysis of the larger mitochondrial enzyme has also been published. The core subunits of the bacterial and mitochondrial enzymes have remarkably similar structures. The proposed mechanism of coupling between electron transfer and proton translocation involves long-range conformational changes, coordinated in part by a long α-helix, akin to the coupling rod of a steam engine. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Molecular Dynamic Studies of the Complex Polyethylenimine and Glucose Oxidase.
Szefler, Beata; Diudea, Mircea V; Putz, Mihai V; Grudzinski, Ireneusz P
2016-10-27
Glucose oxidase (GOx) is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state) to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H₂O₂). GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI). The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD) study of the PEI ligand (C14N8_07_B22) and the GOx enzyme (3QVR) was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1) of -5.8 kcal/mol and (LIG2) of -4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1) and on its surface (LIG2) were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase.
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Effect of posttranslational modifications on enzyme function and assembly.
Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej
2013-10-30
The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.
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Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.
Boehlein, Susan K; Shaw, Janine R; Hwang, Seon K; Stewart, Jon D; Curtis Hannah, L
2013-07-15
ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis. Copyright © 2013 Elsevier Inc. All rights reserved.
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Conformational Sub-states and Populations in Enzyme Catalysis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Agarwal, Pratul K; Doucet, Nicholas; Chennubholta, C
reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surroundingmore » environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.« less
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System for thermochemical hydrogen production
Werner, R.W.; Galloway, T.R.; Krikorian, O.H.
1981-05-22
Method and apparatus are described for joule boosting a SO/sub 3/ decomposer using electrical instead of thermal energy to heat the reactants of the high temperature SO/sub 3/ decomposition step of a thermochemical hydrogen production process driven by a tandem mirror reactor. Joule boosting the decomposer to a sufficiently high temperature from a lower temperature heat source eliminates the need for expensive catalysts and reduces the temperature and consequent materials requirements for the reactor blanket. A particular decomposer design utilizes electrically heated silicon carbide rods, at a temperature of 1250/sup 0/K, to decompose a cross flow of SO/sub 3/ gas.
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Aridity and decomposition processes in complex landscapes
NASA Astrophysics Data System (ADS)
Ossola, Alessandro; Nyman, Petter
2015-04-01
Decomposition of organic matter is a key biogeochemical process contributing to nutrient cycles, carbon fluxes and soil development. The activity of decomposers depends on microclimate, with temperature and rainfall being major drivers. In complex terrain the fine-scale variation in microclimate (and hence water availability) as a result of slope orientation is caused by differences in incoming radiation and surface temperature. Aridity, measured as the long-term balance between net radiation and rainfall, is a metric that can be used to represent variations in water availability within the landscape. Since aridity metrics can be obtained at fine spatial scales, they could theoretically be used to investigate how decomposition processes vary across complex landscapes. In this study, four research sites were selected in tall open sclerophyll forest along a aridity gradient (Budyko dryness index ranging from 1.56 -2.22) where microclimate, litter moisture and soil moisture were monitored continuously for one year. Litter bags were packed to estimate decomposition rates (k) using leaves of a tree species not present in the study area (Eucalyptus globulus) in order to avoid home-field advantage effects. Litter mass loss was measured to assess the activity of macro-decomposers (6mm litter bag mesh size), meso-decomposers (1 mm mesh), microbes above-ground (0.2 mm mesh) and microbes below-ground (2 cm depth, 0.2 mm mesh). Four replicates for each set of bags were installed at each site and bags were collected at 1, 2, 4, 7 and 12 months since installation. We first tested whether differences in microclimate due to slope orientation have significant effects on decomposition processes. Then the dryness index was related to decomposition rates to evaluate if small-scale variation in decomposition can be predicted using readily available information on rainfall and radiation. Decomposition rates (k), calculated fitting single pool negative exponential models, generally decreased with increasing aridity with k going from 0.0025 day-1 on equatorial (dry) facing slopes to 0.0040 day-1 on polar (wet) facing slopes. However, differences in temperature as a result of morning vs afternoon sun on east and west aspects, respectively, (not captured in the aridity metric) resulted in poor prediction of decomposition for the sites located in the intermediate aridity range. Overall the results highlight that relatively small differences in microclimate due to slope orientation can have large effects on decomposition. Future research will aim to refine the aridity metric to better resolve small scale variation in surface temperature which is important when up-scaling decomposition processes to landscapes.
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Introducing Michaelis-Menten Kinetics through Simulation
ERIC Educational Resources Information Center
Halkides, Christopher J.; Herman, Russell
2007-01-01
We describe a computer tutorial that introduces the concept of the steady state in enzyme kinetics. The tutorial allows students to produce graphs of the concentrations of free enzyme, enzyme-substrate complex, and product versus time in order to learn about the approach to steady state. By using a range of substrate concentrations and rate…
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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.
Kürten, Charlotte; Syrén, Per-Olof
2016-01-16
Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.
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Gold nanoparticles bound on microgel particles and their application as an enzyme support
NASA Astrophysics Data System (ADS)
Xu, Jing; Zeng, Fang; Wu, Shuizhu; Liu, Xinxing; Hou, Chao; Tong, Zhen
2007-07-01
Submicron-sized poly(N-isopropyl acrylamide)/polyethyleneimine core-shell microgels were prepared in aqueous media by using tert-butyl hydroperoxide (TBHP) as an initiator, and then the gold nanoparticles (~8 nm) were formed on the surface of the microgels. The amino groups on the polyethyleneimine (PEI) chains act as the binder for the assembly of the gold nanoparticles/microgel complex. In aqueous media the microgels are highly stable with the gold nanoparticles on their extended PEI chains, and this multi-scale nanoparticle complex can be recovered from water and redispersed in water. The nanogold/microgel particles were conjugated with the enzymes horseradish peroxidase (HRP) and urease. It is found that under identical assay conditions the enzyme/nanogold/microgel systems exhibit enhanced biocatalytic activity over free enzymes in solution, especially at lower enzyme concentrations. In addition, compared to free HRP, the HRP/nanogold/microgel systems show higher activity at varied pHs and temperatures, as well as higher storage stability. Thus the novel nanogold/microgel particles can serve as an excellent support for enzymes.
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Shin, W; Lindahl, P A
1992-12-29
Adding 1,10-phenanthroline to carbon monoxide dehydrogenase from Clostridium thermoaceticum results in the complete loss of the NiFeC EPR signal and the CO/acetyl-CoA exchange activity. Other EPR signals characteristic of the enzyme (the gav = 1.94 and gav = 1.86 signals) and the CO oxidation activity are completely unaffected by the 1,10-phenanthroline treatment. This indicates that there are two catalytic sites on the enzyme; the NiFe complex is required for catalyzing the exchange and acetyl-CoA synthase reactions, while some other site is responsible for CO oxidation. The strength of CO binding to the NiFe complex was examined by titrating dithionite-reduced enzyme with CO. During the titration, the NiFeC EPR signal developed to a final spin intensity of 0.23 spin/alpha beta. The resulting CO titration curve (NiFeC spins/alpha beta vs CO pha beta) was fitted using two reactions: binding of CO to the oxidized NiFe complex, and reduction of the CO-bound species to a form that exhibits the NiFeC signal. Best fits yielded apparent binding constants between 6000 and 14,000 M-1 (Kd = 70-165 microM). This sizable range is due to uncertainty whether CO binds to all or only a small fraction (approximately 23%) of the NiFe complexes. Reduction of the CO-bound NiFe complex is apparently required to activate it for catalysis. The electron used for this reduction originates from the CO oxidation site, suggesting that delivery of a low-potential electron to the CO-bound NiFe complex is the physiological function of the CO oxidation reaction catalyzed by this enzyme.
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Reducing the complexity of the software design process with object-oriented design
NASA Technical Reports Server (NTRS)
Schuler, M. P.
1991-01-01
Designing software is a complex process. How object-oriented design (OOD), coupled with formalized documentation and tailored object diagraming techniques, can reduce the complexity of the software design process is described and illustrated. The described OOD methodology uses a hierarchical decomposition approach in which parent objects are decomposed into layers of lower level child objects. A method of tracking the assignment of requirements to design components is also included. Increases in the reusability, portability, and maintainability of the resulting products are also discussed. This method was built on a combination of existing technology, teaching experience, consulting experience, and feedback from design method users. The discussed concepts are applicable to hierarchal OOD processes in general. Emphasis is placed on improving the design process by documenting the details of the procedures involved and incorporating improvements into those procedures as they are developed.
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Strategies for concurrent processing of complex algorithms in data driven architectures
NASA Technical Reports Server (NTRS)
Stoughton, John W.; Mielke, Roland R.
1988-01-01
The purpose is to document research to develop strategies for concurrent processing of complex algorithms in data driven architectures. The problem domain consists of decision-free algorithms having large-grained, computationally complex primitive operations. Such are often found in signal processing and control applications. The anticipated multiprocessor environment is a data flow architecture containing between two and twenty computing elements. Each computing element is a processor having local program memory, and which communicates with a common global data memory. A new graph theoretic model called ATAMM which establishes rules for relating a decomposed algorithm to its execution in a data flow architecture is presented. The ATAMM model is used to determine strategies to achieve optimum time performance and to develop a system diagnostic software tool. In addition, preliminary work on a new multiprocessor operating system based on the ATAMM specifications is described.
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Structure of choline oxidase in complex with the reaction product glycine betaine.
Salvi, Francesca; Wang, Yuan-Fang; Weber, Irene T; Gadda, Giovanni
2014-02-01
Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Å resolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.
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Rodriguez-Maciá, Patricia; Dutta, Arnab; Lubitz, Wolfgang; Shaw, Wendy J; Rüdiger, Olaf
2015-10-12
The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sullivan, Sarah M.; Holyoak, Todd
2008-09-17
The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sullivan, S.M.; Holyoak, T.
2009-05-26
The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less
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Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization
2015-01-01
A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628
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NASA Astrophysics Data System (ADS)
Lloret Sevilla, E.; Brodie, E.; Bouskill, N.; Hao, Z.
2016-12-01
Phosphorus is an essential nutrient with a reduced availability in tropical forests. In these ecosystems, P is recycled highly efficiently through resorption and mineralization and P immobilization in the microbial biomass prevents its loss through occlusion in the soil mineral fraction. To improve models of ecosystem response to global change, further studies of the above and belowground plant and microbial traits related to P availability and uptake, are required. In tropical forests, high temperature and rainfall lead to some of the highest rates of litter decomposition on earth. Litter decomposition is a complex process mediated by a range of trophic groups: meso and microfauna initiate litter turnover through litter fragmentation facilitating colonization by fungi, and bacteria mediate the mineralization of organic matter and release of nutrients. To determine the important functional traits of these players in the efficient cycling of P in soils with low P availability, we are performing a leaf litter decomposition experiment in a humid tropical forest in Puerto Rico. Nylon litterbags with three mesh sizes (2mm, 20 μm and 0.45 μm) containing litter with different chemistry (tabonuco and palm) will be deployed on soil surface and sampled 6 times throughout 12 months. The use of different mesh sizes will allow us to identify the leading roles in litter turnover by physical allowance and/or exclusion of the decomposers. The 2 mm bags allow meso and microfauna, roots, fungi and bacteria. 20 μm bags will exclude fauna and roots and 0.45 μm only allow some bacteria. We hypothesize that fungi will dominate over bacteria in earlier stages of the decomposition with a higher production of extracellular hydrolytic enzymes. On the other hand, bacterial biomass is expected to increase with time. Qualitative changes in both fungal and bacterial communities along the decomposition process are also expected leading to changes in enzyme activity. We also postulate an enhanced microbial communities abundance and activity in litter with higher nutrient content. Regarding the microarthropods, we hypothesize that their diversity and abundance will be inversely related to mass loss.
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The Aminoacyl-tRNA Synthetase Complex.
Mirande, Marc
2017-01-01
Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.
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Bawa, Priya; Choonara, Yahya E; du Toit, Lisa C; Kumar, Pradeep; Ndesendo, Valence M K; Meyer, Leith C R; Pillay, Viness
2013-03-28
The study focussed on designing a Stimuli-Synchronized Matrix (SSM) for space-defined colonic delivery of the anti-inflammatory drug mesalamine. The configured matrix provided time-independent delivery and stimuli targeting. Formulations were optimized according to a Box-Behnken experimental design that constituted mesalamine-loaded BaSO4-crosslinked chitosan dispersed within a pectin, carboxymethylcellulose and xanthan gum complex. The complex was compressed into matrices and subsequently alloy-treated with pectin and ethylcellulose. In vitro drug release was determined in the presence and absence of colonic enzymes and the mean dissolution time was used for formulation optimization. To mechanistically elucidate the synchronous catalytic action of the enzymes pectinase and glucosidase on the matrix, computer-aided 3D modelling of active fractions of the enzyme-substrate complexes was generated to predict the orientation of residues affecting the substrate domain. Drug release profiles revealed distinct colonic enzyme responsiveness with fractions of 0.402 and 0.152 of mesalamine released in the presence and absence of enzymes, respectively after 24h. The commercial comparator product showed irreproducible release profiles over the same period (SD=0.550) compared to the SSM formulation (SD=0.037). FTIR spectra of alloy-treated matrices showed no peaks from 1589 to 1512cm(-1) after colonic enzyme exposure. With increasing enzyme exposure there were also no peaks between 1646 and 1132cm(-1). This indicated polymeric enzyme cleavage for controlled and space-defined release of mesalamine. Plasma concentration profiles in the Large White pig model produced a Cmax of 3.77±1.375μg/mL compared to 10.604±2.846μg/mL for the comparator formulation. The SSM formulation proved superior over the comparator product by providing superiorly controlled enzyme-responsive colonic drug delivery. Copyright © 2012 Elsevier B.V. All rights reserved.
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Protein dynamics and enzyme catalysis: insights from simulations.
McGeagh, John D; Ranaghan, Kara E; Mulholland, Adrian J
2011-08-01
The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.
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Herschlag, Daniel; Natarajan, Aditya
2013-01-01
Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multi-faceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis. PMID:23488725
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Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification.
Guan, Weihua; Chen, Liben; Rane, Tushar D; Wang, Tza-Huei
2015-09-03
We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.
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Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification
Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei
2015-01-01
We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples. PMID:26333806
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Herschlag, Daniel; Natarajan, Aditya
2013-03-26
Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.
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Rocchitta, Gaia; Spanu, Angela; Babudieri, Sergio; Latte, Gavinella; Madeddu, Giordano; Galleri, Grazia; Nuvoli, Susanna; Bagella, Paola; Demartis, Maria Ilaria; Fiore, Vito; Manetti, Roberto; Serra, Pier Andrea
2016-01-01
Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented. PMID:27249001
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Ilag, Leopold L; Westblade, Lars F; Deshayes, Caroline; Kolb, Annie; Busby, Stephen J W; Robinson, Carol V
2004-02-01
The E. coli RNA polymerase core enzyme is a multisubunit complex of 388,981 Da. To initiate transcription at promoters, the core enzyme associates with a sigma subunit to form holo RNA polymerase. Here we have used nanoflow electrospray mass spectrometry, coupled with tandem mass spectrometry, to probe the interaction of the RNA polymerase core enzyme with the most abundant sigma factor, sigma70. The results show remarkably well-resolved spectra for both the core and holo RNA polymerases. The regulator of sigma70, Rsd protein, has previously been identified as a protein that binds to free sigma70. We show that Rsd also interacts with core enzyme. In addition, by adding increasing amounts of Rsd, we show that sigma70 is displaced from holo RNA polymerase, resulting in complexes of Rsd with core and sigma70. The results argue for a model in which Rsd not only sequesters sigma70, but is also an effector of core RNA polymerase.
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Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification
NASA Astrophysics Data System (ADS)
Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei
2015-09-01
We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.
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Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals
Kostic, Nenad M.; Chen, Jian
1991-03-05
Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme.
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Rhodes, D.; Gage, D. A.; Cooper, AJL.; Hanson, A. D.
1997-01-01
Leaves of Wollastonia biflora (L.) DC. synthesize the osmoprotectant 3-dimethylsulfoniopropionate (DMSP) from methionine via S-methylmethionine (SMM) and 3-dimethylsulfoniopropionaldehyde (DMSP-ald); no other intermediates have been detected. To test whether the amino group of SMM is lost by transamination or deamination, [methyl-2H3,15N]SMM was supplied to leaf discs, and 15N-labeling of amino acids was monitored, along with synthesis of [2H3]DMSP. After short incubations more 15N was incorporated into glutamate than into other amino acids, and the 15N abundance in glutamate exceeded that in the amide group of glutamine (Gln). This is more consistent with transamination than deamination, because deamination would be predicted to give greater labeling of Gln amide N due to reassimilation, via Gln synthetase, of the 15NH4+ released. This prediction was borne out by control experiments with 15NH4Cl. The transamination product of SMM, 4-dimethylsulfonio-2-oxobutyrate (DMSOB), is expected to be extremely unstable. This was confirmed by attempting to synthesize it enzymatically from SMM using L-amino acid oxidase or Gln transaminase K and from 4-methylthio-2-oxobutyrate using methionine S-methyltransferase. In each case, the reaction product decomposed rapidly, releasing dimethylsulfide. The conversion of SMM to DMSP-ald is therefore unlikely to involve a simple transamination that generates free DMSOB. Plausible alternatives are that DMSOB is channeled within a specialized transaminase-decarboxylase complex or that it exists only as the bound intermediate of a single enzyme catalyzing an unusual transamination-decarboxylation reaction. PMID:12223879
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Chen, Ying; Liu, Yu Xue; Chen, Chong Jun; Lyu, Hao Hao; Wa, Yu Ying; He, Li Li; Yang, Sheng Mao
2018-01-01
In recent years, studies on carbon sequestration of biochar in soil has been in spotlight owing to the specific characteristics of biochar such as strong carbon stability and well developed pore structure. However, whether biochar will ultimately increase soil carbon storage or promote soil carbon emissions when applied into the soil? This question remains controversial in current academic circles. Further research is required on priming effect of biochar on mineralization of native soil organic carbon and its mechanisms. Based on the analysis of biochar characteristics, such as its carbon composition and stability, pore structure and surface morphology, research progress on the priming effect of biochar on the decomposition of native soil organic carbon was reviewed in this paper. Furthermore, possible mechanisms of both positive and negative priming effect, that is promoting and suppressing the mineralization, were put forward. Positive priming effect is mainly due to the promotion of soil microbial activity caused by biochar, the preferential mineralization of easily decomposed components in biochar, and the co-metabolism of soil microbes. While negative priming effect is mainly based on the encapsulation and adsorption protection of soil organic matter due to the internal pore structure and the external surface of biochar. Other potential reasons for negative priming effect can be the stabilization resulted from the formation of organic-inorganic complex promoted by biochar in the soil, and the inhibition of activity of soil microbes and its enzymes by biochar. Finally, future research directions were proposed in order to provide theoretical basis for the application of biochar in soil carbon sequestration.
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Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi
2003-01-01
Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859
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NASA Astrophysics Data System (ADS)
Kaiser, Christina; Evans, Sarah; Dieckmann, Ulf; Widder, Stefanie
2016-04-01
At the μm-scale, soil is a highly structured and complex environment, both in physical as well as in biological terms, characterized by non-linear interactions between microbes, substrates and minerals. As known from mathematics and theoretical ecology, spatial structure significantly affects the system's behaviour by enabling synergistic dynamics, facilitating diversity, and leading to emergent phenomena such as self-organisation and self-regulation. Such phenomena, however, are rarely considered when investigating mechanisms of microbial soil organic matter turnover. Soil organic matter is the largest terrestrial reservoir for organic carbon (C) and nitrogen (N) and plays a pivotal role in global biogeochemical cycles. Still, the underlying mechanisms of microbial soil organic matter buildup and turnover remain elusive. We explored mechanisms of microbial soil organic matter turnover using an individual-based, stoichiometrically and spatially explicit computer model, which simulates the microbial de-composer system at the soil microscale (i.e. on a grid of 100 x 100 soil microsites). Soil organic matter dynamics in our model emerge as the result of interactions among individual microbes with certain functional traits (f.e. enzyme production rates, growth rates, cell stoichiometry) at the microscale. By degrading complex substrates, and releasing labile substances microbes in our model continusly shape their environment, which in turn feeds back to spatiotemporal dynamics of the microbial community. In order to test the effect of microbial functional traits and organic matter input rate on soil organic matter turnover and C and N storage, we ran the model into steady state using continuous inputs of fresh organic material. Surprisingly, certain parameter settings that induce resource limitation of microbes lead to regular spatial pattern formation (f.e. moving spiral waves) of microbes and substrate at the μm-scale at steady-state. The occurrence of these pattern can be explained by the Turing mechanism. These pattern formation had strong consequences for process rates, as well as for C and N storage in the soil at the steady state: Scenarios that exhibited pattern formation were generally associated with higher C storage at steady state compared to those without pattern formation (i.e. at non-limiting conditions for microbes). Moreover, pattern formation lead to a spatial decoupling of C and N turnover processes, and to a spatial decoupling of microbial N mineralization and N immobilization. Taken together, our theoretical analysis shows that self-organisation may be a feature of the soil decomposer system, with consequences for process rates of microbial C and N turnover. Pattern formation through spatial self-organization, which has been observed on larger spatial scales in other resource-limited communities (e.g., vegetation patterns in arid or wetland eco-systems), may also occur at the soil microscale, leaving its mark on the soil's storage capacity for C and N.
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Predicting the response of soil organic matter microbial decomposition to moisture
NASA Astrophysics Data System (ADS)
Chenu, Claire; Garnier, Patricia; Monga, Olivier; Moyano, Fernando; Pot, Valérie; Nunan, Naoise; Coucheney, Elsa; Otten, Wilfred
2014-05-01
Next to temperature, soil moisture is a main driver of soil C and N transformations in soils, because it affects microbial activity and survival. The moisture sensitivity of soil organic matter decay may be a source of uncertainty of similar magnitude to that of the temperature sensitivity and receives much less attention. The basic concepts and mechanisms relating soil water to microorganisms were identified early (i.e. in steady state conditions : direct effects on microbial physiology, diffusion substrates, nutrients, extracellular enzymes, diffusion of oxygen, movement of microorganisms). However, accounting for how moisture controls soil microbial activity remains essentially empirical and poorly accounts for soil characteristics. Soil microorganisms live in a complex 3-D framework of mineral and organic particles defining pores of various sizes, connections with adjacent pores, and with pore walls of contrasted nature, which result in a variety of microhabitats. The water regime to which microorganisms are exposed can be predicted to depend the size and connectivity of pores in which they are located. Furthermore, the spatial distribution of microorganisms as well as that of organic matter is very heterogeneous, determining the diffusion distances between substrates and decomposers. A new generation of pore scale models of C dynamics in soil may challenge the difficulty of modelling such a complex system. These models are based on an explicit representation of soil structure (i.e. soil particles and voids), microorganisms and organic matter localisation. We tested here the ability of such a model to account for changes in microbial respiration with soil moisture. In the model MOSAIC II, soil pore space is described using a sphere network coming from a geometrical modelling algorithm. MicroCT tomography images were used to implement this representation of soil structure. A biological sub-model describes the hydrolysis of insoluble SOM into dissolved organic matter, its assimilation, respiration and microbial mortality. A recent improvement of the model was the description of the diffusion of soluble organic matter. We tested the model using the results from an experiment where a simple substrate (fructose) was decomposed by bacteria within a simple media (sand). Separate incubations in microcosms were carried out using five different bacterial communities at two different moisture conditions corresponding to water potentials of -0.01 and -0.1 bars. We calibrated the biological parameters using the experimental data obtained at high water content and we tested the model without any parameters change at low water content. Both the experiments and simulations showed a decrease in mineralisation with a decrease of water content, of which pattern depended on the bacterial species and its physiological characteristics. The model was able to correctly simulate the decrease of connectivity between substrate and microorganism due the decrease of water content. The potential and required developments of such models in describing how heterotrophic respiration is affected by micro-scale distribution and processes in soils and in testing scenarios regarding water regimes in a changing climate is discussed.
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Al-Otaiba, Amna; John, Annie; Al-Belooshi, Thekra; Raza, Haider
2010-11-01
We have previously reported the occurrence of multiple forms of drug-metabolizing enzymes in camel tissues. Here, we investigate glutathione (GSH)-dependent redox homeostasis, reactive oxygen species (ROS) production and mitochondrial respiratory functions in camel tissues and compare them with imported domestic goats and laboratory rats and mice. Cytochrome P450 2E1 (CYP 2E1) and GSH-metabolizing enzymes were differentially expressed in the liver and kidney of these animals. Camel liver has significantly lower GSH pool than that in goats, rats and mice. Mitochondria isolated from the tissues of these animals showed a comparable ability to metabolize specific substrates for respiratory enzyme complexes I, II/III and IV. These complexes were metabolically more active in the kidney than in the liver of all the species. Furthermore, the activity of complex IV in camel tissues was significantly lower than in other species. On the other hand, complex II/III activity in camel kidney was higher compared to the other species. In addition, as expected, we observed that inhibitors of these enzyme complexes augment the production of mitochondrial ROS in camel and goat tissues. These results help to better understand the metabolic ability and adaptation in desert camels in comparison with domestic goats and laboratory rats and mice since they are exposed to different environmental and dietary conditions. Our study may also have implications in the pharmacology and toxicology of drugs and pollutants in these species.
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ERIC Educational Resources Information Center
Cieslicka, Anna B.
2013-01-01
The purpose of this study was to explore possible cerebral asymmetries in the processing of decomposable and nondecomposable idioms by fluent nonnative speakers of English. In the study, native language (Polish) and foreign language (English) decomposable and nondecomposable idioms were embedded in ambiguous (neutral) and unambiguous (biasing…
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Modeling and optimum time performance for concurrent processing
NASA Technical Reports Server (NTRS)
Mielke, Roland R.; Stoughton, John W.; Som, Sukhamoy
1988-01-01
The development of a new graph theoretic model for describing the relation between a decomposed algorithm and its execution in a data flow environment is presented. Called ATAMM, the model consists of a set of Petri net marked graphs useful for representing decision-free algorithms having large-grained, computationally complex primitive operations. Performance time measures which determine computing speed and throughput capacity are defined, and the ATAMM model is used to develop lower bounds for these times. A concurrent processing operating strategy for achieving optimum time performance is presented and illustrated by example.
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Crossing symmetry in alpha space
NASA Astrophysics Data System (ADS)
Hogervorst, Matthijs; van Rees, Balt C.
2017-11-01
We initiate the study of the conformal bootstrap using Sturm-Liouville theory, specializing to four-point functions in one-dimensional CFTs. We do so by decomposing conformal correlators using a basis of eigenfunctions of the Casimir which are labeled by a complex number α. This leads to a systematic method for computing conformal block decompositions. Analyzing bootstrap equations in alpha space turns crossing symmetry into an eigenvalue problem for an integral operator K. The operator K is closely related to the Wilson transform, and some of its eigenfunctions can be found in closed form.
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Fracturing fluid cleanup by controlled release of enzymes from polyelectrolyte complex nanoparticles
NASA Astrophysics Data System (ADS)
Barati Ghahfarokhi, Reza
Guar-based polymer gels are used in the oil and gas industry to viscosify fluids used in hydraulic fracturing of production wells, in order to reduce leak-off of fluids and pressure, and improve the transport of proppants. After fracturing, the gel and associated filter cake must be degraded to very low viscosities using breakers to recover the hydraulic conductivity of the well. Enzymes are widely used to achieve this but injecting high concentrations of enzyme may result in premature degradation, or failure to gel; denaturation of enzymes at alkaline pH and high temperature conditions can also limit their applicability. In this study, application of polyelectrolyte nanoparticles for entrapping, carrying, releasing and protecting enzymes for fracturing fluids was examined. The objective of this research is to develop nano-sized carriers capable of carrying the enzymes to the filter cake, delaying the release of enzyme and protecting the enzyme against pH and temperature conditions inhospitable to native enzyme. Polyethylenimine-dextran sulfate (PEI-DS) polyelectrolyte complexes (PECs) were used to entrap two enzymes commonly used in the oil industry in order to obtain delayed release and to protect the enzyme from conditions inhospitable to native enzyme. Stability and reproducibility of PEC nanoparticles was assured over time. An activity measurement method was used to measure the entrapment efficiency of enzyme using PEC nanoparticles. This method was confirmed using a concentration measurement method (SDS-PAGE). Entrapment efficiencies of pectinase and a commercial high-temperature enzyme mixture in polyelectrolyte complex nanoparticles were maximized. Degradation, as revealed by reduction in viscoelastic moduli of borate-crosslinked hydroxypropyl guar (HPG) gel by commercial enzyme loaded in polyelectrolyte nanoparticles, was delayed, compared to equivalent systems where the enzyme mixture was not entrapped. This indicates that PEC nanoparticles delay the activity of enzymes by entrapping them. It was also observed that control PEC nanoparticles decreased both viscoelastic moduli, but with a slower rate compared to the PEC nanoparticles loaded with enzyme. Preparation shear and applied shear showed no significant effect on activity of enzyme-loaded PEC nanoparticles mixed with HPG solutions. However, fast addition of chemicals during the preparations showed smaller particle size compared to the drop-wise method. PEC nanoparticles (PECNPs) also protected both enzymes from denaturation at elevated temperature and pH. Following preparation, enzyme-loaded PEC nanoparticles were mixed with borate crosslinked HPG and the mixture was injected through a shear loop. Pectinase-loaded nanoparticles mixed with gelled HPG showed no sensitivity to shear applied along the shear loop at 25 °C. However, EL2X-loaded PEC nanoparticles showed sensitivity to shear applied along the shear loop at 40 °C. Filter cake was formed and degraded in a fluid loss cell for borate crosslinked HPG solutions mixed with either enzymes or enzyme-loaded PEC nanoparticles. Cleanup slopes of filter cake degraded using enzyme-loaded PEC nanoparticles and systems with enzymes mixed with HPG gel were significantly higher than for the filter cake formed with HPG gel mixed with no enzyme. In a different application, enzyme-loaded PEC nanoparticles showed significantly slower reduction in viscosity of HPG solution over time compared to the HPG systems mixed with enzyme. Increasing the viscosity of low concentration HPG, used as slick-water, decreases the proppant settling velocity. This is of specific interest in fracturing fluids used for unconventional reservoirs.
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Phung, Thu-Ha; Jung, Sunyo
2015-04-03
This study focuses on differential molecular mechanisms of antioxidant and detoxification systems in rice plants under two different types of photodynamic stress imposed by porphyrin deregulators, 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). The ALA-treated plants with white necrosis exhibited a greater decrease in photochemical quantum efficiency, Fv/Fm, as well as a greater increase in activity of superoxide dismutase, compared to the OF-treated plants. By contrast, the brown necrosis in OF-treated plants resulted in not only more widely dispersed H2O2 production and greater increases in H2O2-decomposing enzymes, catalase and peroxidase, but also lower ascorbate redox state. In addition, ALA- and OF-treated plants markedly up-regulated transcript levels of genes involved in detoxification processes including transport and movement, cellular homeostasis, and xenobiotic conjugation, with prominent up-regulation of serine/threonine kinase and chaperone only in ALA-treated plants. Our results demonstrate that different photodynamic stress imposed by ALA and OF developed differential actions of antioxidant enzymes and detoxification. Particularly, detoxification system may play potential roles in plant protection against photodynamic stress imposed by porphyrin deregulators, thereby contributing to alleviation of photodynamic damage. Copyright © 2015 Elsevier Inc. All rights reserved.
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Multiphoton manipulations of enzymatic photoactivity in aspartate aminotransferase.
Hill, Melissa P; Freer, Lucy H; Vang, Mai C; Carroll, Elizabeth C; Larsen, Delmar S
2011-04-21
The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal 5'-phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc.2010, 132 (47), 16953-16961]. This increases the net catalytic activity (k(cat)) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.
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Yu, Huanzhou; Shimakawa, Ann; Hines, Catherine D. G.; McKenzie, Charles A.; Hamilton, Gavin; Sirlin, Claude B.; Brittain, Jean H.; Reeder, Scott B.
2011-01-01
Multipoint water–fat separation techniques rely on different water–fat phase shifts generated at multiple echo times to decompose water and fat. Therefore, these methods require complex source images and allow unambiguous separation of water and fat signals. However, complex-based water–fat separation methods are sensitive to phase errors in the source images, which may lead to clinically important errors. An alternative approach to quantify fat is through “magnitude-based” methods that acquire multiecho magnitude images. Magnitude-based methods are insensitive to phase errors, but cannot estimate fat-fraction greater than 50%. In this work, we introduce a water–fat separation approach that combines the strengths of both complex and magnitude reconstruction algorithms. A magnitude-based reconstruction is applied after complex-based water–fat separation to removes the effect of phase errors. The results from the two reconstructions are then combined. We demonstrate that using this hybrid method, 0–100% fat-fraction can be estimated with improved accuracy at low fat-fractions. PMID:21695724
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NASA Astrophysics Data System (ADS)
Watson, Brett; Yeo, Leslie; Friend, James
2010-06-01
Making use of mechanical resonance has many benefits for the design of microscale devices. A key to successfully incorporating this phenomenon in the design of a device is to understand how the resonant frequencies of interest are affected by changes to the geometric parameters of the design. For simple geometric shapes, this is quite easy, but for complex nonlinear designs, it becomes significantly more complex. In this paper, two novel modeling techniques are demonstrated to extract the axial and torsional resonant frequencies of a complex nonlinear geometry. The first decomposes the complex geometry into easy to model components, while the second uses scaling techniques combined with the finite element method. Both models overcome problems associated with using current analytical methods as design tools, and enable a full investigation of how changes in the geometric parameters affect the resonant frequencies of interest. The benefit of such models is then demonstrated through their use in the design of a prototype piezoelectric ultrasonic resonant micromotor which has improved performance characteristics over previous prototypes.
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A-Kinase Anchoring Proteins: From protein complexes to physiology and disease
Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.
2009-01-01
Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965
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A-kinase anchoring proteins: from protein complexes to physiology and disease.
Carnegie, Graeme K; Means, Christopher K; Scott, John D
2009-04-01
Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review, we focus on recent advances in the elucidation of AKAP function.
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Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes
Smith, R.E.; Dolbeare, F.A.
1980-10-21
Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings
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Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes
Smith, Robert E. [557 Escondido Cir., Livermore, CA 94550; Dolbeare, Frank A. [5178 Diane La., Livermore, CA 94550
1980-10-21
Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.
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Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes
Smith, Robert E.; Dolbeare, Frank A.
1979-01-01
Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 5-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.
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NASA Astrophysics Data System (ADS)
Hall, S. J.; Silver, W. L.
2011-12-01
Anaerobic conditions have been proposed to impose a "latch" on soil organic matter decomposition by inhibiting the activity of extracellular enzymes that catalyze the transformation of organic polymers into monomers for microbial assimilation. Here, we tested the hypothesis that anaerobiosis inhibits soil hydrolytic enzyme activity in a humid tropical forest ecosystem in Puerto Rico. We sampled surface and sub-surface soil from each of 59 plots (n = 118) stratified across distinct topographical zones (ridges, slopes, and valleys) known to vary in soil oxygen (O2) concentrations, and measured the potential activity of five hydrolytic enzymes that decompose carbon (C), nitrogen (N), and phosphorus (P) substrates. We measured reduced iron (Fe (II)) concentrations in soil extractions to provide a spatially and temporally integrated index of anaerobic microbial activity, since iron oxides constitute the dominant anaerobic terminal electron acceptor in this ecosystem. Surprisingly, we observed positive relationships between Fe (II) concentrations and the activity of all enzymes that we assayed. Linear mixed effects models that included Fe (II) concentration, topographic position, and their interaction explained between 30 to 70 % of the variance of enzyme activity of β-1,4-glucosidase, β-cellobiohydrolase, β-xylosidase, N-acetylglucosaminidase, and acid phosphatase. Soils from ridges and slopes contained between 10 and 800 μg Fe (II) g-1 soil, and exhibited consistently positive relationships (p < 0.0001) between Fe (II) and enzyme activity. Valley soils did not display significant relationships between enzyme activity and Fe (II), although they displayed variation in soil Fe (II) concentrations similar to ridges and slopes. Overall, valleys exhibited lower enzyme activity and lower Fe (II) concentrations than ridges or slopes, possibly related to decreased root biomass and soil C. Our data provide no indication that anaerobiosis suppresses soil enzyme activity, but rather that high rates of decomposition induce a higher proportion of anaerobiosis soil microsites. The spatial patterns of Fe (II) concentrations that we observed also support this hypothesis. Soil Fe (II) concentrations were significantly greater in ridges than in slopes or valleys, in spite of the fact that slopes and valleys tend to experience higher soil moisture and lower bulk soil O2 concentrations. In our samples, Fe (II) concentrations correlated only weakly with ambient soil moisture, suggesting the importance of biological demand in controlling O2 availability as opposed to physical limitations on O2 diffusion imposed by soil moisture. In sum, our data suggest that anaerobic conditions do not necessarily constrain enzyme activity in humid tropical forest soils, and may not provide a proximate control on soil C storage in these ecosystems as has been recently proposed.
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North Carolina Biomolecular Engineering and Materials Applications Center (NC-BEMAC).
1987-12-29
enzyme has been replaced with cobalt(II). A further objective was to investigate Co2 activation by low molecular weight transition metal complexes as...Characterization of Low Molecular Weight Metal Complexes as Potential Models for IBio-Catalytic Processes. A number of transit ion met~~il oom~pi cxe; hive...binding, the enzyme suffered loss of activity during radiation polymerization. When covalent binding was u:sed it was necessary to introduce suitably
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Kela, U; Vijayvargiya, R
1981-03-01
Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), as determined by Subhrama Rao & Raghav Rao's [(1955) J. Sci. Chem. Ind. Res. 143, 278], method is found to be 6.74. It is suggested that the formation of a stable complex between 6-mercaptopurine molecules and Cu2+ may be an additional mechanism of action of 6-mercaptopurine, particularly with reference to its anti-inflammatory properties.
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Kela, U; Vijayvargiya, R
1981-01-01
Interaction between 6-mercaptopurine, Cu2+ and the enzyme xanthine oxidase (EC 1.2.3.2.) was examined. Whereas Cu2+ was found to inhibit the enzyme, 6-mercaptopurine could protect as well as reverse the enzyme inhibition produced by the metal ion. The formation of a complex between 6-mercaptopurine and Cu2+ seems to be responsible for the observed effect. Job's [(1928) Ann. Chem. 9, 113] method has shown the composition of the complex to be 1:1. The apparent stability constant (log K value), as determined by Subhrama Rao & Raghav Rao's [(1955) J. Sci. Chem. Ind. Res. 143, 278], method is found to be 6.74. It is suggested that the formation of a stable complex between 6-mercaptopurine molecules and Cu2+ may be an additional mechanism of action of 6-mercaptopurine, particularly with reference to its anti-inflammatory properties. PMID:6895465
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Elegheert, Jonathan; Brigé, Ann; Van Beeumen, Jozef; Savvides, Savvas N
2017-10-01
Shewanella oneidensis, a Gram-negative γ-proteobacterium with an extensive redox capacity, possesses four old yellow enzyme (OYE) homologs. Of these, Shewanella yellow enzyme 4 (SYE4) is implicated in resistance to oxidative stress. Here, we present a series of high-resolution crystal structures for SYE4 in the oxidized and reduced states, and in complex with phenolic ligands and the nitro-aromatic explosive picric acid. The structures unmask new features, including the identification of a binding platform for long-chain hydrophobic molecules. Furthermore, we present the first structural observation of a hydride-Meisenheimer complex of picric acid with a flavoenzyme. Overall, our study exposes the binding promiscuity of SYE4 toward a variety of electrophilic substrates and is consistent with a general detoxification function for SYE4. © 2017 Federation of European Biochemical Societies.
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Zhang, K. Y.; Cascio, D.; Eisenberg, D.
1994-01-01
The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6. PMID:8142899
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Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics
NASA Astrophysics Data System (ADS)
Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing
2014-03-01
Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
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Nonaka, Gen; Takumi, Kazuhiro
2017-12-01
Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.
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Characterization of Trapped Lignin-Degrading Microbes in Tropical Forest Soil
DeAngelis, Kristen M.; Allgaier, Martin; Chavarria, Yaucin; Fortney, Julian L.; Hugenholtz, Phillip; Simmons, Blake; Sublette, Kerry; Silver, Whendee L.; Hazen, Terry C.
2011-01-01
Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition. PMID:21559391
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Edwards, J C; Quinn, P J
1982-09-01
The unsaturated fatty acyl residues of egg yolk lecithin are selectively removed when bilayer dispersions of the lipid are exposed to decomposing peroxychromate at pH 7.6 or pH 9.0. Mannitol (50 mM or 100 mM)partially prevents the oxidation of the phospholipid due to decomposing peroxychromate at pH 7.6 and the amount of lipid lost is inversely proportional to the concentration of mannitol. N,N-Dimethyl-p-nitrosoaniline, mixed with the lipid in a molar ratio of 1.3:1, completely prevents the oxidation of lipid due to decomposing peroxychromate at pH 9.0, but some linoleic acid is lost if the incubation is done at pH 7.6. If the concentration of this quench reagent is reduced tenfold, oxidation of linoleic acid by decomposing peroxychromate at pH 9.0 is observed. Hydrogen peroxide is capable of oxidizing the unsaturated fatty acids of lecithin dispersions. Catalase or boiled catalase (2 mg/ml) protects the lipid from oxidation due to decomposing peroxychromate at pH 7.6 to approximately the same extent, but their protective effect is believed to be due to the non-specific removal of .OH. It is concluded that .OH is the species responsible for the lipid oxidation caused by decomposing peroxychromate. This is consistent with the observed bleaching of N,N-dimethyl-p-nitrosoanaline and the formation of a characteristic paramagnetic .OH adduct of the spin trap, 5,5-dimethylpyrroline-1-oxide.
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NASA Astrophysics Data System (ADS)
Liu, Yang; Du, Juanjuan; Yan, Ming; Lau, Mo Yin; Hu, Jay; Han, Hui; Yang, Otto O.; Liang, Sheng; Wei, Wei; Wang, Hui; Li, Jianmin; Zhu, Xinyuan; Shi, Linqi; Chen, Wei; Ji, Cheng; Lu, Yunfeng
2013-03-01
Organisms have sophisticated subcellular compartments containing enzymes that function in tandem. These confined compartments ensure effective chemical transformation and transport of molecules, and the elimination of toxic metabolic wastes. Creating functional enzyme complexes that are confined in a similar way remains challenging. Here we show that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomplexes. These nanocomplexes exhibit improved catalytic efficiency and enhanced stability when compared with free enzymes. Furthermore, the co-localized enzymes display complementary functions, whereby toxic intermediates generated by one enzyme can be promptly eliminated by another enzyme. We show that nanocomplexes containing alcohol oxidase and catalase could reduce blood alcohol levels in intoxicated mice, offering an alternative antidote and prophylactic for alcohol intoxication.
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Cross-linking proteins with bimetallic tetracarboxylate compounds of transition metals
Kostic, N.M.; Chen, J.
1991-03-05
Stable cross-linked complexes of transition-metal tetracarboxylates and proteins are formed. The preferred transition-metal is rhodium. The protein may be collagen or an enzyme such as a proteolytic enzyme. No Drawings
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NASA Astrophysics Data System (ADS)
Wlodarczyk, Jakub; Kierdaszuk, Borys
2005-08-01
Decays of tyrosine fluorescence in protein-ligand complexes are described by a model of continuous distribution of fluorescence lifetimes. Resulted analytical power-like decay function provides good fits to highly complex fluorescence kinetics. Moreover, this is a manifestation of so-called Tsallis q-exponential function, which is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence. The proposed decay functions were applied to analysis of fluorescence decays of tyrosine in a protein, i.e. the enzyme purine nucleoside phosphorylase from E. coli (the product of the deoD gene), free in aqueous solution and in a complex with formycin A (an inhibitor) and orthophosphate (a co-substrate). The power-like function provides new information about enzyme-ligand complex formation based on the physically justified heterogeneity parameter directly related to the lifetime distribution. A measure of the heterogeneity parameter in the enzyme systems is provided by a variance of fluorescence lifetime distribution. The possible number of deactivation channels and excited state mean lifetime can be easily derived without a priori knowledge of the complexity of studied system. Moreover, proposed model is simpler then traditional multi-exponential one, and better describes heterogeneous nature of studied systems.