Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

PubMed

Atack, John M; Srikhanta, Yogitha N; Djoko, Karrera Y; Welch, Jessica P; Hasri, Norain H M; Steichen, Christopher T; Vanden Hoven, Rachel N; Grimmond, Sean M; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

2013-06-01

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.

PubMed

Roberts, Richard J; Vincze, Tamas; Posfai, Janos; Macelis, Dana

2015-01-01

REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Biocatalysis engineering: the big picture.

PubMed

Sheldon, Roger A; Pereira, Pedro C

2017-05-22

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

Enzyme Amplified Detection of Microbial Cell Wall Components

NASA Technical Reports Server (NTRS)

Wainwright, Norman R.

2004-01-01

This proposal is MBL's portion of NASA's Johnson Space Center's Astrobiology Center led by Principal Investigator, Dr. David McKay, entitled: 'Institute for the Study of Biomarkers in Astromaterials.' Dr. Norman Wainwright is the principal investigator at MBL and is responsible for developing methods to detect trace quantities of microbial cell wall chemicals using the enzyme amplification system of Limulus polyphemus and other related methods.

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

PubMed

Amid, Mehrnoush; Manap, Mohd Yazid; Hussin, Muhaini; Mustafa, Shuhaimi

2015-06-17

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

Biochemical characterization and synergism of cellulolytic enzyme system from Chaetomium globosum on rice straw saccharification.

PubMed

Wanmolee, Wanwitoo; Sornlake, Warasirin; Rattanaphan, Nakul; Suwannarangsee, Surisa; Laosiripojana, Navadol; Champreda, Verawat

2016-11-21

Efficient hydrolysis of lignocellulosic materials to sugars for conversion to biofuels and chemicals is a key step in biorefinery. Designing an active saccharifying enzyme system with synergy among their components is considered a promising approach. In this study, a lignocellulose-degrading enzyme system of Chaetomium globosum BCC5776 (CG-Cel) was characterized for its activity and proteomic profiles, and synergism with accessory enzymes. The highest cellulase productivity of 0.40 FPU/mL was found for CG-Cel under the optimized submerged fermentation conditions on 1% (w/v) EPFB (empty palm fruit bunch), 2% microcrystalline cellulose (Avicel®) and 1% soybean meal (SBM) at 30 °C, pH 5.8 for 6 d. CG-Cel worked optimally at 50-60 °C in an acidic pH range. Proteomics analysis by LC/MS/MS revealed a complex enzyme system composed of core cellulases and accessory hydrolytic/non-hydrolytic enzymes attacking plant biopolymers. A synergistic enzyme system comprising the CG-Cel, a β-glucosidase (Novozyme® 188) and a hemicellulase Accellerase® XY was optimized on saccharification of alkaline-pretreated rice straw by a mixture design approach. Applying a full cubic model, the optimal ratio of ternary enzyme mixture containing CG-Cel: Novozyme® 188: Accellerase® XY of 44.4:20.6:35.0 showed synergistic enhancement on reducing sugar yield with a glucose releasing efficiency of 256.4 mg/FPU, equivalent to a 2.9 times compared with that from CG-Cel alone. The work showed an approach for developing an active synergistic enzyme system based on the newly characterized C. globosum for lignocellulose saccharification and modification in bio-industries.

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

PubMed

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

2016-01-01

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

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

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

Rajagopal, P.; Klevit, R.E.

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residuemore » at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.« less

[Study on relationship between effective components and soil enzyme activity in different growth patterns of Panax ginseng].

PubMed

Yang, Yan-Wen; Jiang, Yuan-Tong

2016-08-01

Study on 5 effective components and 6 soil enzyme activities of 2 different growth patterns, analyse the dates with the canonical correlation analysis, In order to reveal the relations between the effective components and soil enzyme activities. The result showed that they had a great relation between the effective components and soil enzyme activities, the activity of the same enzyme in humus soil was higher than that in farmland soil. Growth pattern of farmland soil, if the invertase and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside, water-miscible total proteins and total amino acid; Growth pattern of humus soil, if the invertase, urease and phosphatase activity were too high, which would inhibit the accumulation of total ginsenoside and the total essential oils. Integral soil enzyme activity can be used as a index of soil quality, which, together with other growth factors. The appropriate enzyme activity can accelerate the circulation and transformation of all kinds of material in the soil, improve effectively components accumulation. Copyright© by the Chinese Pharmaceutical Association.

[The digestive characteristics of fishes (the biocenotic aspects)].

PubMed

Kuz'mina, V V

1996-03-01

The cavity, membrane and intracellular digestion is added by two additional types of digestion in the fish: a symbiont digestion and an induced autolysis. As the intensity of processes maintained by the enzyme systems of the victim and a symbiont depends upon the structure and conditions of the biocenosis, the latter are regarded as an ecological component. The enzymes of microflora and objects of feeding contribute much to digestive processes in fish and take part in the adaptation of their digestive system to the character of the food and specifics of the biocenosis.

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

PubMed Central

van Heeswijk, Wally C.; Westerhoff, Hans V.

2013-01-01

SUMMARY We present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism in Escherichia coli. The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system. First, the structural and molecular knowledge on these proteins is reviewed. Thereafter, the activities of the components as they engage together in transport, metabolism, signal transduction, and transcription and their regulation are discussed. Next, old and new molecular data and physiological data are put into a common perspective on integral cellular functioning, especially with the aim of resolving counterintuitive or paradoxical processes featured in nitrogen assimilation. Finally, we articulate what still remains to be discovered and what general lessons can be learned from the vast amounts of data that are available now. PMID:24296575

Toxin detection using a tyrosinase-coupled oxygen electrode.

PubMed

Smit, M H; Rechnitz, G A

1993-02-15

An enzyme-based "electrochemical canary" is described for the detection of cyanide. The sensing system imitates cyanide's site of toxicity in the mitochondria. The terminal sequence of electron transfer in aerobic respiration is mimicked by mediator coupling of tyrosinase catalysis to an electro-chemical system. An enzyme-coupled oxygen electrode is created which is sensitive to selective poisoning. Biocatalytic reduction of oxygen is promoted by electrochemically supplying tyrosinase with electrons. Thus, ferrocyanide is generated at a cathode and mediates the enzymatic reduction of oxygen to water. An enzyme-dependent reductive current can be monitored which is inhibited by cyanide in a concentration-dependent manner. Oxygen depletion in the reaction layer can be minimized by addressing enzyme activity using a potential pulsing routine. Enzyme activity is electrochemically initiated and terminated and the sensor becomes capable of continuous monitoring. Cyanide poisoning of the biological component is reversible, and it can be reused after rinsing. The resulting sensor detects cyanide based on its biological activity rather than its physical or chemical properties.

Analysis of enzyme production by submerged culture of Aspergillus oryzae using whole barley.

PubMed

Masuda, Susumu; Kikuchi, Kaori; Matsumoto, Yuko; Sugimoto, Toshikazu; Shoji, Hiroshi; Tanabe, Masayuki

2009-10-01

We have reported on high enzyme production by submerged culture of Aspergillus kawachii using barley with the husk (whole barley). To elucidate the mechanism underlying this high enzyme production, we performed a detailed analysis. Aspergillus oryzae RIB40 was submerged-cultured using whole barley and milled whole barley. Enzyme production was analyzed in terms of changes in medium components and gene expression levels. When whole barley was used, high production of glucoamylase and alpha-amylase and high gene expression levels of these enzymes were observed. Low ammonium concentrations were maintained with nitrate ion uptake continuing into the late stage using whole barley. These findings suggest that the sustainability of nitrogen metabolism is related to high enzyme production, and that a mechanism other than that associated with the conventional amylase expression system is involved in this relationship.

INTERRELATION BETWEEN ACTIVATION AND POLYMERIZATION IN GRAMICIDIN S BIOSYNTHESIS*

PubMed Central

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

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Electrochemical biosensor based on immobilized enzymes and redox polymers

DOEpatents

Skotheim, Terje A.; Okamoto, Yoshiyuki; Hale, Paul D.

1992-01-01

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

Impact on enzyme activity as a new quality index of wastewater.

PubMed

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

2013-03-15

The aim of this study was to define a new indicator for the quality of wastewaters that are released into the environment. A quality index is proposed for wastewater samples in terms of the inertness of wastewater samples toward enzyme activity. This involves taking advantage of the sensitivity of enzymes to pollutants that may be present in the waste samples. The effect of wastewater samples on the rate of a number of different enzyme-catalyzed reactions was measured, and the results for all the selected enzymes were analyzed in an integrated fashion (multi-enzymatic sensor). This approach enabled us to define an overall quality index, the "Impact on Enzyme Function" (IEF-index), which is composed of three indicators: i) the Synoptic parameter, related to the average effect of the waste sample on each component of the enzymatic sensor; ii) the Peak parameter, related to the maximum effect observed among all the effects exerted by the sample on the sensor components; and, iii) the Interference parameter, related to the number of sensor components that are affected less than a fixed threshold value. A number of water based samples including public potable tap water, fluids from urban sewage systems, wastewater disposal from leather, paper and dye industries were analyzed and the IEF-index was then determined. Although the IEF-index cannot discriminate between different types of wastewater samples, it could be a useful parameter in monitoring the improvement of the quality of a specific sample. However, by analyzing an adequate number of waste samples of the same type, even from different local contexts, the profile of the impact of each component of the multi-enzymatic sensor could be typical for specific types of waste. The IEF-index is proposed as a supplementary qualification score for wastewaters, in addition to the certification of the waste's conformity to legal requirements. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro interactions of thallium with components of the glutathione-dependent antioxidant defence system.

PubMed

Villaverde, Marcela S; Hanzel, Cecilia E; Verstraeten, Sandra V

2004-09-01

We investigated the hypothesis that thallium (Tl) interactions with the glutathione-dependent antioxidant defence system could contribute to the oxidative stress associated with Tl toxicity. Working in vitro with reduced glutathione (GSH), glutathione reductase (GR) or glutathione peroxidase (GPx) in solution, we studied the effects of Tl+ and Tl3+ (1-25 microM) on: (a) the amount of free GSH, investigating whether the metal binds to GSH and/or oxidizes it; (b) the activity of the enzyme GR, that catalyzes GSH regeneration; and (c) the enzyme GPx, that reduces hydroperoxide at expense of GSH oxidation. We found that, while Tl+ had no effect on GSH concentration, Tl3+ oxidized it. Both cations inhibited the reduction of GSSG by GR and the diaphorase activity of this enzyme. In addition, Tl3+ per se oxidized NADPH, the cofactor of GR. The effects of Tl on GPx activity depended on the metal charge: Tl+ inhibited GPx when cumene hydroperoxide (CuOOH) was the substrate, while Tl(3+)-mediated GPx inhibition occurred with both substrates. The present results show that Tl interacts with all the components of GSH/GSSG antioxidant defence system. Alterations of this protective pathway could be partially responsible for the oxidative stress associated with Tl toxicity.

A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

PubMed

Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

PubMed Central

Irie, S; Doi, S; Yorifuji, T; Takagi, M; Yano, K

1987-01-01

The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed. Images PMID:3667527

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

PubMed Central

2010-01-01

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

Function of Coenzyme F420 in Aerobic Catabolism of 2,4,6-Trinitrophenol and 2,4-Dinitrophenol by Nocardioides simplex FJ2-1A

PubMed Central

Ebert, Sybille; Rieger, Paul-Gerhard; Knackmuss, Hans-Joachim

1999-01-01

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these π-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme. PMID:10217752

Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases. Formation of a hybrid-type heptaprenyl diphosphate synthase.

PubMed

Koike-Takeshita, A; Koyama, T; Ogura, K

1998-10-01

Among prenyltransferases that catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components. These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself. In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26. As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity. The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B. subtilis and B. stearothermophilus. There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate synthases.

Expression profiles of genes for mitochondrial respiratory energy-dissipating systems and antioxidant enzymes in wheat leaves during de-etiolation.

PubMed

Garmash, Elena V; Velegzhaninov, Ilya O; Grabelnych, Olga I; Borovik, Olga A; Silina, Ekaterina V; Voinikov, Victor K; Golovko, Tamara K

2017-08-01

Mitochondrial respiratory components participate in the maintenance of chloroplast functional activity. This study investigates the effects 48h de-etiolation of spring wheat seedlings (Triticum aestivum L., var. Irgina) on the expression of genes that encode energy-dissipating respiratory components and antioxidant enzymes under continuous light conditions. The expression of AOX1a following the prolonged darkness exhibited a pattern indicating a prominent dependence on light. The expression of other respiratory genes, including NDA2, NDB2, and UCP1b, increased during de-etiolation and dark-to-light transition; however, changes in the expression of these genes occurred later than those in AOX1a expression. A high expression of NDA1 was detected after 12h of de-etiolation. The suppression of AOX1a, NDA2, NDB2, and UCP1b was observed 24h after de-etiolation when the photosynthetic apparatus and its defence systems against excess light were completely developed. The expression patterns of the respiratory genes and several genes encoding antioxidant enzymes (MnSOD, Cu-ZnSOD, t-APX, GR, and GRX) were quite similar. Our data indicate that the induction of nuclear genes encoding respiratory and antioxidant enzymes allow the plants to control reactive oxygen species (ROS) production and avoid oxidative stress during de-etiolation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Enzymatic Removal of Diacetyl from Beer 1

PubMed Central

Thompson, Janet W.; Shovers, J.; Sandine, W. E.; Elliker, P. R.

1970-01-01

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at — 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors. PMID:4318450

Surface Plasmon Resonance Imaging of the Enzymatic Degradation of Cellulose Microfibrils

NASA Astrophysics Data System (ADS)

Reiter, Kyle; Raegen, Adam; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2012-02-01

As the largest component of biomass on Earth, cellulose represents a significant potential energy reservoir. Enzymatic hydrolysis of cellulose into fermentable sugars, an integral step in the production of biofuel, is a challenging problem on an industrial scale. More efficient conversion processes may be developed by an increased understanding of the action of the cellulolytic enzymes involved in cellulose degradation. We have used our recently developed quantitative, angle-scanning surface plasmon resonance imaging (SPRi) device to study the degradation of cellulose microfibrils upon exposure to cellulosic enzymes. In particular, we have studied the action of individual enzymes, and combinations of enzymes, from the Hypocrea Jecorina cellulase system on heterogeneous, industrially-relevant cellulose substrates. This has allowed us to define a characteristic time of action for the enzymes for different degrees of surface coverage of the cellulose microfibrils.

In search of mitochondrial mechanisms: interfield excursions between cell biology and biochemistry.

PubMed

Bechtel, William; Abrahamsen, Adele

2007-01-01

Developing models of biological mechanisms, such as those involved in respiration in cells, often requires collaborative effort drawing upon techniques developed and information generated in different disciplines. Biochemists in the early decades of the 20th century uncovered all but the most elusive chemical operations involved in cellular respiration, but were unable to align the reaction pathways with particular structures in the cell. During the period 1940-1965 cell biology was emerging as a new discipline and made distinctive contributions to understanding the role of the mitochondrion and its component parts in cellular respiration. In particular, by developing techniques for localizing enzymes or enzyme systems in specific cellular components, cell biologists provided crucial information about the organized structures in which the biochemical reactions occurred. Although the idea that biochemical operations are intimately related to and depend on cell structures was at odds with the then-dominant emphasis on systems of soluble enzymes in biochemistry, a reconceptualization of energetic processes in the 1960s and 1970s made it clear why cell structure was critical to the biochemical account. This paper examines how numerous excursions between biochemistry and cell biology contributed a new understanding of the mechanism of cellular respiration.

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

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

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

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

Mathematical modeling of physiological systems: an essential tool for discovery.

PubMed

Glynn, Patric; Unudurthi, Sathya D; Hund, Thomas J

2014-08-28

Mathematical models are invaluable tools for understanding the relationships between components of a complex system. In the biological context, mathematical models help us understand the complex web of interrelations between various components (DNA, proteins, enzymes, signaling molecules etc.) in a biological system, gain better understanding of the system as a whole, and in turn predict its behavior in an altered state (e.g. disease). Mathematical modeling has enhanced our understanding of multiple complex biological processes like enzyme kinetics, metabolic networks, signal transduction pathways, gene regulatory networks, and electrophysiology. With recent advances in high throughput data generation methods, computational techniques and mathematical modeling have become even more central to the study of biological systems. In this review, we provide a brief history and highlight some of the important applications of modeling in biological systems with an emphasis on the study of excitable cells. We conclude with a discussion about opportunities and challenges for mathematical modeling going forward. In a larger sense, the review is designed to help answer a simple but important question that theoreticians frequently face from interested but skeptical colleagues on the experimental side: "What is the value of a model?" Copyright © 2014 Elsevier Inc. All rights reserved.

Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

PubMed

Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

2016-10-01

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Production of cellulase from Pestalotiopsis versicolor

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

Rao, M.N.A.; Mithal, B.M.; Thakur, R.N.

1983-01-01

Production of cellulase from Pestalotiopsis versicolor was studied in a shake flask culture. The cellulase system was found to be rich in beta-glucosidase. Kinetic parameters such as pH and temperature have been optimized for the various enzyme components. 9 references.

Angiotensin-converting enzyme in epithelial and neuroepithelial cells.

PubMed

Defendini, R; Zimmerman, E A; Weare, J A; Alhenc-Gelas, F; Erdös, E G

1983-07-01

Angiotensin-converting enzyme (CE) occurs in three types of cell: endothelial, epithelial, and neuroepithelial. In all three, it appears to be bound to plasma membrane. With antisera to the human enzyme, CE is demonstrated in paraffin sections on the apical surface of epithelial cells in the proximal tubule of the kidney, the mucosa of the small intestine, the syncytial trophoblast of the placenta, and the choroid plexus. Epithelial CE is characteristically found on microvillous surfaces in contact with an effluent, well placed to act on substrate in flux. In the brain, CE occurs in nerve fibers and terminals, mainly mesiobasally and in basal ganglia. Mesiobasal CE coincides with other components of the renin-angiotensin system (RAS) in the choroid/ventricular fluid, the subfornical organ, and the magnocellular neurosecretory system of the hypothalamus. Extrapyramidal CE, however, may not be related to the RAS. In the substantia nigra and the globus pallidus, the enzyme has the same cellular distribution as two putative neuromodulators, substance P and enkephalin, the latter a known substrate of CE.

Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs)

PubMed Central

Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; Padilla, María N.; Corpas, Francisco J.; Barroso, Juan B.

2016-01-01

Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance. PMID:26909095

Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

DOEpatents

Collart, Frank R.; Huberman, Eliezer

2000-11-28

This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

RCAD/BiP pathway is necessary for the proper synthesis of digestive enzymes and secretory function of the exocrine pancreas.

PubMed

Miller, Camille; Cai, Yafei; Patton, Tadd; Graves, Sarai Hacker; Li, Honglin; Sabbatini, Maria Eugenia

2017-03-01

Alcoholism causes an imbalance of endoplasmic reticulum (ER) homeostasis in pancreatic acini. In those cells, the ER is involved in the synthesis and folding of pancreatic enzymes. Ubiquitin-fold modifier 1 (Ufm1) is part of a novel ubiquitin-like modification system involved in maintaining ER homeostasis. Among the components of the Ufm1 system, Regulator of C53 and DDRGK1 (RCAD) has recently been identified as a Ufm1-specific E3 ligase that promotes ufmylation of DDRGK1, an RCAD-interacting protein. We determined the importance of RCAD in the proper synthesis and secretion of pancreatic enzymes using mice with genetically deleted RCAD. The pancreas of RCAD-deficient mice was of normal size and histology. Using quantitative PCR and Western blotting, we found that amylase was upregulated in pancreas organs from RCAD-knockout (KO) mice. Constitutive amylase secretion was much higher in isolated pancreatic acini from RCAD KO mice, whereas CCK-stimulated amylase secretion was disturbed. RCAD deficiency caused a downregulation in expression of ER chaperone BiP, which affected ER homeostasis and activated both apoptosis and trypsin. We also found that both RCAD and DDRGK1 transcript levels were upregulated in pancreatic acini from alcohol-preferring rats. Elevated expression of RCAD and DDRGK1 was associated with increased ER stress and UPR activation. Because of the lack of BiP expression, caspase 3 and trypsin activation we enhanced in RCAD-deficient pancreatic acini upon treatment with ethanol and CCK. In conclusion, the RCAD/BiP pathway is required for proper synthesis and secretion of pancreatic enzymes. In alcoholism, increased levels of components of the Ufm1 system could prevent the deleterious effects of alcohol in the pancreas by regulating BiP levels. NEW & NOTEWORTHY RCAD/BiP pathway is required for the proper synthesis and secretion of amylase from pancreatic acini, as well as for the maintenance of the ER homeostasis. In alcoholism, the exocrine pancreas could increase the levels of components of the Ufm1 system to protect itself from alcohol's deleterious effects by regulating the expression of ER chaperone BiP. Copyright © 2017 the American Physiological Society.

Quantitative evolutionary design

PubMed Central

Diamond, Jared

2002-01-01

The field of quantitative evolutionary design uses evolutionary reasoning (in terms of natural selection and ultimate causation) to understand the magnitudes of biological reserve capacities, i.e. excesses of capacities over natural loads. Ratios of capacities to loads, defined as safety factors, fall in the range 1.2-10 for most engineered and biological components, even though engineered safety factors are specified intentionally by humans while biological safety factors arise through natural selection. Familiar examples of engineered safety factors include those of buildings, bridges and elevators (lifts), while biological examples include factors of bones and other structural elements, of enzymes and transporters, and of organ metabolic performances. Safety factors serve to minimize the overlap zone (resulting in performance failure) between the low tail of capacity distributions and the high tail of load distributions. Safety factors increase with coefficients of variation of load and capacity, with capacity deterioration with time, and with cost of failure, and decrease with costs of initial construction, maintenance, operation, and opportunity. Adaptive regulation of many biological systems involves capacity increases with increasing load; several quantitative examples suggest sublinear increases, such that safety factors decrease towards 1.0. Unsolved questions include safety factors of series systems, parallel or branched pathways, elements with multiple functions, enzyme reaction chains, and equilibrium enzymes. The modest sizes of safety factors imply the existence of costs that penalize excess capacities. Those costs are likely to involve wasted energy or space for large or expensive components, but opportunity costs of wasted space at the molecular level for minor components. PMID:12122135

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

PubMed

Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E

2010-03-01

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

A Novel Aqueous Micellar Two-Phase System Composed of Surfactant and Sorbitol for Purification of Pectinase Enzyme from Psidium guajava and Recycling Phase Components

PubMed Central

Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

2015-01-01

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme. PMID:25756051

The protective arm of the renin-angiotensin system may counteract the intense inflammatory process in fetuses with posterior urethral valves.

PubMed

Rocha, Natalia P; Bastos, Fernando M; Vieira, Érica L M; Prestes, Thiago R R; Silveira, Katia D da; Teixeira, Mauro M; Simões E Silva, Ana Cristina

2018-03-11

Posterior urethral valve is the most common lower urinary tract obstruction in male children. A high percentage of patients with posterior urethral valve evolve to end-stage renal disease. Previous studies showed that cytokines, chemokines, and components of the renin-angiotensin system contribute to the renal damage in obstructive uropathies. The authors recently found that urine samples from fetuses with posterior urethral valve have increased levels of inflammatory molecules. The aim of this study was to measure renin-angiotensin system molecules and to investigate their correlation with previously detected inflammatory markers in the same urine samples of fetuses with posterior urethral valve. Urine samples from 24 fetuses with posterior urethral valve were collected and compared to those from 22 healthy male newborns at the same gestational age (controls). Renin-angiotensin system components levels were measured by enzyme-linked immunosorbent assay. Fetuses with posterior urethral valve presented increased urinary levels of angiotensin (Ang) I, Ang-(1-7) and angiotensin-converting enzyme 2 in comparison with controls. ACE levels were significantly reduced and Ang II levels were similar in fetuses with posterior urethral valve in comparison with controls. Increased urinary levels of angiotensin-converting enzyme 2 and of Ang-(1-7) in fetuses with posterior urethral valve could represent a regulatory response to the intense inflammatory process triggered by posterior urethral valve. Copyright © 2018 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Novel biocatalytic systems for maintaining the nucleotide balance based on adenylate kinase immobilized on carbon nanostructures.

PubMed

Hetmann, Anna; Wujak, Magdalena; Bolibok, Paulina; Zięba, Wojciech; Wiśniewski, Marek; Roszek, Katarzyna

2018-07-01

In this study graphene oxide (GO), carbon quantum dots (CQD) and carbon nanoonions (CNO) have been characterized and applied for the first time as a matrix for recombinant adenylate kinase (AK, EC 2.7.4.3) immobilization. AK is an enzyme fulfilling a key role in metabolic processes. This phosphotransferase catalyzes the interconversion of adenine nucleotides (ATP, ADP and AMP) and thereby participates in nucleotide homeostasis, monitors a cellular energy charge as well as acts as a component of purinergic signaling system. The AK activity in all obtained biocatalytic systems was higher as compared to the free enzyme. We have found that the immobilization on carbon nanostructures increased both activity and stability of AK. Moreover, the biocatalytic systems consisting of AK immobilized on carbon nanostructures can be easily and efficiently lyophilized without risk of desorption or decrease in the catalytic activity of the investigated enzyme. The positive action of AK-GO biocatalytic system in maintaining the nucleotide balance in in vitro cell culture was proved. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.

PubMed

Dongowski, G; Sembries, S

2001-09-01

The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.

The Use of Enzyme Hydrolysis to Assess the Seasonal Mobility and Bioavailability of Organic Phosphorus in Lake Sediments

NASA Astrophysics Data System (ADS)

Giles, C. D.; Lee, L. G.; Cade-Menun, B. J.; Rutila, B. C.; Schroth, A. W.; Xu, Y.; Hill, J. E.; Druschel, G.

2013-12-01

Lake sediments represent a significant internal source of phosphorus (P) in eutrophic freshwater systems during periods of high biological activity and oxygen depletion in sediments. Enzyme-labile and redox-sensitive P fractions may be a major component of the mobile sediment P pool which contributes to the development of harmful algal blooms. We present a high-through-put enzyme-based method for assessing potentially bioavailable (enzyme-labile) P in lake sediments and describe the relationship between enzyme-labile P, ascorbate-extractable (reactive) P and metals (Fe, Mn, Al, Ca), and P species identified using solution 31-P NMR spectroscopy. Sediment cores (0-10 cm) were collected from Lake Champlain over multiple years (Missisquoi Bay, VT, USA; 2007-2013). A principal components analysis of sediment properties suggests that enzyme-labile and reactive P, Mn, and Fe concentrations were more effective than the 31-P NMR methodology alone for differentiating algal bloom stage associated with periods of sediment anoxia. Bloom onset (July 2008) and peak bloom (August 2008, 2012) periods corresponded to the highest enzyme-labile P and lowest reactive P and metals proportions, despite 31-P NMR profiles which did not change significantly with respect to time and depth. High levels of reduced Fe and Mn ions were also detected in pore-water during this period, confirming previous reports that organic P bioavailability is linked to the redox status of sediments. High through-put analysis of enzyme-labile P fractions will provide spatially and temporally resolved information on bioavailable P pools at lower cost than traditional methods (i.e., 31-P NMR), and provide much-needed detail on aquatic P cycles during discrete stages of algal bloom development and sediment anoxia.

Hydrolytic enzymes expressivity in different parts of the Rapana digestive system.

PubMed

Toptikov, V A; Totsky, V N; Alieksieieva, T G; Kovtun, O A

2016-01-01

The relevance of comprehensive studies of the Rapana vital functions is determined by its considerab­le negative impact on the ecosystem of the Black Sea. The aim of the work was to find out the polymorphism and activity of the main hydrolases in the different parts of the digestive system of Rapana. Hydrolases (proteases, amylases, esterases, lipases and phosphatases) in glandular structures of the Rapana digestive system were studied by electrophoresis. It was found that different sets of hydrolytic enzymes are functioning in certain parts of the Rapana digestive tract. The gland of Leiblein and hepatopancreas played the most important role in the digestion of food components. The salivary glands had the significant influence on proteolysis.

Influence of the incubation temperature and the batch components on the sensitivity of an enzyme-linked immunosorbent assay to detect Aujeszky's disease virus glycoprotein E (gE).

PubMed

Cay, A B; Van der Stede, Y

2010-12-01

Although licensed batches of an enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease virus (ADV) were used, and the assays were performed within an ISO/IEC 17025 accredited quality control system, certain routine runs of the ADV ELISA were not validated using the quality system criteria, even when all technical parameters were controlled. Incubation at different temperatures and batch composition were identified as parameters that could result in non-validated assays/runs. Therefore, the effect of incubation temperature and batch composition on the analytical sensitivity of the ELISA was investigated. The World Organisation for Animal Health (OIE) standard reference serum ADV1 was diluted 1:8 and tested in 94 different glycoprotein E ELISA runs performed with different batches and different incubation temperatures. The incubation temperature and batch components had a significant influence on the qualitative result for the OIE standard reference serum. An incubation temperature of at least 22 degrees C was recommended, based on the results of this analysis. Which of the batch components caused these differences in sensitivity was not investigated further.

Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

PubMed

de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

2017-11-01

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

Turning tryptophanase into odor-generating biosensors.

PubMed

Xu, Yaqin; Zhang, Zhuyuan; Ali, M Monsur; Sauder, Joanna; Deng, Xudong; Giang, Karen; Aguirre, Sergio D; Pelton, Robert; Li, Yingfu; Filipe, Carlos D M

2014-03-03

An odor-based sensor system that exploits the metabolic enzyme tryptophanase (TPase) as the key component is reported. This enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. To make a biosensor, TPase was biotinylated so that it could be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method was used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low μM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrical contacting of an assembly of pseudoazurin and nitrite reductase using DNA-directed immobilization.

PubMed

Tepper, Armand W J W

2010-05-12

A method for the electrical contacting of redox enzymes that obtain oxidizing or reducing equivalents from small electron-transfer proteins is demonstrated. The electrochemical contacting of redox enzymes through their immobilization onto electrode supports offers great potential for technological applications and for fundamental studies, but finding appropriate methods to immobilize the enzymes in an orientation allowing rapid electron transfer with the electrode has proven difficult. The copper enzyme nitrite reductase (NiR) and its natural electron-exchange partner pseudoazurin (Paz) are conjugated to a specific DNA tag and immobilized to a gold electrode into a stoichiometrically defined assembly. The DNA tethered to the electrode surface acts as flexible place-holder for the protein components, allowing both proteins to move within the construct. It is shown that Paz efficiently shuttles electrons between the electrode and the NiR enzyme, allowing the electrochemically driven NiR catalysis to be monitored. The activity of the NiR enzyme remains unperturbed by the immobilization. The rate-limiting step of the system is tentatively ascribed to the dissociation of the Paz/NiR complex. The electrochemical response of the system reports not only on the NiR catalysis and on interfacial electron transfer but also on the interaction between NiR and Paz.

An Oxidase-Based Electrochemical Fluidic Sensor with High-Sensitivity and Low-Interference by On-Chip Oxygen Manipulation

PubMed Central

Radhakrishnan, Nitin; Park, Jongwon; Kim, Chang-Soo

2012-01-01

Utilizing a simple fluidic structure, we demonstrate the improved performance of oxidase-based enzymatic biosensors. Electrolysis of water is utilized to generate bubbles to manipulate the oxygen microenvironment close to the biosensor in a fluidic channel. For the proper enzyme reactions to occur, a simple mechanical procedure of manipulating bubbles was developed to maximize the oxygen level while minimizing the pH change after electrolysis. The sensors show improved sensitivities based on the oxygen dependency of enzyme reaction. In addition, this oxygen-rich operation minimizes the ratio of electrochemical interference signal by ascorbic acid during sensor operation (i.e., amperometric detection of hydrogen peroxide). Although creatinine sensors have been used as the model system in this study, this method is applicable to many other biosensors that can use oxidase enzymes (e.g., glucose, alcohol, phenol, etc.) to implement a viable component for in-line fluidic sensor systems. PMID:23012527

Integrated microdroplet-based system for enzyme synthesis and sampling

NASA Astrophysics Data System (ADS)

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

2013-12-01

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

In-depth investigation of enzymatic hydrolysis of biomass wastes based on three major components: Cellulose, hemicellulose and lignin.

PubMed

Lin, Lili; Yan, Rong; Liu, Yongqiang; Jiang, Wenju

2010-11-01

The artificial biomass based on three biomass components (cellulose, hemicellulose and lignin) were developed on the basis of a simplex-lattice approach. Together with a natural biomass sample, they were employed in enzymatic hydrolysis researches. Different enzyme combines of two commercial enzymes (ACCELLERASE 1500 and OPTIMASH BG) showed a potential to hydrolyze hemicellulose completely. Negligible interactions among the three components were observed, and the used enzyme ACCELLERASE 1500 was proven to be weak lignin-binding. On this basis, a multiple linear-regression equation was established for predicting the reducing sugar yield based on the component proportions in a biomass. The hemicellulose and cellulose in a biomass sample were found to have different contributions in staged hydrolysis at different time periods. Furthermore, the hydrolysis of rice straw was conducted to validate the computation approach through considerations of alkaline solution pretreatment and combined enzymes function, so as to understand better the nature of biomass hydrolysis, from the aspect of three biomass components.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

PubMed

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L; Sopory, Sudhir K

2017-03-30

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni 2+ - and Zn 2+ -dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

PubMed Central

Bourand, A.; Yebra, M. J.; Boël, G.; Mazé, A.

2013-01-01

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD+ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation. PMID:23564164

Characterization of the Enzymatic Component of the ADP-Ribosyltransferase Toxin CDTa from Clostridium difficile

PubMed Central

Gülke, Irene; Pfeifer, Gunther; Liese, Jan; Fritz, Michaela; Hofmann, Fred; Aktories, Klaus; Barth, Holger

2001-01-01

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1–240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244–263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208–413 but loss of activity of several N-terminally deleted C2I proteins including C2I103–431, C2I190–431, and C2I30–431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity. PMID:11553537

Development of portable health monitoring system for automatic self-blood glucose measurement

NASA Astrophysics Data System (ADS)

Kim, Huijun; Mizuno, Yoshihumi; Nakamachi, Eiji; Morita, Yusuke

2010-02-01

In this study, a new HMS (Health Monitoring System) device is developed for diabetic patient. This device mainly consists of I) 3D blood vessel searching unit and II) automatic blood glucose measurement (ABGM) unit. This device has features such as 1)3D blood vessel location search 2) laptop type, 3) puncturing a blood vessel by using a minimally invasive micro-needle, 4) very little blood sampling (10μl), and 5) automatic blood extraction and blood glucose measurement. In this study, ABGM unit is described in detail. It employs a syringe type's blood extraction mechanism because of its high accuracy. And it consists of the syringe component and the driving component. The syringe component consists of a syringe itself, a piston, a magnet, a ratchet and a micro-needle whose inner diameter is about 80μm. And the syringe component is disposable. The driving component consists of body parts, a linear stepping motor, a glucose enzyme sensor and a slider for accurate positioning control. The driving component has the all-in-one mechanism with a glucose enzyme sensor for compact size and stable blood transfer. On designing, required thrust force to drive the slider is designed to be greater than the value of the blood extraction force. Further, only one linear stepping motor is employed for blood extraction and transportation processes. The experimental result showed more than 80% of volume ratio under the piston speed 2.4mm/s. Further, the blood glucose was measured successfully by using the prototype unit. Finally, the availability of our ABGM unit was confirmed.

EFICAz2: enzyme function inference by a combined approach enhanced by machine learning.

PubMed

Arakaki, Adrian K; Huang, Ying; Skolnick, Jeffrey

2009-04-13

We previously developed EFICAz, an enzyme function inference approach that combines predictions from non-completely overlapping component methods. Two of the four components in the original EFICAz are based on the detection of functionally discriminating residues (FDRs). FDRs distinguish between member of an enzyme family that are homofunctional (classified under the EC number of interest) or heterofunctional (annotated with another EC number or lacking enzymatic activity). Each of the two FDR-based components is associated to one of two specific kinds of enzyme families. EFICAz exhibits high precision performance, except when the maximal test to training sequence identity (MTTSI) is lower than 30%. To improve EFICAz's performance in this regime, we: i) increased the number of predictive components and ii) took advantage of consensual information from the different components to make the final EC number assignment. We have developed two new EFICAz components, analogs to the two FDR-based components, where the discrimination between homo and heterofunctional members is based on the evaluation, via Support Vector Machine models, of all the aligned positions between the query sequence and the multiple sequence alignments associated to the enzyme families. Benchmark results indicate that: i) the new SVM-based components outperform their FDR-based counterparts, and ii) both SVM-based and FDR-based components generate unique predictions. We developed classification tree models to optimally combine the results from the six EFICAz components into a final EC number prediction. The new implementation of our approach, EFICAz2, exhibits a highly improved prediction precision at MTTSI < 30% compared to the original EFICAz, with only a slight decrease in prediction recall. A comparative analysis of enzyme function annotation of the human proteome by EFICAz2 and KEGG shows that: i) when both sources make EC number assignments for the same protein sequence, the assignments tend to be consistent and ii) EFICAz2 generates considerably more unique assignments than KEGG. Performance benchmarks and the comparison with KEGG demonstrate that EFICAz2 is a powerful and precise tool for enzyme function annotation, with multiple applications in genome analysis and metabolic pathway reconstruction. The EFICAz2 web service is available at: http://cssb.biology.gatech.edu/skolnick/webservice/EFICAz2/index.html.

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

PubMed

Barter, P J; Hopkins, G J; Gorjatschko, L

1984-01-17

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Further Studies on the Lyo and Desmo Components of Several Hydrolytic Enzymes and Their Histochemical Significance

PubMed Central

Hannibal, Mark J.; Nachlas, Marvin M.

1959-01-01

This report describes additional studies of the lyo and desmo components of esterase, alkaline phosphatase, acid phosphatase, leucine aminopeptidase, and β-glucuronidase. The techniques used have already been reported (7). Enzyme diffusion occurs to different degrees in different fixatives, and varies somewhat with each enzyme. Loss of enzymatic activity during fixation occurs as a result of both inactivation due to the chemical reaction of the fixative with the enzymic protein, and diffusion of the lyo component into the fixative. The amount of diffusion into formalin can be reduced by the addition of salts, sucrose, or methocel. The pH of the aqueous medium significantly influences the removal of the lyo fraction from the tissue section. A striking similarity can be noted in the proportions of each fraction of enzyme present in the kidney of the rat, dog, and man. The procedure of fixation and paraffin embedding of tissue blocks does not wholly prevent the diffusion of the lyo component from the tissue sections when they are subsequently immersed in the aqueous incubation medium. PMID:13654449

Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases.

PubMed

Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

2014-08-01

The only known function of the Nox family of NADPH oxidases is the production of reactive oxygen species (ROS). Some Nox enzymes show high tissue-specific expression and the ROS locally produced are required for synthesis of hormones or tissue components. In the cardiovascular system, Nox enzymes are low abundant and function as redox-modulators. By reacting with thiols, nitric oxide (NO) or trace metals, Nox-derived ROS elicit a plethora of cellular responses required for physiological growth factor signaling and the induction and adaptation to pathological processes. The interactions of Nox-derived ROS with signaling elements in the cardiovascular system are highly diverse and will be detailed in this article, which is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System". Copyright © 2014 Elsevier Ltd. All rights reserved.

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

PubMed Central

Walton, Jonathan; Banerjee, Goutami; Car, Suzana

2011-01-01

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations. GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com). Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid. All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase). PMID:22042431

GENPLAT: an automated platform for biomass enzyme discovery and cocktail optimization.

PubMed

Walton, Jonathan; Banerjee, Goutami; Car, Suzana

2011-10-24

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations. GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com). Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid. All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).

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

PubMed

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

2014-02-01

In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease

PubMed Central

Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug

2012-01-01

Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD. PMID:22577256

Effects of different components of Mao Dongqing's total flavonoids and total saponins on transient ischemic attack (TIA) model of rats.

PubMed

Miao, Ming-San; Peng, Meng-Fan; Ma, Rui-Juan; Bai, Ming; Liu, Bao-Song

2018-03-01

Objective: To study the effects of the different components of the total flavonoids and total saponins from Mao Dongqing's active site on the rats of TIA model, determine the optimal reactive components ratio of Mao Dongqing on the rats of TIA. Methods: TIA rat model was induced by tail vein injection of tert butyl alcohol, the blank group was injected with the same amount of physiological saline, then behavioral score wasevaluated. Determination the level of glutamic acid in serum, the activity of Na+-K+-ATP enzyme, CA ++ -ATP enzyme and Mg ++ -ATP enzyme in Brain tissue, observe the changes of hippocampus in brain tissue, the comprehensive weight method was used to evaluate the efficacy of each component finally. Results: The contents of total flavonoids and total saponins in the active part of Mao Dongqing can significantly improve the pathological changes of brain tissue in rats, improve the activity of Na + -K + -ATP enzyme, Ca ++ -ATP enzyme and Mg ++ -ATP enzyme in the brain of rats, and reduce the level of glutamic acid in serum. The most significant of the contents was the ratio of 10:6. The different proportions of total flavonoids and total saponins in the active part of Mao Dongqing all has a better effect on the rats with TIA, and the ratio of 10:6 is the best active component for preventing and controlling TIA.

Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures

PubMed Central

Fu, Jinglin; Liu, Minghui; Liu, Yan; Yan, Hao

2013-01-01

Conspectus Living systems have evolved a variety of nanostructures to control the molecular interactions that mediate many functions including the recognition of targets by receptors, the binding of enzymes to substrates, and the regulation of enzymatic activity. Mimicking these structures outside of the cell requires methods that offer nanoscale control over the organization of individual network components. Advances in DNA nanotechnology have enabled the design and fabrication of sophisticated one-, two- and three-dimensional (1D, 2D and 3D) nanostructures that utilize spontaneous and sequence specific DNA hybridization. Compared to other self-assembling biopolymers, DNA nanostructures offer predictable and programmable interactions, and surface features to which other nanoparticles and bio-molecules can be precisely positioned. The ability to control the spatial arrangement of the components while constructing highly-organized networks will lead to various applications of these systems. For example, DNA nanoarrays with surface displays of molecular probes can sense noncovalent hybridization interactions with DNA, RNA, and proteins and covalent chemical reactions. DNA nanostructures can also align external molecules into well-defined arrays, which may improve the resolution of many structural determination methods, such as X-ray diffraction, cryo-EM, NMR, and super-resolution fluorescence. Moreover, by constraining target entities to specific conformations, self-assembled DNA nanostructures can serve as molecular rulers to evaluate conformation-dependent activities. This Account describes the most recent advances in the DNA nanostructure directed assembly of biomolecular networks and explores the possibility of applying this technology to other fields of study. Recently, several reports have demonstrated the DNA nanostructure directed assembly of spatially-interactive biomolecular networks. For example, researchers have constructed synthetic multi-enzyme cascades by organizing the position of the components using DNA nanoscaffolds in vitro, or by utilizing RNA matrices in vivo. These structures display enhanced efficiency compared to the corresponding unstructured enzyme mixtures. Such systems are designed to mimic cellular function, where substrate diffusion between enzymes is facilitated and reactions are catalyzed with high efficiency and specificity. In addition, researchers have assembled multiple choromophores into arrays using a DNA nanoscaffold that optimizes the relative distance between the dyes and their spatial organization. The resulting artificial light harvesting system exhibits efficient cascading energy transfers. Finally, DNA nanostructures have been used as assembly templates to construct nanodevices that execute rationally-designed behaviors, including cargo loading, transportation and route control. PMID:22642503

Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

PubMed

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

2011-02-17

Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

PubMed Central

Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H.

2011-01-01

Background Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management. PMID:21379573

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

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.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

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.

A multi-step chromatographic strategy to purify three fungal endo-β-glucanases.

PubMed

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.

Aptamer- and nucleic acid enzyme-based systems for simultaneous detection of multiple analytes

DOEpatents

Lu, Yi [Champaign, IL; Liu, Juewen [Albuquerque, NM

2011-11-15

The present invention provides aptamer- and nucleic acid enzyme-based systems for simultaneously determining the presence and optionally the concentration of multiple analytes in a sample. Methods of utilizing the system and kits that include the sensor components are also provided. The system includes a first reactive polynucleotide that reacts to a first analyte; a second reactive polynucleotide that reacts to a second analyte; a third polynucleotide; a fourth polynucleotide; a first particle, coupled to the third polynucleotide; a second particle, coupled to the fourth polynucleotide; and at least one quencher, for quenching emissions of the first and second quantum dots, coupled to the first and second reactive polynucleotides. The first particle includes a quantum dot having a first emission wavelength. The second particle includes a second quantum dot having a second emission wavelength different from the first emission wavelength. The third polynucleotide and the fourth polynucleotide are different.

Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

NASA Astrophysics Data System (ADS)

Garber, M. B.; Agalarov, S. Ch.; Eliseikina, I. A.; Sedelnikova, S. E.; Tishchenko, S. V.; Shirokov, V. A.; Yusupov, M. M.; Reshetnikova, L. S.; Trakhanov, S. D.; Tukalo, M. A.; Yaremchuk, A. D.

1991-03-01

An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

Regulatory components of carbon concentrating mechanisms in aquatic unicellular photosynthetic organisms.

PubMed

Tomar, Vandana; Sidhu, Gurpreet Kaur; Nogia, Panchsheela; Mehrotra, Rajesh; Mehrotra, Sandhya

2017-11-01

This review provides an insight into the regulation of the carbon concentrating mechanisms (CCMs) in lower organisms like cyanobacteria, proteobacteria, and algae. CCMs evolved as a mechanism to concentrate CO 2 at the site of primary carboxylating enzyme Ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), so that the enzyme could overcome its affinity towards O 2 which leads to wasteful processes like photorespiration. A diverse set of CCMs exist in nature, i.e., carboxysomes in cyanobacteria and proteobacteria; pyrenoids in algae and diatoms, the C 4 system, and Crassulacean acid metabolism in higher plants. Prime regulators of CCM in most of the photosynthetic autotrophs belong to the LysR family of transcriptional regulators, which regulate the activity of the components of CCM depending upon the ambient CO 2 concentrations. Major targets of these regulators are carbonic anhydrase and inorganic carbon uptake systems (CO 2 and HCO 3 - transporters) whose activities are modulated either at transcriptional level or by changes in the levels of their co-regulatory metabolites. The article provides information on the localization of the CCM components as well as their function and participation in the development of an efficient CCM. Signal transduction cascades leading to activation/inactivation of inducible CCM components on perception of low/high CO 2 stimuli have also been brought into picture. A detailed study of the regulatory components can aid in identifying the unraveled aspects of these mechanisms and hence provide information on key molecules that need to be explored to further provide a clear understanding of the mechanism under study.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes

PubMed Central

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L.; Sopory, Sudhir K.

2017-01-01

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni2+- and Zn2+-dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies. PMID:28358304

Modulation of Metabolic Detoxification Pathways Using Foods and Food-Derived Components: A Scientific Review with Clinical Application.

PubMed

Hodges, Romilly E; Minich, Deanna M

2015-01-01

Research into human biotransformation and elimination systems continues to evolve. Various clinical and in vivo studies have been undertaken to evaluate the effects of foods and food-derived components on the activity of detoxification pathways, including phase I cytochrome P450 enzymes, phase II conjugation enzymes, Nrf2 signaling, and metallothionein. This review summarizes the research in this area to date, highlighting the potential for foods and nutrients to support and/or modulate detoxification functions. Clinical applications to alter detoxification pathway activity and improve patient outcomes are considered, drawing on the growing understanding of the relationship between detoxification functions and different disease states, genetic polymorphisms, and drug-nutrient interactions. Some caution is recommended, however, due to the limitations of current research as well as indications that many nutrients exert biphasic, dose-dependent effects and that genetic polymorphisms may alter outcomes. A whole-foods approach may, therefore, be prudent.

Modulation of Metabolic Detoxification Pathways Using Foods and Food-Derived Components: A Scientific Review with Clinical Application

PubMed Central

Hodges, Romilly E.; Minich, Deanna M.

2015-01-01

Research into human biotransformation and elimination systems continues to evolve. Various clinical and in vivo studies have been undertaken to evaluate the effects of foods and food-derived components on the activity of detoxification pathways, including phase I cytochrome P450 enzymes, phase II conjugation enzymes, Nrf2 signaling, and metallothionein. This review summarizes the research in this area to date, highlighting the potential for foods and nutrients to support and/or modulate detoxification functions. Clinical applications to alter detoxification pathway activity and improve patient outcomes are considered, drawing on the growing understanding of the relationship between detoxification functions and different disease states, genetic polymorphisms, and drug-nutrient interactions. Some caution is recommended, however, due to the limitations of current research as well as indications that many nutrients exert biphasic, dose-dependent effects and that genetic polymorphisms may alter outcomes. A whole-foods approach may, therefore, be prudent. PMID:26167297

Evaluation of lenticular antioxidant and redox system components in the lenses of acetyl-L-carnitine treatment in BSO-induced glutathione deprivation.

PubMed

Elanchezhian, R; Sakthivel, M; Isai, M; Geraldine, P; Thomas, P A

2009-07-31

To investigate whether acetyl-L-carnitine (ALCAR) retards L-buthionine-(S,R)-sulfoximine (BSO)-induced cataractogenesis in Wistar rat pups. On postpartum day 3, group I pups received intraperitoneal (ip) saline and group II and group III pups received i.p. injections of BSO once daily for three consecutive days. In addition, group III pups received ip ALCAR once daily from postpartum days 3-15. Both eyes of each pup were examined up from postpartum day 16 to day 30. After sacrifice, extricated pup lenses were analyzed for antioxidant and redox system components. There was dense lenticular opacification in all group II pups, minimal opacification in 40% of group III pups, and no opacification in 60% of group III pups and in all of group I pups. Group II lenses exhibited significantly lower values of antioxidant and redox system components and higher malondialdehyde concentrations than in group I or group III lenses. ALCAR prevents cataractogenesis in the BSO-induced cataract model, possibly by inhibiting depleting antioxidant enzyme and redox system components and inhibiting lipid peroxidation.

Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw

PubMed Central

2013-01-01

Background The use of the enzymatic hydrolysis of lignocellulose with subsequent fermentation to ethanol provides a green alternative for the production of transportation fuels. Because of its recalcitrant nature, the lignocellulosic biomass must be pretreated before enzymatic hydrolysis. However, the pretreatment often results in the formation of compounds that are inhibitory for the enzymes or fermenting organism. Although well recognized, little quantitative information on the inhibition of individual cellulase components by identified inhibitors is available. Results Strong cellulase inhibitors were separated from the liquid fraction of the hydrothermal pretreatment of wheat straw. HPLC and mass-spectroscopy analyses confirmed that the inhibitors were oligosaccharides (inhibitory oligosaccharides, IOS) with a degree of polymerization from 7 to 16. The IOS are composed of a mixture of xylo- (XOS) and gluco-oligosaccharides (GOS). We propose that XOS and GOS are the fragments of the xylan backbone and mixed-linkage β-glucans, respectively. The IOS were approximately 100 times stronger inhibitors for Trichoderma reesei cellobiohydrolases (CBHs) than cellobiose, which is one of the strongest inhibitors of these enzymes reported to date. Inhibition of endoglucanases (EGs) by IOS was weaker than that of CBHs. Most of the tested cellulases and hemicellulases were able to slowly degrade IOS and reduce the inhibitory power of the liquid fraction to some extent. The most efficient single enzyme component here was T. reesei EG TrCel7B. Although reduced by the enzyme treatment, the residual inhibitory power of IOS and the liquid fraction was strong enough to silence the major component of the T. reesei cellulase system, CBH TrCel7A. Conclusions The cellulase inhibitors described here may be responsible for the poor yields from the enzymatic conversion of the whole slurries from lignocellulose pretreatment under conditions that do not favor complete degradation of hemicellulose. Identification of the inhibitory compounds helps to design better enzyme mixtures for their degradation and to optimize the pretreatment regimes to minimize their formation. PMID:24053778

Feasibility Assessment for the Use of Cellulase in Biomass Conversion for Human Application

DTIC Science & Technology

2003-03-25

conclude that there is potential for targeted delivery of enzymes and other functional components (e.g., peptides, probiotics, prebiotics , etc.) to...potential for targeted delivery of enzymes and other functional components (e.g., peptides, probiotics, prebiotics , etc.) to the GI tract. • There is

Transcriptome analysis of the digestive system of a wood-feeding termite (Coptotermes formosanus) revealed a unique mechanism for effective biomass degradation.

PubMed

Geng, Alei; Cheng, Yanbing; Wang, Yongli; Zhu, Daochen; Le, Yilin; Wu, Jian; Xie, Rongrong; Yuan, Joshua S; Sun, Jianzhong

2018-01-01

Wood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive. In order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus , were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment. These findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of future biorefinery.

A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates

ERIC Educational Resources Information Center

Johnson, R. Jeremy; Hoops, Geoffrey C.; Savas, Christopher J.; Kartje, Zachary; Lavis, Luke D.

2015-01-01

Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein…

Energetic and Informative Interaction of Microwaves with Neurons and Enzymes

NASA Astrophysics Data System (ADS)

Maharramov, A. A.; Babazade, S. N.; Yusifov, E. Yu.; Gajiyev, A. M.

2007-04-01

Besides Purkinje Cells (PC) in cat cerebellum, experiments on Microwave-Living System interaction have been performed on some antioxidant enzymes - Super oxide dismutase (SOD), Catalase (C) and Glutathione peroxidase (GP) in the eye structures (pigment epithelium and neuronal structure - retina) in frogs, and on Glucose-6-Phosphatedehydrogenase (GPD) and Pyruvate-kinase (PK) in different organs - cerebellum, hypothalamus, liver and erythrocytes - of wistar albino rats. Exposure parameters of Microwaves of decimetre range (DRM) - total exposure, λ=65 cm, duration of exposition 10-20 minutes. According to the data obtained it may be concluded that, PC increasing their impulse activity irregularity, may react to the energetic (thermal) component of DRM action, whereas the result of informative (subtle) interaction between DRM and PC leads to the increase of regularity in electrophysiological activity of the latter. In the case of enzymes, in identification of the character of interactions, the type of the enzymes, the structure where an enzyme activity is studied and the physiological conditions related to such a factor as hunger, for example, take places. In this paper the effects of DRM on PC, G6PD and PK have been presented.

Sunlight-Dependent Hydrogen Production by Photosensitizer/Hydrogenase Systems.

PubMed

Adam, David; Bösche, Lisa; Castañeda-Losada, Leonardo; Winkler, Martin; Apfel, Ulf-Peter; Happe, Thomas

2017-03-09

We report a sustainable in vitro system for enzyme-based photohydrogen production. The [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii was tested for photohydrogen production as a proton-reducing catalyst in combination with eight different photosensitizers. Using the organic dye 5-carboxyeosin as a photosensitizer and plant-type ferredoxin PetF as an electron mediator, HydA1 achieves the highest light-driven turnover number (TON HydA1 ) yet reported for an enzyme-based in vitro system (2.9×10 6  mol(H 2 ) mol(cat) -1 ) and a maximum turnover frequency (TOF HydA1 ) of 550 mol(H 2 ) mol(HydA1) -1  s -1 . The system is fueled very effectively by ambient daylight and can be further simplified by using 5-carboxyeosin and HydA1 as a two-component photosensitizer/biocatalyst system without an additional redox mediator. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Classical Renin-Angiotensin System in Kidney Physiology

PubMed Central

Sparks, Matthew A.; Crowley, Steven D.; Gurley, Susan B.; Mirotsou, Maria; Coffman, Thomas M.

2014-01-01

The renin-angiotensin system has powerful effects in control of the blood pressure and sodium homeostasis. These actions are coordinated through integrated actions in the kidney, cardio-vascular system and the central nervous system. Along with its impact on blood pressure, the renin-angiotensin system also influences a range of processes from inflammation and immune responses to longevity. Here, we review the actions of the “classical” renin-angiotensin system, whereby the substrate protein angiotensinogen is processed in a two-step reaction by renin and angiotensin converting enzyme, resulting in the sequential generation of angiotensin I and angiotensin II, the major biologically active renin-angiotensin system peptide, which exerts its actions via type 1 and type 2 angiotensin receptors. In recent years, several new enzymes, peptides, and receptors related to the renin-angiotensin system have been identified, manifesting a complexity that was previously unappreciated. While the functions of these alternative pathways will be reviewed elsewhere in this journal, our focus here is on the physiological role of components of the “classical” renin-angiotensin system, with an emphasis on new developments and modern concepts. PMID:24944035

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

PubMed Central

2014-01-01

Background The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Results Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. Conclusion In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus. PMID:24961398

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae.

PubMed

Liu, Lifang; Feizi, Amir; Österlund, Tobias; Hjort, Carsten; Nielsen, Jens

2014-06-24

The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.

Enzyme-free nucleic acid dynamical systems.

PubMed

Srinivas, Niranjan; Parkin, James; Seelig, Georg; Winfree, Erik; Soloveichik, David

2017-12-15

Chemistries exhibiting complex dynamics-from inorganic oscillators to gene regulatory networks-have been long known but either cannot be reprogrammed at will or rely on the sophisticated enzyme chemistry underlying the central dogma. Can simpler molecular mechanisms, designed from scratch, exhibit the same range of behaviors? Abstract chemical reaction networks have been proposed as a programming language for complex dynamics, along with their systematic implementation using short synthetic DNA molecules. We developed this technology for dynamical systems by identifying critical design principles and codifying them into a compiler automating the design process. Using this approach, we built an oscillator containing only DNA components, establishing that Watson-Crick base-pairing interactions alone suffice for complex chemical dynamics and that autonomous molecular systems can be designed via molecular programming languages. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease†

PubMed Central

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E.

2008-01-01

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID:16669630

Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.

PubMed

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E

2006-05-09

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.

High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients.

PubMed

Shero, J H; Bordwell, B; Rothfield, N F; Earnshaw, W C

1986-02-14

Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation.

PubMed

Droux, M; Miginiac-Maslow, M; Jacquot, J P; Gadal, P; Crawford, N A; Kosower, N S; Buchanan, B B

1987-07-01

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

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

Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn,more » reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.« less

Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System*

PubMed Central

Campeotto, Ivan; Percy, Matthew G.; MacDonald, James T.; Förster, Andreas; Freemont, Paul S.; Gründling, Angelika

2014-01-01

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaPLm and LtaSLm. Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaSLm structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain. PMID:25128528

Phylogenomic reconstruction of archaeal fatty acid metabolism

PubMed Central

Dibrova, Daria V.; Galperin, Michael Y.; Mulkidjanian, Armen Y.

2014-01-01

While certain archaea appear to synthesize and/or metabolize fatty acids, the respective pathways still remain obscure. By analyzing the genomic distribution of the key lipid-related enzymes, we were able to identify the likely components of the archaeal pathway of fatty acid metabolism, namely, a combination of the enzymes of bacterial-type β-oxidation of fatty acids (acyl-CoA-dehydrogenase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase) with paralogs of the archaeal acetyl-CoA C-acetyltransferase, an enzyme of the mevalonate biosynthesis pathway. These three β-oxidation enzymes working in the reverse direction could potentially catalyze biosynthesis of fatty acids, with paralogs of acetyl-CoA C-acetyltransferase performing addition of C2 fragments. The presence in archaea of the genes for energy-transducing membrane enzyme complexes, such as cytochrome bc complex, cytochrome c oxidase, and diverse rhodopsins, was found to correlate with the presence of the proposed system of fatty acid biosynthesis. We speculate that because these membrane complexes functionally depend on fatty acid chains, their genes could have been acquired via lateral gene transfer from bacteria only by those archaea that already possessed a system of fatty acid biosynthesis. The proposed pathway of archaeal fatty acid metabolism operates in extreme conditions and therefore might be of interest in the context of biofuel production and other industrial applications. PMID:24818264

Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot-enzyme conjugates.

PubMed

Jang, Eunji; Kim, Sinyoung; Koh, Won-Gun

2012-01-15

This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H(2)O(2)-generating oxidase. The biosensor consists of three components (quantum dot-enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)-enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H(2)O(2), which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50 μM of glucose and 70 μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels. Copyright © 2011 Elsevier B.V. All rights reserved.

A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora.

PubMed

Flego, D; Marits, R; Eriksson, A R; Kõiv, V; Karlsson, M B; Heikinheimo, R; Palva, E T

2000-04-01

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

PubMed Central

Suen, W C; Haigler, B E; Spain, J C

1996-01-01

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor. PMID:8759857

Characterization of a restriction modification system from the commensal Escherichia coli strain A0 34/86 (O83:K24:H31).

PubMed

Weiserová, Marie; Ryu, Junichi

2008-06-27

Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature. The aim of this study was characterisation of a putative R-M system EcoA0ORF42P identified in the commensal Escherichia coli A0 34/86 (O83: K24: H31) strain, which is efficiently used at Czech paediatric clinics for prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants. We have characterised a restriction-modification system EcoA0ORF42P of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system, designated as EcoAO83I, is a new functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as was demonstrated by an immunological cross-reactivity and a complementation assay. Using the plasmid transformation method and the RM search computer program, we identified the DNA recognition sequence of the EcoAO83I as GGA(8N)ATGC. In consistence with the amino acids alignment data, the 3' TRD component of the recognition sequence is identical to the sequence recognized by the EcoEI enzyme. The A-T (modified adenine) distance is identical to that in the EcoAI and EcoEI recognition sites, which also indicates that this system is a Type IB member. Interestingly, the recognition sequence we determined here is identical to the previously reported prototype sequence for Eco377I and its isoschizomers. Putative restriction-modification system EcoA0ORF42P in the commensal Escherichia coli strain A0 34/86 (O83: K24: H31) was found to be a member of the Type IB family and was designated as EcoAO83I. Combination of the classical biochemical and bacterial genetics approaches with comparative genomics might contribute effectively to further classification of many other putative Type-I enzymes, especially in clinical samples.

Structures of ribonucleoprotein particle modification enzymes

PubMed Central

Liang, Bo; Li, Hong

2016-01-01

Small nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo. PMID:21108865

Kdo2-lipid A: structural diversity and impact on immunopharmacology

PubMed Central

Wang, Xiaoyuan; Quinn, Peter J; Yan, Aixin

2015-01-01

3-deoxy-d-manno-octulosonic acid-lipid A (Kdo2-lipid A) is the essential component of lipopolysaccharide in most Gram-negative bacteria and the minimal structural component to sustain bacterial viability. It serves as the active component of lipopolysaccharide to stimulate potent host immune responses through the complex of Toll-like-receptor 4 (TLR4) and myeloid differentiation protein 2. The entire biosynthetic pathway of Escherichia coli Kdo2-lipid A has been elucidated and the nine enzymes of the pathway are shared by most Gram-negative bacteria, indicating conserved Kdo2-lipid A structure across different species. Yet many bacteria can modify the structure of their Kdo2-lipid A which serves as a strategy to modulate bacterial virulence and adapt to different growth environments as well as to avoid recognition by the mammalian innate immune systems. Key enzymes and receptors involved in Kdo2-lipid A biosynthesis, structural modification and its interaction with the TLR4 pathway represent a clear opportunity for immunopharmacological exploitation. These include the development of novel antibiotics targeting key biosynthetic enzymes and utilization of structurally modified Kdo2-lipid A or correspondingly engineered live bacteria as vaccines and adjuvants. Kdo2-lipid A/TLR4 antagonists can also be applied in anti-inflammatory interventions. This review summarizes recent knowledge on both the fundamental processes of Kdo2-lipid A biosynthesis, structural modification and immune stimulation, and applied research on pharmacological exploitations of these processes for therapeutic development. PMID:24838025

The Biogeochemistry of Sulfur in Hydrothermal Systems

NASA Technical Reports Server (NTRS)

Schulte, Mitchell; Rogers, K. L.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. The incorporation of sulfur into many biomolecules likely dates back to the development of the earliest metabolic strategies. Sulfur is common in enzymes and co-enzymes and is an indispensable structural component in many peptides and proteins. Early metabolism may have been heavily influenced by the abundance of sulfide minerals in hydrothermal systems. Understanding how sulfur became prevalent in biochemical processes and many biomolecules requires knowledge of the reaction properties of sulfur-bearing compounds. We have previously estimated thermodynamic data for thiols, the simplest organic sulfur compounds, at elevated temperatures and pressures. If life began in hydrothermal environments, it is especially important to understand reactions at elevated temperatures among sulfur-bearing compounds and other organic molecules essential for the origin and persistence of life. Here we examine reactions that may have formed amino acids with thiols as reaction intermediates in hypothetical early Earth hydrothermal environments. (There are two amino acids, cysteine and methionine, that contain sulfur.) Our calculations suggest that significant amounts of some amino acids were produced in early Earth hydrothermal fluids, given reasonable concentrations H2, NH3, H2S and CO. For example, preliminary results indicate that glycine activities as high as 1 mmol can be reached in these systems at 100 C. Alanine formation from propanethiol is also a favorable reaction. On the other hand, the calculated equilibrium log activities of cysteine and serine from propanethiol are -21 and -19, respectively, at 100 C. These results indicate that while amino acid formation with thiols as intermediates is favored in some cases, other mechanisms may have been necessary to produce significant amounts of other amino acids. Coupled with our previous results for thiols, these studies imply that sulfur may have been easily incorporated into the organic geochemistry of early Earth hydrothermal systems, leading to its widespread use in biomolecules. Formation of more complex biomolecules in hydrothermal systems may have required sulfur-bearing organic compounds as reaction intermediates.

Functional Heterologous Expression of an Engineered Full Length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

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

Currie, Devin; Herring, Christopher; Guss, Adam M

BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to expressmore » the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a cipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.« less

Drug Research

NASA Technical Reports Server (NTRS)

1989-01-01

NBOD2, a program developed at Goddard Space Flight Center to solve equations of motion coupled N-body systems is used by E.I. DuPont de Nemours & Co. to model potential drugs as a series of elements. The program analyses the vibrational and static motions of independent components in drugs. Information generated from this process is used to design specific drugs to interact with enzymes in designated ways.

Biogenesis of glycerol 3-phosphate acyltransferase (GPAT): influence of transmembrane domains and protein-protein interactions on the localization of GPAT to ER subdomains

USDA-ARS?s Scientific Manuscript database

Glycerolipids are the major components of cellular membranes in all plant cells, storage oils in developing seeds, and the cuticular surface of plant organs. Using the tung (Vernicia fordii) triacylglycerol (TAG) biosynthetic enzymes as model system, we previously showed that the type 1 and 2 diacyl...

The antioxidant system of seminal fluid during in vitro storage of sterlet Acipenser ruthenus sperm.

PubMed

Dzyuba, Viktoriya; Cosson, Jacky; Dzyuba, Borys; Yamaner, Gunes; Rodina, Marek; Linhart, Otomar

2016-04-01

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

PubMed

Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A

2015-01-01

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Evaluation of lenticular antioxidant and redox system components in the lenses of acetyl-L-carnitine treatment in BSO-induced glutathione deprivation

PubMed Central

Elanchezhian, R.; Sakthivel, M.; Isai, M.; Thomas, P.A.

2009-01-01

Purpose To investigate whether acetyl-L-carnitine (ALCAR) retards L-buthionine-(S,R)-sulfoximine (BSO)-induced cataractogenesis in Wistar rat pups. Methods On postpartum day 3, group I pups received intraperitoneal (ip) saline and group II and group III pups received i.p. injections of BSO once daily for three consecutive days. In addition, group III pups received ip ALCAR once daily from postpartum days 3–15. Both eyes of each pup were examined up from postpartum day 16 to day 30. After sacrifice, extricated pup lenses were analyzed for antioxidant and redox system components. Results There was dense lenticular opacification in all group II pups, minimal opacification in 40% of group III pups, and no opacification in 60% of group III pups and in all of group I pups. Group II lenses exhibited significantly lower values of antioxidant and redox system components and higher malondialdehyde concentrations than in group I or group III lenses. Conclusions ALCAR prevents cataractogenesis in the BSO-induced cataract model, possibly by inhibiting depleting antioxidant enzyme and redox system components and inhibiting lipid peroxidation. PMID:19649174

Ultrasensitivity by Molecular Titration in Spatially Propagating Enzymatic Reactions

PubMed Central

Semenov, Sergey N.; Markvoort, Albert J.; Gevers, Wouter B.L.; Piruska, Aigars; de Greef, Tom F.A.; Huck, Wilhelm T.S.

2013-01-01

Delineating design principles of biological systems by reconstitution of purified components offers a platform to gauge the influence of critical physicochemical parameters on minimal biological systems of reduced complexity. Here we unravel the effect of strong reversible inhibitors on the spatiotemporal propagation of enzymatic reactions in a confined environment in vitro. We use micropatterned, enzyme-laden agarose gels which are stamped on polyacrylamide films containing immobilized substrates and reversible inhibitors. Quantitative fluorescence imaging combined with detailed numerical simulations of the reaction-diffusion process reveal that a shallow gradient of enzyme is converted into a steep product gradient by addition of strong inhibitors, consistent with a mathematical model of molecular titration. The results confirm that ultrasensitive and threshold effects at the molecular level can convert a graded input signal to a steep spatial response at macroscopic length scales. PMID:23972857

[Effect of population density on enzymatic activity of antioxidative and phenol oxidase systems of imagoes and nymphs of the marble cockroach Nauphoeta cinerea].

PubMed

Murzagulov, G S; Saltykova, E S; Gaĭfullina, L R; Nikolenko, A G

2013-01-01

The work deals with effect of density of population on functional activity of components pf protective system of adult individuals and nymphs of the marble cockroach. The resistance of individuals has been noted to decrease both at individual maintenance and under conditions of overpopulation. Changes in activities of enzymes of antioxidative and phenoloxidase systems are studied ion the insect hemolymph and intestine. Possible consequences of isolation and overpopulation are discussed both for stability and for individual development.

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.

PubMed

Beattie, Thomas R; Bell, Stephen D

2011-01-01

Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.

Long-Term Regulation of the Local Renin-Angiotensin System in the Myocardium of Spontaneously Hypertensive Rats by Feeding Bioactive Peptides Derived from Spirulina platensis.

PubMed

Pan, Huanglei; She, Xingxing; Wu, Hongli; Ma, Jun; Ren, Difeng; Lu, Jun

2015-09-09

This study investigated the long-term (8 weeks) anti-hypertensive effects of 10 mg/kg tripeptides isolated from Spirulina platensis, Ile-Gln-Pro (IQP) and Val-Glu-Pro (VEP), and S. platensis hydrolysates (SH) on spontaneously hypertensive rats. The treatment period was 6 weeks, and observation continued for another 2 weeks. After treatment, weighted systolic blood pressure, weighted diastolic blood pressure, left ventricular mass index, and right ventricular mass index of groups treated with IQP, VEP, and SH were significantly lower than those of the group treated with distilled water, even when the treatments had been withdrawn for 2 weeks. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting showed the mRNA expression levels and protein/peptide concentrations of the main components of the renin angiotensin system in myocardium were significantly affected by treatment: angiotensin converting enzyme, angiotensin II, and angiotensin type 1 receptor were down-regulated, whereas angiotensin type 2 receptor, angiotensin converting enzyme 2, angiotensin-(1-7), and Mas receptor were up-regulated.

A linear framework for time-scale separation in nonlinear biochemical systems.

PubMed

Gunawardena, Jeremy

2012-01-01

Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level.

An In Vitro Enzyme System for the Production of myo-Inositol from Starch

PubMed Central

Fujisawa, Tomoko; Fujinaga, Shohei

2017-01-01

ABSTRACT We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD+, resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD+ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol. PMID:28600316

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

PubMed

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

2016-04-01

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

Computational characterization of DNA/peptide/nanotube self assembly for bioenergy applications

NASA Astrophysics Data System (ADS)

Ortiz, Vanessa; Araki, Ruriko; Collier, Galen

2012-02-01

Multi-enzyme pathways have become a subject of increasing interest for their role in the engineering of biomimetic systems for applications including biosensors, bioelectronics, and bioenergy. The efficiencies found in natural metabolic pathways partially arise from biomolecular self-assembly of the component enzymes in an effort to avoid transport limitations. The ultimate goal of this effort is to design and build biofuel cells with efficiencies similar to those of native systems by introducing biomimetic structures that immobilize multiple enzymes in specific orientations on a bioelectrode. To achieve site-specific immobilization, the specificity of DNA-binding domains is exploited with an approach that allows any redox enzyme to be modified to site-specifically bind to double stranded (ds) DNA while retaining activity. Because of its many desirable properties, the bioelectrode of choice is single-wall carbon nanotubes (SWNTs), but little is known about dsDNA/SWNT assembly and how this might affect the activity of the DNA-binding domains. Here we evaluate the feasibility of the proposed assembly by performing atomistic molecular dynamics simulations to look at the stability and conformations adopted by dsDNA when bound to a SWNT. We also evaluate the effects of the presence of a SWNT on the stability of the complex formed by a DNA-binding domain and DNA.

[Thyroid hormone metabolism and action].

PubMed

Köhrle, Josef

2004-05-01

Reductive deiodination of thyroid hormones at the phenolic and tyrosyl ring leads to the activation or inactivation of the thyromimetic activity inherent to thyroid hormones. Alterations in the activities of the three selenocysteine-containing enzymes, the iodothyronine deiodinases, have been reported during development and in specific cells and tissues of the adult organism. Furthermore, pathophysiological changes in the deiodinase expression lead to therapeutically relevant disturbances of the homeostasis of thyroid hormones. Metabolisation of thyroid hormones by conjugation of their phenolic 4'-OH group, their alanine side chain or cleavage of their diphenylether bridge also contributes to both local and systemic supply of thyromimetic activity or hormone degradation. Further components mediating the pleiotropic action of thyroid hormones in part include redundant T3 receptors, binding and transport proteins, metabolising enzymes and T3-regulated gene products. This is achieved in a finely tuned manner with multiple feedback control, malfunction or complete failure of individual components and networks involved in the iodothyronine metabolism and thyroid hormone action can thus be compensated or prevented.

Design of experiments and principal component analysis as approaches for enhancing performance of gas-diffusional air-breathing bilirubin oxidase cathode

NASA Astrophysics Data System (ADS)

Babanova, Sofia; Artyushkova, Kateryna; Ulyanova, Yevgenia; Singhal, Sameer; Atanassov, Plamen

2014-01-01

Two statistical methods, design of experiments (DOE) and principal component analysis (PCA) are employed to investigate and improve performance of air-breathing gas-diffusional enzymatic electrodes. DOE is utilized as a tool for systematic organization and evaluation of various factors affecting the performance of the composite system. Based on the results from the DOE, an improved cathode is constructed. The current density generated utilizing the improved cathode (755 ± 39 μA cm-2 at 0.3 V vs. Ag/AgCl) is 2-5 times higher than the highest current density previously achieved. Three major factors contributing to the cathode performance are identified: the amount of enzyme, the volume of phosphate buffer used to immobilize the enzyme, and the thickness of the gas-diffusion layer (GDL). PCA is applied as an independent confirmation tool to support conclusions made by DOE and to visualize the contribution of factors in individual cathode configurations.

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

PubMed

Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

2015-01-01

Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

Preliminary studies on immobilization of lipase using chicken eggshell

NASA Astrophysics Data System (ADS)

Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

2016-06-01

A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

ERIC Educational Resources Information Center

Saperas, Nuria; Fonfria-Subiros, Elsa

2011-01-01

This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase

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.

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

PubMed Central

Kurkcuoglu, Zeynep; Bakan, Ahmet; Kocaman, Duygu; Bahar, Ivet; Doruker, Pemra

2012-01-01

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. PMID:23028297

Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

PubMed Central

Ymele-Leki, Patrick; Houot, Laetitia

2013-01-01

Vibrio cholerae is a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera. V. cholerae use a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport by V. cholerae requires the mannitol-specific EII (EIIMtl), which is expressed only in the presence of mannitol. Here we show that mannitol activates V. cholerae biofilm formation and transcription of the vps biofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EIIMtl is sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability of V. cholerae to respond to environmental mannitol by forming a biofilm may play an important role in habitat selection. PMID:23728818

Genetically Engineered Materials for Biofuels Production

NASA Astrophysics Data System (ADS)

Raab, Michael

2012-02-01

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

4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2.

PubMed Central

Locher, H H; Leisinger, T; Cook, A M

1991-01-01

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol. Images Fig. 3. PMID:2012609

Development of electrochemical biosensors with various types of zeolites

NASA Astrophysics Data System (ADS)

Soldatkina, O. V.; Kucherenko, I. S.; Soldatkin, O. O.; Pyeshkova, V. M.; Dudchenko, O. Y.; Akata Kurç, B.; Dzyadevych, S. V.

2018-03-01

In the work, different types of zeolites were used for the development of enzyme-based electrochemical biosensors. Zeolites were added to the biorecognition elements of the biosensors and served as additional components of the biomembranes or adsorbents for enzymes. Three types of biosensors (conductometric, amperometric and potentiometric) were studied. The developed biosensors were compared with the similar biosensors without zeolites. The biosensors contained the following enzymes: urease, glucose oxidase, glutamate oxidase, and acetylcholinesterase and were intended for the detection of urea, glucose, glutamate, and acetylcholine, respectively. Construction of the biosensors using the adsorption of enzymes on zeolites has several advantages: simplicity, good reproducibility, quickness, absence of toxic compounds. These benefits are particularly important for the standardization and further mass production of the biosensors. Furthermore, a biosensor for the sucrose determination contained a three-enzyme system (invertase/mutatorase/glucose oxidase), immobilized by a combination of adsorption on silicalite and cross-linking via glutaraldehyde; such combined immobilization demonstrated better results as compared with adsorption or cross-linking separately. The analysis of urea and sucrose concentrations in the real samples was carried out. The results, obtained with biosensors, had high correlation with the results of traditional analytical methods, thus the developed biosensors are promising for practical applications.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea:Polyopisthocotylea).

PubMed

Cable, J; Marks, N J; Halton, D W; Shaw, C; Johnston, C F; Tinsley, R C; Gannicott, A M

1996-12-01

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the FMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (lateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the level of the haptor, the ventral cords provide nerve roots which innervate each of the 9 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones.

Genes of the antioxidant system of the honey bee: annotation and phylogeny.

PubMed

Corona, M; Robinson, G E

2006-10-01

Antioxidant enzymes perform a variety of vital functions including the reduction of life-shortening oxidative damage. We used the honey bee genome sequence to identify the major components of the honey bee antioxidant system. A comparative analysis of honey bee with Drosophila melanogaster and Anopheles gambiae shows that although the basic components of the antioxidant system are conserved, there are important species differences in the number of paralogs. These include the duplication of thioredoxin reductase and the expansion of the thioredoxin family in fly; lack of expansion of the Theta, Delta and Omega GST classes in bee and no expansion of the Sigma class in dipteran species. The differential expansion of antioxidant gene families among honey bees and dipteran species might reflect the marked differences in life history and ecological niches between social and solitary species.

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies.

PubMed

Prince, H E; Ernst, C E; Hogrefe, W R

2000-01-01

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies. Copyright 2000 Wiley-Liss, Inc.

A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

PubMed

Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

2015-11-10

In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

Flume Computer-Aided Design (CAD): Integrated CAD for Microflume Components and Systems

DTIC Science & Technology

2002-04-01

31 3.3: Matching the Mix Ratio (Part B...sizes) will be optimized based on the required flow rates and mixing ratios of the different species. The influence of etch depth is investigated on a...Inhibition Study In this network, the target protein is mixed with protease (i.e. enzyme that cleaves the target protein) and the protease inhibitor (the

Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis.

PubMed

Huang, Chun-Yen; Lin, Hui-Chen; Lin, Cheng-Huang

2015-01-01

We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determine whether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12h of hypoxia and 12h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na(+)/K(+)-ATPase enzyme activity did not decrease, but increases in Na(+)/K(+)-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments. Copyright © 2014 Elsevier Inc. All rights reserved.

Remote Sensing of a Manipulated Prairie Grassland Experiment to Predict Belowground Processes

NASA Astrophysics Data System (ADS)

Cavender-Bares, J.; Schweiger, A. K.; Hobbie, S. E.; Madritch, M. D.; Wang, Z.; Couture, J. J.; Gamon, J. A.; Townsend, P. A.

2017-12-01

Given the importance of plant biodiversity for providing the ecosystem functions and services on which humans depend, rapid and remote methods of monitoring plant biodiversity across large spatial extents and biological scales are increasingly critical. In North American prairie systems, the ecosystem benefits of diversity are a subject of ongoing investigation and relevance to policy. However, detecting belowground components of ecosystem biodiversity, composition and associated functions are not possible directly through remote sensing. Nevertheless, belowground components of diversity may be linked to aboveground components allowing indirect inferences. Here we test a series of hypotheses about how aboveground functional and chemical diversity and composition of plant communities drive belowground functions, including N mineralization, enzyme activity and microbial biomass, as well as microbial diversity and composition. We hypothesize that the quantity and chemical composition of aboveground inputs to soil drive belowground processes, including decomposition and microbial enzyme activity. We use plant spectra (400 nm to 2500 nm) measured at the leaf and airborne level to determine chemical and functional composition of leaves and canopies in a long-term grassland experiment where diversity is manipulated at the Cedar Creek Ecosystem Science Reserve. We then assess the extent to which belowground chemistry, microbial diversity and composition are predicted from aboveground plant diversity, biomass and chemical composition. We find strong associations between aboveground inputs and belowground enzyme activity and microbial biomass but only weak linkages between aboveground diversity and belowground diversity. We discuss the potential for such approaches and the caveats related to the spatial scale of measurements and spatial resolution of airborne detection.

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes▿

PubMed Central

Schilhabel, Anke; Studenik, Sandra; Vödisch, Martin; Kreher, Sandra; Schlott, Bernhard; Pierik, Antonio Y.; Diekert, Gabriele

2009-01-01

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases. PMID:19011025

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

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

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source.

PubMed

Kumura, H; Ishido, T; Shimazaki, K

2011-02-01

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of β-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

PubMed Central

Lea, Wendy A.; Jadhav, Ajit; Rai, Ganesha; Sayed, Ahmed A.; Cass, Cynthia L.; Inglese, James; Williams, David L.; Austin, Christopher P.

2008-01-01

Abstract Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-μl final volume assay is based on the Ellman reagent [5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate. PMID:18665782

The transcription factor Mlc promotes Vibrio cholerae biofilm formation through repression of phosphotransferase system components.

PubMed

Pickering, Bradley S; Lopilato, Jane E; Smith, Daniel R; Watnick, Paula I

2014-07-01

The phosphoenol phosphotransferase system (PTS) is a multicomponent signal transduction cascade that regulates diverse aspects of bacterial cellular physiology in response to the availability of high-energy sugars in the environment. Many PTS components are repressed at the transcriptional level when the substrates they transport are not available. In Escherichia coli, the transcription factor Mlc (for makes large colonies) represses transcription of the genes encoding enzyme I (EI), histidine protein (HPr), and the glucose-specific enzyme IIBC (EIIBC(Glc)) in defined media that lack PTS substrates. When glucose is present, the unphosphorylated form of EIIBC(Glc) sequesters Mlc to the cell membrane, preventing its interaction with DNA. Very little is known about Vibrio cholerae Mlc. We found that V. cholerae Mlc activates biofilm formation in LB broth but not in defined medium supplemented with either pyruvate or glucose. Therefore, we questioned whether V. cholerae Mlc functions differently than E. coli Mlc. Here we have shown that, like E. coli Mlc, V. cholerae Mlc represses transcription of PTS components in both defined medium and LB broth and that E. coli Mlc is able to rescue the biofilm defect of a V. cholerae Δmlc mutant. Furthermore, we provide evidence that Mlc indirectly activates transcription of the vps genes by repressing expression of EI. Because activation of the vps genes by Mlc occurs under only a subset of the conditions in which repression of PTS components is observed, we conclude that additional inputs present in LB broth are required for activation of vps gene transcription by Mlc. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Recreating the synthesis of starch granules in yeast

PubMed Central

Pfister, Barbara; Sánchez-Ferrer, Antoni; Diaz, Ana; Lu, Kuanjen; Otto, Caroline; Holler, Mirko; Shaik, Farooque Razvi; Meier, Florence; Mezzenga, Raffaele; Zeeman, Samuel C

2016-01-01

Starch, as the major nutritional component of our staple crops and a feedstock for industry, is a vital plant product. It is composed of glucose polymers that form massive semi-crystalline granules. Its precise structure and composition determine its functionality and thus applications; however, there is no versatile model system allowing the relationships between the biosynthetic apparatus, glucan structure and properties to be explored. Here, we expressed the core Arabidopsis starch-biosynthesis pathway in Saccharomyces cerevisiae purged of its endogenous glycogen-metabolic enzymes. Systematic variation of the set of biosynthetic enzymes illustrated how each affects glucan structure and solubility. Expression of the complete set resulted in dense, insoluble granules with a starch-like semi-crystalline organization, demonstrating that this system indeed simulates starch biosynthesis. Thus, the yeast system has the potential to accelerate starch research and help create a holistic understanding of starch granule biosynthesis, providing a basis for the targeted biotechnological improvement of crops. DOI: http://dx.doi.org/10.7554/eLife.15552.001 PMID:27871361

Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

PubMed

Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

2016-06-01

In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular lysis of Streptococcus faecalis induced with triton X-100.

PubMed Central

Cornett, J B; Shockman, G D

1978-01-01

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed. PMID:97265

Subcellular characteristics of functional intracellular renin–angiotensin systems☆

PubMed Central

Abadir, Peter M.; Walston, Jeremy D.; Carey, Robert M.

2013-01-01

The renin–angio tensin system (RAS) is now regarded as an integral component in not only the development of hypertension, but also in physiologic and pathophysiologic mechanisms in multiple tissues and chronic disease states. While many of the endocrine (circulating), paracrine (cell-to-different cell) and autacrine (cell-to-same cell) effects of the RAS are believed to be mediated through the canonical extracellular RAS, a complete, independent and differentially regulated intracellular RAS (iRAS) has also been proposed. Angiotensinogen, the enzymes renin and angiotensin-converting enzyme (ACE) and the angiotensin peptides can all be synthesized and retained intracellularly. Angiotensin receptors (types I and 2) are also abundant intracellularly mainly at the nuclear and mitochondrial levels. The aim of this review is to focus on the most recent information concerning the subcellular localization, distribution and functions of the iRAS and to discuss the potential consequences of activation of the subcellular RAS on different organ systems. PMID:23032352

Mechanism of energy conversion and transfer in bioluminescence. Final report. [Sea pansy Renilla reniformis

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

Cormier, M.J.

1979-01-01

Bioluminescence in the sea pansy, Renilla reniformis, a marine anthozoan coelenterate, is a complex process involving the participation of three proteins specific to anthozoan coelenterate-type systems. These are: (1) the luciferin binding protein, (2) the enzyme luciferase, and (3) the green-fluorescent protein. Each of these have been purified and characterized and the structure of luciferin has been confirmed by synthesis. Luciferin binding protein (BP-LH/sub 2/) is a specific substrate binding protein which binds one molecule of coelenterate-type luciferin per molecule of protein and which then releases luciferin in the presence of Ca/sup + +/. Luciferase is the enzyme which catalyzesmore » oxidation (by O/sub 2/) of coelenterate-type luciferin, leading to the production of CO/sub 2/ and enzyme-bound, excited-state oxyluciferin. Oxyluciferin may then emit blue light by a direct de-excitation pathway or may transfer excitation energy to the green-fluorescent protein (GFP). GFP is a non-catalytic accessory protein which accepts excitation energy from oxyluciferin, by radiationless energy transfer, and then emits green bioluminescence. The Renilla bioluminescence system is thus the first radiationless energy transfer system the individual components of which have been purified to homogeneity, characterized, and then reassembled in vitro with restoration of the energy transfer function.« less

Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

PubMed

Tsfasman, Irina M; Lapteva, Yulia S; Krasovskaya, Ludmila A; Kudryakova, Irina V; Vasilyeva, Natalia V; Granovsky, Igor E; Stepnaya, Olga A

2015-01-01

Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms. © 2015 S. Karger AG, Basel.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

PubMed Central

Fagerlund, Robert D.; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S.

2015-01-01

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA–RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P. PMID:26135751

Target-induced formation of neuraminidase inhibitors from in vitro virtual combinatorial libraries

PubMed Central

Hochgürtel, Matthias; Kroth, Heiko; Piecha, Dorothea; Hofmann, Michael W.; Nicolau, Claude; Krause, Sonja; Schaaf, Otmar; Sonnenmoser, Gabriele; Eliseev, Alexey V.

2002-01-01

Neuraminidase, a key enzyme responsible for influenza virus propagation, has been used as a template for selective synthesis of small subsets of its own inhibitors from theoretically highly diverse dynamic combinatorial libraries. We show that the library building blocks, aldehydes and amines, form significant amounts of the library components resulting from their coupling by reductive amination only in the presence of the enzyme. The target amplifies the best hits at least 120-fold. The dynamic libraries synthesized and screened in such an in vitro virtual mode form the components that possess high inhibitory activity, as confirmed by enzyme assays with independently synthesized individual compounds. PMID:11891312

Enzyme-modified starch as an oil delivery system for bake-only chicken nuggets.

PubMed

Purcell, Sarah; Wang, Ya-Jane; Seo, Han-Seok

2014-05-01

This study investigated the effects of enzyme modification on starch as an effective oil delivery system for bake-only chicken nuggets. Various native starches were hydrolyzed by amyloglucosidase to a hydrolysis degree of 20% to 25% and plated with 50% (w/w, starch dry basis) with canola oil to create a starch-oil matrix. This matrix was then blended into a dry ingredient blend for batter and breader components. Nuggets were prepared by coated with predust, hydrated batter, and breader, and the coated nuggets were steam-baked until fully cooked and then frozen until texture and sensory analyses. The enzyme-modified starches showed a significant decrease in pasting viscosities for all starch types. For textural properties of nuggets, no clear relationship was found between peak force and starch source or amylose content. Sensory attributes related to fried foods (for example, crispness and mouth-coating) did not significantly differ between bake-only nuggets formulated using the enzyme-modified starches and the partially fried and baked ones. The present findings suggest that enzyme-modified starches can deliver sufficient quantity of oil to create sensory attributes similar to those of partially fried chicken nuggets. Further study is needed to optimize the coating formulation of bake-only chicken nugget to become close to the fried one in sensory aspects. The food industry has become increasingly focused on healthier items. Frying imparts several critical and desirable product functionalities, such as developing texture and color, and providing mouth-feel and flavor. The food industry has yet to duplicate all of the unique characteristics of fried chicken nuggets with a baking process. This study investigated the application of enzyme-modified starch as an oil delivery system in bake-only chicken nugget formulation in attempts to provide characteristics of fried items. This information is useful to improve the nutritional value of fried food by eliminating the frying process while preserving the desired characteristics of fried products. © 2014 Institute of Food Technologists®

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

PubMed

Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

2015-01-01

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus.

PubMed

Amid, Mehrnoush; Manap, Yazid; Azmira, Farhana; Hussin, Muhaini; Sarker, Zaidul Islam

2015-07-01

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme. Copyright © 2015 Elsevier B.V. All rights reserved.

Reduction of N-hydroxy-sulfonamides, including N-hydroxy-valdecoxib, by the molybdenum-containing enzyme mARC.

PubMed

Havemeyer, Antje; Grünewald, Sanja; Wahl, Bettina; Bittner, Florian; Mendel, Ralf; Erdélyi, Péter; Fischer, János; Clement, Bernd

2010-11-01

Purification of the mitochondrial enzyme responsible for reduction of N-hydroxylated amidine prodrugs led to the identification of two newly discovered mammalian molybdenum-containing proteins, the mitochondrial amidoxime reducing components mARC-1 and mARC-2 (Gruenewald et al., 2008). These 35-kDa proteins represent a novel group of molybdenum proteins in eukaryotes as they form a molybdenum cofactor-dependent enzyme system consisting of three separate proteins (Havemeyer et al., 2006). Each mARC protein reduces N-hydroxylated compounds after reconstitution with the electron transport proteins cytochrome b(5) and b(5) reductase. In continuation of our drug metabolism investigations (Havemeyer et al., 2006; Gruenewald et al., 2008), we present data from reconstituted enzyme systems with recombinant human and native porcine enzymes showing the reduction of N-hydroxy-sulfonamides (sulfohydroxamic acids) to sulfonamides: the N-hydroxy-sulfonamide N-hydroxy-valdecoxib (N-hydroxy-4-[5-methyl-3-phenyl-4-isoxazolyl]-benzenesulfonamide) represents a novel cyclooxygenase (COX)-2 inhibitor and is therefore a drug candidate in the treatment of diseases associated with rheumatic inflammation, pain, and fever. It was synthesized as an analog of the known COX-2 inhibitor valdecoxib (4-[5-methyl-3-phenyl-4-isoxazolyl]-benzenesulfonamide) (Talley et al., 2000). N-Hydroxy-valdecoxib had low in vitro COX-2 activity but showed significant analgesic activity in vivo and a prolonged therapeutic effect compared with valdecoxib (Erdélyi et al., 2008). In this report, we demonstrate that N-hydroxy-valdecoxib is enzymatically reduced to its pharmacologically active metabolite valdecoxib. Thus, N-hydroxy-valdecoxib acts as prodrug that is activated by the molybdenum-containing enzyme mARC.

[Influence of extracellular polymeric substance on enzyme hydrolysis of sludge under anaerobic condition].

PubMed

Chen, Wei; Jia, Yuan-Yuan; Zheng, Wei; Li, Xiao-Ming; Zhou, Jun; Yang, Qi; Luo, Kun

2011-08-01

The effect of extracellular polymeric substance (EPS) on the enzymatic solubilisation of sludge and the changes of chemical components was investigated. Sludge solubilization with and without EPS was studied in the enzymatic system, and in the normal system without enzyme addition, respectively. The result indicated that only EPS could be hydrolyzed when the enzyme addition less than 20 mg/g, while the cell lysis occurred significantly with the doses of enzymes increasing. Treatment with lysozyme for the original sludge was proved to have a higher hydrolysis efficiency, and the SCOD/TCOD rate reached up to 28.14%. And at the enzyme dosage of 60 mg/g, the VSS removal rate increased to 51.66% and the concentration of DNA attained 68.34 mg/g (calculated by VSS) after 48 h reaction, which were 29.01% and 59.63 mg/g higher than the control test, respectively, and were 24.86% and 53.39 mg/g higher than that with EPS removed in advance, respectively. Meanwhile, NH4+ -N, PO4(3-)-P and SCOD showed high dissolution efficiency, and the maximal concentrations achieved to 503 mg/L, 78.9 mg/L and 3171 mg/L, respectively. After removal of extracellular polymers, higher lysis efficiency was also observed by protease and cellulose, by which VSS reduction rate reached to 49.95% and 39.85%, respectively. The concentration of DNA showed a correlation coefficient of more than 0.9 with the concentrations of SCOD, NH4+ -N and PO4(3-)-P. And the highest hydrolysis rate obtained in 6 hours, which was about 3 hours earlier than the control test. Moreover, under those condition, sludge hydrolyzation could be well realized by only small amount of the enzyme addition.

Upregulation of circulating components of the alternative renin-angiotensin system in inflammatory bowel disease: A pilot study.

PubMed

Garg, Mayur; Burrell, Louise M; Velkoska, Elena; Griggs, Karen; Angus, Peter W; Gibson, Peter R; Lubel, John S

2015-09-01

The relationship between intestinal inflammation and circulating components of the renin-angiotensin system (RAS) is poorly understood. Demographic and clinical data were obtained from healthy controls and patients with inflammatory bowel disease (IBD). Plasma concentrations of the classical RAS components (angiotensin-converting enzyme (ACE) and angiotensin II (Ang II)) and alternative RAS components (ACE2 and angiotensin (1-7) (Ang (1-7))) were analysed by radioimmuno- and enzymatic assays. Systemic inflammation was assessed using serum C-reactive protein (CRP), white cell count, platelet count and albumin, and intestinal inflammation by faecal calprotectin. Nineteen healthy controls (11 female; mean age 38 years, range 23-68), 19 patients with Crohn's disease (11 female; aged 45 years, range 23-76) and 15 patients with ulcerative colitis (6 female; aged 42 years, 26-64) were studied. Circulating classical RAS component levels were similar across the three groups, whereas ACE2 activity and Ang (1-7) concentrations were higher in patients with IBD compared to controls (ACE2: 21.5 vs 13.3 pmol/ml/min, p<0.05; Ang (1-7): 22.8 vs 14.1 pg/ml, p<0.001). Ang (1-7) correlated weakly with platelet and white cell counts, but not calprotectin or CRP, in patients with IBD. Circulating components of the alternative RAS are increased in patients with IBD. © The Author(s) 2014.

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study.

PubMed

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger . This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger , we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

PubMed Central

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi. PMID:29623069

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation.

PubMed

Klingaman, Chase A; Wagner, Matthew J; Brown, Justin R; Klecker, John B; Pauley, Ethan H; Noldner, Colin J; Mays, Jared R

2017-01-01

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis-Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts. Copyright © 2016 Elsevier Inc. All rights reserved.

A rational workflow for sequential virtual screening of chemical libraries on searching for new tyrosinase inhibitors.

PubMed

Le-Thi-Thu, Huong; Casanola-Martín, Gerardo M; Marrero-Ponce, Yovani; Rescigno, Antonio; Abad, Concepcion; Khan, Mahmud Tareq Hassan

2014-01-01

The tyrosinase is a bifunctional, copper-containing enzyme widely distributed in the phylogenetic tree. This enzyme is involved in the production of melanin and some other pigments in humans, animals and plants, including skin pigmentations in mammals, and browning process in plants and vegetables. Therefore, enzyme inhibitors has been under the attention of the scientist community, due to its broad applications in food, cosmetic, agricultural and medicinal fields, to avoid the undesirable effects of abnormal melanin overproduction. However, the research of novel chemical with antityrosinase activity demands the use of more efficient tools to speed up the tyrosinase inhibitors discovery process. This chapter is focused in the different components of a predictive modeling workflow for the identification and prioritization of potential new compounds with activity against the tyrosinase enzyme. In this case, two structure chemical libraries Spectrum Collection and Drugbank are used in this attempt to combine different virtual screening data mining techniques, in a sequential manner helping to avoid the usually expensive and time consuming traditional methods. Some of the sequential steps summarize here comprise the use of drug-likeness filters, similarity searching, classification and potency QSAR multiclassifier systems, modeling molecular interactions systems, and similarity/diversity analysis. Finally, the methodologies showed here provide a rational workflow for virtual screening hit analysis and selection as a promissory drug discovery strategy for use in target identification phase.

Preparation of cellulase concoction using differential adsorption phenomenon.

PubMed

Birhade, Sachinkumar; Pednekar, Mukesh; Sagwal, Shilpa; Odaneth, Annamma; Lali, Arvind

2017-05-28

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37

PubMed Central

Bennici, Carmelo; Quatrini, Paola; Catania, Valentina; Mazzola, Salvatore; Ghersi, Giulio; Cuttitta, Angela

2015-01-01

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended. PMID:26162075

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Activity of components of the antioxidant system in the roots of potato plants at short-term temperature drop and invasion with parasitic nematodes.

PubMed

Lavrova, V V; Matveeva, E M; Zinovieva, S V

2017-09-01

The activity of catalase and superoxide dismutase in the roots of susceptible plants and plants exposed to alternating temperatures, which were infected with the phytoparasitic nematode G. rostochiensis, was studied. It was found that, throughout the invasion period, the plants susceptible to invasion exhibited a high activity of these enzymes, which allowed them to maintain an active defense against the oxidative stress caused by the invasion and subsequent life activity of larvae. For the plants exposed to alternating temperatures, a decrease in the activity of catalase and superoxide dismutase at the early stages of invasion and an increase in the activity of these enzymes at the later stages was detected.

Co-encapsulation of enzyme and sensitive dye as a tool for fabrication of microcapsule based sensor for urea measuring.

PubMed

Kazakova, Lyubov I; Shabarchina, Lyudmila I; Sukhorukov, Gleb B

2011-06-21

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARF-1 into polyelectrolyte multilayer capsules. Co-precipitation of calcium carbonate, urease and dextran followed up by multilayer film coating and Ca-extracting by EDTA resulted in the formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10(-6) to 10(-1) M. The presence of urea can be monitored on a single capsule level as illustrated by confocal fluorescent microscopy. Variations in urease:dye ratio in capsules, applicability and limits of use of that type multi-component microencapsulated sensors are discussed.

Production of digestive enzymes along the gut of the giant keyhole limpet Megathura crenulata (Mollusca: Vetigastropoda).

PubMed

Martin, Gary G; Martin, Alanna; Tsai, Whitney; Hafner, John C

2011-11-01

The esophagus and intestine form the longest regions of the digestive tract in the giant keyhole limpet and are lined by epithelial cells sharing a common morphology and releasing materials into the gut lumen by apocrine secretion. The purpose of this study was to determine if these morphologically similar regions release similar digestive enzymes and compare their contributions to digestive enzymes released from other regions of the gut. Principal component analysis of enzymes detected by the API ZYM system for 19 enzymes plus EnzChek assays for protease, α-amylase, lipase, cellulase, and lysozyme identify four distinct regions of the gut: 1) crystalline style and style sac, 2) digestive gland, 3) salivary glands, and 4) esophagus and intestine. Heterogeneity in enzymatic activity was observed in regions of the gut with similar cell morphology (middle and posterior esophagus and intestine) as well as regions with different cell morphology (salivary glands, digestive gland and crystalline style). Enzyme activity in each of these regions is compared to other gastropods, in particular the abalone. Although much of the length of the digestive tract is lined by a morphologically similar epithelium, different regions of the alimentary tract produce a different suite of enzymes which may contribute to the digestive process. These data will help enhance our limited understanding of the digestive physiology of Megathura crenulata and lead to improvement of its culture for clinical research. Copyright © 2011 Elsevier Inc. All rights reserved.

A cascade reaction network mimicking the basic functional steps of adaptive immune response

NASA Astrophysics Data System (ADS)

Han, Da; Wu, Cuichen; You, Mingxu; Zhang, Tao; Wan, Shuo; Chen, Tao; Qiu, Liping; Zheng, Zheng; Liang, Hao; Tan, Weihong

2015-10-01

Biological systems use complex ‘information-processing cores’ composed of molecular networks to coordinate their external environment and internal states. An example of this is the acquired, or adaptive, immune system (AIS), which is composed of both humoral and cell-mediated components. Here we report the step-by-step construction of a prototype mimic of the AIS that we call an adaptive immune response simulator (AIRS). DNA and enzymes are used as simple artificial analogues of the components of the AIS to create a system that responds to specific molecular stimuli in vitro. We show that this network of reactions can function in a manner that is superficially similar to the most basic responses of the vertebrate AIS, including reaction sequences that mimic both humoral and cellular responses. As such, AIRS provides guidelines for the design and engineering of artificial reaction networks and molecular devices.

Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.

PubMed

Banerjee, Goutami; Car, Suzana; Scott-Craig, John S; Borrusch, Melissa S; Walton, Jonathan D

2010-10-12

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h. The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

The enzymic hydrolysis of amygdalin

PubMed Central

Haisman, D. R.; Knight, D. J.

1967-01-01

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond β-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the β-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. PMID:4291788

Regulation of Ubiquitin Enzymes in the TGF-β Pathway.

PubMed

Iyengar, Prasanna Vasudevan

2017-04-20

The transforming growth factor-β (TGF-β) pathway has a tumor suppressor role in normal and premalignant cells but promotes oncogenesis in advanced cancer cells. Components of the pathway are tightly controlled by ubiquitin modifying enzymes and aberrations in these enzymes are frequently observed to dysregulate the pathway causing diseases such as bone disorders, cancer and metastasis. These enzymes and their counterparts are increasingly being tested as druggable targets, and thus a deeper understanding of the enzymes is required. This review summarizes the roles of specific ubiquitin modifying enzymes in the TGF-β pathway and how they are regulated.

A kinetic study on the Novozyme 435-catalyzed esterification of free fatty acids with octanol to produce octyl esters.

PubMed

Chowdhury, Avisha; Mitra, Debarati

2015-01-01

Octyl esters can serve as an important class of biolubricant components replacing their mineral oil counterparts. The purpose of the current work was to investigate the enzymatic esterification reaction of free fatty acids (FFA, from waste cooking oil) with octanol in a solvent-free system using a commercial lipase Novozyme 435. It was found that the esterificaton reaction followed the Ping-pong bi-bi kinetics with no inhibition by substrates or products within the studied concentration range. The maximum reaction rate was estimated to be 0.041 mol L(-1) g(-1) h(-1) . Additionally, the stability of Novozyme 435 in the current reaction system was studied by determining its activity and final conversion of FFA to esters after 12 successive utilizations. Novozyme 435 exhibited almost 100% enzyme activity up to 7 cycles of reaction and gradually decreased (by 5%) thereafter. The kinetic parameters evaluated from the study shall assist in the design of reactors for large-scale production of octyl esters from a cheap biomass source. The enzyme reusability data can further facilitate mass production by curtailing the cost of expensive enzyme consumption. © 2015 American Institute of Chemical Engineers.

Ancient Venom Systems: A Review on Cnidaria Toxins

PubMed Central

Jouiaei, Mahdokht; Yanagihara, Angel A.; Madio, Bruno; Nevalainen, Timo J.; Alewood, Paul F.; Fry, Bryan G.

2015-01-01

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or “venom” that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design. PMID:26094698

Ancient Venom Systems: A Review on Cnidaria Toxins.

PubMed

Jouiaei, Mahdokht; Yanagihara, Angel A; Madio, Bruno; Nevalainen, Timo J; Alewood, Paul F; Fry, Bryan G

2015-06-18

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or "venom" that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

Molecular cloning and nucleotide sequences of the genes for two essential proteins constituting a novel enzyme system for heptaprenyl diphosphate synthesis.

PubMed

Koike-Takeshita, A; Koyama, T; Obata, S; Ogura, K

1995-08-04

The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexparenyl-diphosphate:isopentenyl-diphosphate hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and their nucleotide sequences were determined. Sequence analyses revealed the presence of three open reading frames within 2,350 base pairs, designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence, which encode proteins of 220, 234, and 323 amino acids, respectively. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not essential. As a result, this enzyme was proved genetically to consist of two different protein compounds with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. The protein encoded by ORF-1 has no similarity to any protein so far registered. However, the protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363).

Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling

PubMed Central

Burroughs, A. Maxwell; Zhang, Dapeng; Schäffer, Daniel E.; Iyer, Lakshminarayan M.; Aravind, L.

2015-01-01

Cyclic di- and linear oligo-nucleotide signals activate defenses against invasive nucleic acids in animal immunity; however, their evolutionary antecedents are poorly understood. Using comparative genomics, sequence and structure analysis, we uncovered a vast network of systems defined by conserved prokaryotic gene-neighborhoods, which encode enzymes generating such nucleotides or alternatively processing them to yield potential signaling molecules. The nucleotide-generating enzymes include several clades of the DNA-polymerase β-like superfamily (including Vibrio cholerae DncV), a minimal version of the CRISPR polymerase and DisA-like cyclic-di-AMP synthetases. Nucleotide-binding/processing domains include TIR domains and members of a superfamily prototyped by Smf/DprA proteins and base (cytokinin)-releasing LOG enzymes. They are combined in conserved gene-neighborhoods with genes for a plethora of protein superfamilies, which we predict to function as nucleotide-sensors and effectors targeting nucleic acids, proteins or membranes (pore-forming agents). These systems are sometimes combined with other biological conflict-systems such as restriction-modification and CRISPR/Cas. Interestingly, several are coupled in mutually exclusive neighborhoods with either a prokaryotic ubiquitin-system or a HORMA domain-PCH2-like AAA+ ATPase dyad. The latter are potential precursors of equivalent proteins in eukaryotic chromosome dynamics. Further, components from these nucleotide-centric systems have been utilized in several other systems including a novel diversity-generating system with a reverse transcriptase. We also found the Smf/DprA/LOG domain from these systems to be recruited as a predicted nucleotide-binding domain in eukaryotic TRPM channels. These findings point to evolutionary and mechanistic links, which bring together CRISPR/Cas, animal interferon-induced immunity, and several other systems that combine nucleic-acid-sensing and nucleotide-dependent signaling. PMID:26590262

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase*

PubMed Central

Omi, Rie; Kurokawa, Suguru; Mihara, Hisaaki; Hayashi, Hideyuki; Goto, Masaru; Miyahara, Ikuko; Kurihara, Tatsuo; Hirotsu, Ken; Esaki, Nobuyoshi

2010-01-01

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system. PMID:20164179

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

PubMed

Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

2015-01-01

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

Manganese Superoxide Dismutase: Guardian of the Powerhouse

PubMed Central

Holley, Aaron K.; Bakthavatchalu, Vasudevan; Velez-Roman, Joyce M.; St. Clair, Daret K.

2011-01-01

The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component. PMID:22072939

Biomedical engineering strategies in system design space.

PubMed

Savageau, Michael A

2011-04-01

Modern systems biology and synthetic bioengineering face two major challenges in relating properties of the genetic components of a natural or engineered system to its integrated behavior. The first is the fundamental unsolved problem of relating the digital representation of the genotype to the analog representation of the parameters for the molecular components. For example, knowing the DNA sequence does not allow one to determine the kinetic parameters of an enzyme. The second is the fundamental unsolved problem of relating the parameters of the components and the environment to the phenotype of the global system. For example, knowing the parameters does not tell one how many qualitatively distinct phenotypes are in the organism's repertoire or the relative fitness of the phenotypes in different environments. These also are challenges for biomedical engineers as they attempt to develop therapeutic strategies to treat pathology or to redirect normal cellular functions for biotechnological purposes. In this article, the second of these fundamental challenges will be addressed, and the notion of a "system design space" for relating the parameter space of components to the phenotype space of bioengineering systems will be focused upon. First, the concept of a system design space will be motivated by introducing one of its key components from an intuitive perspective. Second, a simple linear example will be used to illustrate a generic method for constructing the design space in which qualitatively distinct phenotypes can be identified and counted, their fitness analyzed and compared, and their tolerance to change measured. Third, two examples of nonlinear systems from different areas of biomedical engineering will be presented. Finally, after giving reference to a few other applications that have made use of the system design space approach to reveal important design principles, some concluding remarks concerning challenges and opportunities for further development will be made.

A Two-Component Monooxygenase Catalyzes Both the Hydroxylation of p-Nitrophenol and the Oxidative Release of Nitrite from 4-Nitrocatechol in Bacillus sphaericus JS905

PubMed Central

Kadiyala, Venkateswarlu; Spain, Jim C.

1998-01-01

Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2,6-dichlorophenolindophenol and nitroblue tetrazolium. Nitrite release from either PNP or 4-nitrocatechol was inhibited by the flavoprotein inhibitor methimazole. Our results indicate that the two monooxygenations of PNP to THB are catalyzed by a single two-component enzyme system comprising a flavoprotein reductase and an oxygenase. PMID:9647818

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

PubMed

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.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

NASA Astrophysics Data System (ADS)

Radhakrishnan, Krishna; Thomas, Midhun B.; Pulakkat, Sreeranjini; Gnanadhas, Divya P.; Chakravortty, Dipshikha; Raichur, Ashok M.

2015-08-01

Enzyme- and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 ± 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Degradative pathways for p-toluenecarboxylate and p-toluenesulfonate and their multicomponent oxygenases in Comamonas testosteroni strains PSB-4 and T-2.

PubMed

Junker, F; Saller, E; Schläfli Oppenberg, H R; Kroneck, P M; Leisinger, T; Cook, A M

1996-09-01

Three multicomponent oxygenases involved in the degradation of p-toluenesulfonate and p-toluenecarboxylate and the regulation of their synthesis have been examined in three strains (T-2, PSB-4 and TER-1) of Comamonas testosteroni. Strain T-2 utilizes p-toluenesulfonate as a source of carbon and energy for growth via p-sulfobenzoate and protocatechuate, and p-toluenecarboxylate via terephthalate and protocatechuate, and has the unusual property of requiring the reductase (TsaB) of the toluenesulfonate methyl monooxygenase system (TsaMB) in an incompletely expressed sulfobenzoate dioxygenase system (PsbAC) [Schläfli Oppenberg, H.R., Chen, G., Leisinger, T. & Cook, A. M. (1995). Microbiology 141, 1891-1899]. The independently isolated C. testosteroni PSB-4 utilized only sulfobenzoate and terephthalate via protocatechuate. Mutant TER-1, derived from strain T-2, utilized only terephthalate via protocatechuate. We detected no enzymes of the pathway from toluenesulfonate to sulfobenzoate in strains PSB-4 and TER-1, and confirmed by PCR and Southern blot analysis that the genes (tsaMB) encoding toluenesulfonate monooxygenase were absent. We concluded that, in strain PSB-4, the regulatory unit encoding the genes for the conversion of toluenesulfonate to sulfobenzoate was missing, and that generation of mutant TER-1 involved deletion of this regulatory unit and of the regulatory unit encoding desulfonation of sulfobenzoate. The degradation of sulfobenzoate in strain PSB-4 was catalysed by a fully inducible sulfobenzoate dioxygenase system (PsbACPSB-4), which, after purification of the oxygenase component (PsbAPSB-4), turned out to be indistinguishable from the corresponding component from strain T-2 (PsbAT-2). Reductase PsbCPSB-4, which we could separate but not purify, was active with oxygenase PsbAPSB-4 and PsbAT-2. Oxygenase PsbAPSB-4 was shown by electron paramagnetic resonance spectroscopy to contain a Rieske [2Fe-2S] centre. The enzyme system oxygenating terephthalate was examined and the oxygenase component purified and characterized. The oxygenase component in strains T-2 (and mutant TER-1) and PSB-4 were indistinguishable. The reductase component, which we separated but failed to purify, was active with the oxygenase from all strains. Gains and losses of blocks of genes in evolution is discussed.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components.

PubMed

Fagerlund, Robert D; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2015-09-01

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA-RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P. © 2015 Fagerlund et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Phosphodiesterase from Daboia russelli russelli venom: purification, partial characterization and inhibition of platelet aggregation.

PubMed

Mitra, Jyotirmoy; Bhattacharyya, Debasish

2014-09-01

Phosphodiesterases (PDEs) belong to a super-family of enzymes that have multiple roles in the metabolism of extracellular nucleotides and regulation of nucleotide-based intercellular signalling. A PDE from Russell's viper (Daboia russelli russelli) venom (DR-PDE) was purified by gel filtration, ion exchange and affinity chromatographies. Homogeneity of the preparation was verified by SDS-PAGE, SE-HPLC and mass spectrometry. It was free from 5'-nucleotidase, alkaline phosphatase and protease activities. Identity of the enzyme was ensured from partial sequence homology with other PDEs. DR-PDE was inactivated by polyvalent anti-venom serum and metal chelators. The enzyme was partially inhibited by the root extracts of four medicinal plants but remained unaffected by inhibitors of intracellular PDEs. DR-PDE hydrolyses ADP and thus, strongly inhibits ADP-induced platelet aggregation in human platelet rich plasma. This study leads to better understanding of a component of Russell's viper venom that affects homoeostatic system of the victim. Copyright © 2014 Elsevier Ltd. All rights reserved.

The structure and function of Alzheimer's gamma secretase enzyme complex.

PubMed

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.

Genetics Home Reference: multicentric osteolysis, nodulosis, and arthropathy

MedlinePlus

... to cut (cleave) a protein called type IV collagen. Type IV collagen is a major structural component of basement membranes, ... enzyme, preventing the normal cleavage of type IV collagen. It is unclear how a loss of enzyme ...

Specialized Genetic Recombination Systems in Bacteria: Their Involvement in Gene Expression and Evolution,

DTIC Science & Technology

1980-01-01

genetics (Hayes 1968). This marvelous process is important in providing us with the breadth of phenotypic diversity that one sees within a single plant or...separate overall pro- cesses, but may share common components of DNA metabolism, such as winding/unwinding enzymes, ligase, polymerases , various nucle...incorpuoted DNA segmnent are re- paired by DNA polymerase and ligase. Any diffoernces (base mispairing’S, nil- cleotide additions or deletions) between

Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

PubMed

Kim, Youngmi; Ximenes, Eduardo; Mosier, Nathan S; Ladisch, Michael R

2011-04-07

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion. Copyright © 2011 Elsevier Inc. All rights reserved.

A definite bite by the Ornamental Snake (Denisonia maculata) causing mild envenoming.

PubMed

Isbister, Geoffrey K; Gault, Alan; Tasoulis, Theo; O'Leary, Margaret A

2016-03-01

Many bites from mildly venomous elapids occur but identification or presence of systemic envenoming is rarely confirmed. To confirm systemic envenoming and binding of venom components to a commercial antivenom in a definite bite by the Ornamental Snake (Denisonia maculata) using enzyme immunoassays. A 9-year old boy was bitten by an identified Ornamental Snake. He developed nausea, vomiting, local pain, and swelling. He had a leucocytosis (white cell count, 20.8 × 10(9)/L), an elevated international normalised ratio (INR) of 1.6, but otherwise normal blood tests including D-Dimer and activated partial thromboplastin time. He was treated with Australian Black Snake antivenom because the commercial venom detection kit was positive for Black snake. He was admitted for 36 h with continuing local pain and swelling requiring parenteral analgesia. Blood samples were collected with informed consent for measurement of venom and antivenom concentrations. Venom-specific enzyme immunoassays were developed using the closely related D. devisi venom with Rabbit anti-Notechis (Tiger Snake) and anti-Tropidechis (Rough-scaled Snake) IgG antibodies to detect venom in serum. Standard curves for measured venom versus actual venom concentrations were made to interpolate Denisonia venom concentrations. In vitro procoagulant and anticoagulant activity of venom was assayed. Denisonia venom was detected in the pre-antivenom sample as 9.6 ng/mL D. devisi venom. No antigenic venom components were detected in post-antivenom samples and there were high antivenom concentrations. D. devisi venom had mild in vitro procoagulant activity with a minimum concentration required to clot after 5 min of 2.5-5 μg/mL and even weaker anticoagulant activity. Denisonia bites appear to cause local effects and possibly mild systemic envenoming (with only non-specific systemic symptoms and leucocytosis), confirmed by detection of antigenic venom components in blood. A significant coagulopathy does not appear to occur.

OCAM - A CELSS modeling tool: Description and results. [Object-oriented Controlled Ecological Life Support System Analysis and Modeling

NASA Technical Reports Server (NTRS)

Drysdale, Alan; Thomas, Mark; Fresa, Mark; Wheeler, Ray

1992-01-01

Controlled Ecological Life Support System (CELSS) technology is critical to the Space Exploration Initiative. NASA's Kennedy Space Center has been performing CELSS research for several years, developing data related to CELSS design. We have developed OCAM (Object-oriented CELSS Analysis and Modeling), a CELSS modeling tool, and have used this tool to evaluate CELSS concepts, using this data. In using OCAM, a CELSS is broken down into components, and each component is modeled as a combination of containers, converters, and gates which store, process, and exchange carbon, hydrogen, and oxygen on a daily basis. Multiple crops and plant types can be simulated. Resource recovery options modeled include combustion, leaching, enzyme treatment, aerobic or anaerobic digestion, and mushroom and fish growth. Results include printouts and time-history graphs of total system mass, biomass, carbon dioxide, and oxygen quantities; energy consumption; and manpower requirements. The contributions of mass, energy, and manpower to system cost have been analyzed to compare configurations and determine appropriate research directions.

Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

NASA Technical Reports Server (NTRS)

Reinecke, D. M.; Bandurski, R. S.

1988-01-01

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

Linear and Branched PEIs (Polyethylenimines) and Their Property Space.

PubMed

Lungu, Claudiu N; Diudea, Mircea V; Putz, Mihai V; Grudziński, Ireneusz P

2016-04-13

A chemical property space defines the adaptability of a molecule to changing conditions and its interaction with other molecular systems determining a pharmacological response. Within a congeneric molecular series (compounds with the same derivatization algorithm and thus the same brute formula) the chemical properties vary in a monotonic manner, i.e., congeneric compounds share the same chemical property space. The chemical property space is a key component in molecular design, where some building blocks are functionalized, i.e., derivatized, and eventually self-assembled in more complex systems, such as enzyme-ligand systems, of which (physico-chemical) properties/bioactivity may be predicted by QSPR/QSAR (quantitative structure-property/activity relationship) studies. The system structure is determined by the binding type (temporal/permanent; electrostatic/covalent) and is reflected in its local electronic (and/or magnetic) properties. Such nano-systems play the role of molecular devices, important in nano-medicine. In the present article, the behavior of polyethylenimine (PEI) macromolecules (linear LPEI and branched BPEI, respectively) with respect to the glucose oxidase enzyme GOx is described in terms of their (interacting) energy, geometry and topology, in an attempt to find the best shape and size of PEIs to be useful for a chosen (nanochemistry) purpose.

Linear and Branched PEIs (Polyethylenimines) and Their Property Space

PubMed Central

Lungu, Claudiu N.; Diudea, Mircea V.; Putz, Mihai V.; Grudziński, Ireneusz P.

2016-01-01

A chemical property space defines the adaptability of a molecule to changing conditions and its interaction with other molecular systems determining a pharmacological response. Within a congeneric molecular series (compounds with the same derivatization algorithm and thus the same brute formula) the chemical properties vary in a monotonic manner, i.e., congeneric compounds share the same chemical property space. The chemical property space is a key component in molecular design, where some building blocks are functionalized, i.e., derivatized, and eventually self-assembled in more complex systems, such as enzyme-ligand systems, of which (physico-chemical) properties/bioactivity may be predicted by QSPR/QSAR (quantitative structure-property/activity relationship) studies. The system structure is determined by the binding type (temporal/permanent; electrostatic/covalent) and is reflected in its local electronic (and/or magnetic) properties. Such nano-systems play the role of molecular devices, important in nano-medicine. In the present article, the behavior of polyethylenimine (PEI) macromolecules (linear LPEI and branched BPEI, respectively) with respect to the glucose oxidase enzyme GOx is described in terms of their (interacting) energy, geometry and topology, in an attempt to find the best shape and size of PEIs to be useful for a chosen (nanochemistry) purpose. PMID:27089324

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

PubMed

Haroune, Lounès; Saibi, Sabrina; Cabana, Hubert; Bellenger, Jean-Philippe

2017-01-17

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Toying with Enzyme Catalysis.

ERIC Educational Resources Information Center

Richards, Debbie

1998-01-01

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

Expression of classical components of the renin-angiotensin system in the human eye.

PubMed

White, Andrew J R; Cheruvu, Sarat C; Sarris, Maria; Liyanage, Surabhi S; Lumbers, Eugenie; Chui, Jeanie; Wakefield, Denis; McCluskey, Peter J

2015-03-01

The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions. © The Author(s) 2014.

Screening method for inhibitors against formosan subterranean termite beta-glucosidases in vivo.

PubMed

Zhu, Betty C R; Henderson, Gregg; Laine, Roger A

2005-02-01

Cellulose, a main structural constituent of plants, is the major nutritional component for wood-feeding termites. Enzymatic hydrolysis of cellulose to glucose occurs by the action of cellulases, a mixture of the three major classes of enzymes including endo-1,4-beta-glucanases, exo-1,4-beta-glucanases, and beta-glucosidase. Lower termites, such as the Formosan subterranean termite, Coptotermes formosanus Shiraki, require cellulolytic protozoa to efficiently digest cellulose for survival. Inhibitors developed against any of these cellulase system enzymes would be a potential termite treatment avenue. Our effort was to develop a screening system to determine whether termites could be controlled by administration of cellulase system inhibitors. Some reported compounds such as gluconolactone, conduritol B epoxide, and 1-deoxynojirimycin are potential beta-glucosidase inhibitors, but they have only been tested in vitro. We describe an in vivo method to test the inhibitory ability of the designated chemicals to act on beta-1,4-glucosidases, one member of the cellulase system that is the key step that releases glucose for use as an energy and carbon source for termites. Inhibition in releasing glucose from cellooligosaccharides might be sufficient to starve termites. Fluorescein di-beta-D-glucopyranoside was used as the artificial enzyme substrate and the fluorescent intensity of the reaction product (fluorescein) quantified with an automated fluorescence plate reader. Several known in vitro beta-1,4-glucosidase inhibitors were tested in vivo, and their inhibitory potential was determined. Endogenous and protozoan cellulase activities are both assumed to play a role.

Purification and characterization of an endoglucanase from the marine rotifer, Brachionus plicatilis.

PubMed

Chun, C Z; Hur, S B; Kim, Y T

1997-10-01

The marine rotifer, Brachionus plicatilis, is able to digest Chlorella efficiently, suggesting that the rotifer contains a powerful cellulolytic enzyme system. A multi-component cellulolytic complex, including endoglucanase (CM-cellulase), cellobiohydrolase and beta-glucosidase, was found in Brachionus plicatilis. Endoglucanase (endo-beta-1,4 glucanase) was purified to homogeneity from rotifer homogenates using a sequential chromatographic method. The purified enzyme exhibits a strong hydrolytic activity with carboxymethyl(CM)-cellulose. The optimum temperature and pH for the endoglucanase activity were 37 degrees C and 7.0, respectively. 80% of the CM-cellulase activity was retained in salt mixture that ranged from 150 to 500 mM NaCl equivalent. The purified protein was isolated with a molecular weight of approximately 62 kDa estimated by SDS-polyacrylamide gel electrophoresis.

Allosteric Inhibition of the Neuropeptidase Neurolysin*

PubMed Central

Hines, Christina S.; Ray, Kallol; Schmidt, Jack J.; Xiong, Fei; Feenstra, Rolf W.; Pras-Raves, Mia; de Moes, Jan Peter; Lange, Jos H. M.; Melikishvili, Manana; Fried, Michael G.; Mortenson, Paul; Charlton, Michael; Patel, Yogendra; Courtney, Stephen M.; Kruse, Chris G.; Rodgers, David W.

2014-01-01

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases. PMID:25378390

Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

PubMed

Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

2015-04-14

The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of a soil enzyme on iron-cyanide complex speciation and mineral adsorption.

PubMed

Zimmerman, Andrew R; Kang, Dong-Hee; Ahn, Mi-Youn; Hyun, Seunghun; Banks, M Katherine

2008-01-01

Cyanide is commonly found as ferrocyanide [Fe(II)(CN)(6)](-4) and in the more mobile form, ferricyanide [Fe(III)(CN)(6)](-3) in contaminated soils and sediments. Although soil minerals may influence ferrocyanide speciation, and thus mobility, the possible influence of soil enzymes has not been examined. In a series of experiments conducted under a range of soil-like conditions, laccase, a phenoloxidase enzyme derived from the fungi Trametes versicolor, was found to exert a large influence on iron-cyanide speciation and mobility. In the presence of laccase, up to 93% of ferrocyanide (36-362ppm) was oxidized to ferricyanide within 4h. No significant effect of pH (3.6 and 6.2) or initial ferrocyanide concentration on the extent or rate of oxidation was found and ferrocyanide oxidation did not occur in the absence of laccase. Relative to iron-cyanide-mineral systems without laccase, ferrocyanide adsorption to aluminum hydroxide and montmorillonite decreased in the presence of laccase and was similar to or somewhat greater than that of ferricyanide without laccase. Laccase-catalyzed conversion of ferrocyanide to ferricyanide was extensive though up to 33% of the enzyme was mineral-bound. These results demonstrate that soil enzymes can play a major role in ferrocyanide speciation and mobility. Biotic soil components must be considered as highly effective oxidation catalysts that may alter the mobility of metals and metal complexes in soil. Immobilized enzymes should also be considered for use in soil metal remediation efforts.

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

PubMed

Lam, Charlton; Jamerson, Melissa; Cabral, Guy; Carlesso, Ana Maris; Marciano-Cabral, Francine

2017-10-01

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

NASA Astrophysics Data System (ADS)

Venditti, Vincenzo; Tugarinov, Vitali; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius

2015-01-01

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

[The genetic determination and function of RR-proteins--the regulators of photoperiodic reaction and circadian rhythms in plants].

PubMed

Tots'kyĭ, V M; D'iachenko, L F; Muterko, O F; Balashova, I A; Toptikov, V A

2012-01-01

The present review devoted to the analysis of recent literature on genetic determination and the domain organization of the newly discovered two-component signaling systems in pro- and eukaryotes. These structures are involved in the regulation of numerous morphological and physiological processes in plants. RR-proteins, it the key elements of signaling systems, they launch a cascade of phosphotransferase reactions and directly or indirectly regulate the transcription and activity other proteins, including enzymes, in response to hormones or environmental factors. Modern views on the molecular and genetic mechanisms of photoperiodic response, circadian rhythms and anti-stress responses in plants are set out in these positions. The relationship between gene expression and photoreceptor sensitivity of plants to photoperiod traced. We present our own data obtained on the isogenic lines of wheat, where been showed dependence expression of structural genes of enzymes on the allelic composition of individual PRR-loci and the duration action of low temperature.

Elucidating light-induced charge accumulation in an artificial analogue of methane monooxygenase enzymes using time-resolved X-ray absorption spectroscopy

DOE PAGES

Moonshiram, Dooshaye; Picon, Antonio; Vazquez-Mayagoitia, Alvaro; ...

2017-02-08

Here, we report the use of time-resolved X-ray absorption spectroscopy in the ns–μs time scale to track the light induced two electron transfer processes in a multi-component photocatalytic system, consisting of [Ru(bpy) 3] 2+/ a diiron(III,III) model/triethylamine. EXAFS analysis with DFT calculations confirms the structural configurations of the diiron(III,III) and reduced diiron(II,II) states.

Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes

Treesearch

Claudia Cano-Ramirez; Maria Fernanda Lopez; Ana K. Cesar-Ayala; Veronica Pineda-Martinez; Brian T. Sullivan; Gerardo and Zuniga

2013-01-01

Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i....

Elucidating light-induced charge accumulation in an artificial analogue of methane monooxygenase enzymes using time-resolved X-ray absorption spectroscopy

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

Moonshiram, Dooshaye; Picon, Antonio; Vazquez-Mayagoitia, Alvaro

Here, we report the use of time-resolved X-ray absorption spectroscopy in the ns–μs time scale to track the light induced two electron transfer processes in a multi-component photocatalytic system, consisting of [Ru(bpy) 3] 2+/ a diiron(III,III) model/triethylamine. EXAFS analysis with DFT calculations confirms the structural configurations of the diiron(III,III) and reduced diiron(II,II) states.

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas.

PubMed

Chamizo-Ampudia, Alejandro; Sanz-Luque, Emanuel; Llamas, Ángel; Ocaña-Calahorro, Francisco; Mariscal, Vicente; Carreras, Alfonso; Barroso, Juan B; Galván, Aurora; Fernández, Emilio

2016-10-01

Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. N\\R and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it. © 2016 John Wiley & Sons Ltd.

Physiological Response of Plants Grown on Porous Ceramic Tubes

NASA Technical Reports Server (NTRS)

Tsao, David; Okos, Martin

1997-01-01

This research involves the manipulation of the root-zone water potential for the purposes of discriminating the rate limiting step in the inorganic nutrient uptake mechanism utilized by higher plants. This reaction sequence includes the pathways controlled by the root-zone conditions such as water tension and gradient concentrations. Furthermore, plant based control mechanisms dictated by various protein productions are differentiated as well. For the nutrients limited by the environmental availability, the kinetics were modeled using convection and diffusion equations. Alternatively, for the nutrients dependent upon enzyme manipulations, the uptakes are modeled using Michaelis-Menten kinetics. In order to differentiate between these various mechanistic steps, an experimental apparatus known as the Porous Ceramic Tube - Nutrient Delivery System (PCT-NDS) was used. Manipulation of the applied suction pressure circulating a nutrient solution through this system imposes a change in the matric component of the water potential. This compensates for the different osmotic components of water potential dictated by nutrient concentration. By maintaining this control over the root-zone conditions, the rate limiting steps in the uptake of the essential nutrients into tomato plants (Lycopersicon esculentum cv. Cherry Elite) were differentiated. Results showed that the uptake of some nutrients were mass transfer limited while others were limited by the enzyme kinetics. Each of these were adequately modeled with calculations and discussions of the parameter estimations provided.

An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

PubMed

Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

2014-01-01

Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

Transporter-Enzyme Interplay: Deconvoluting Effects of Hepatic Transporters and Enzymes on Drug Disposition Using Static and Dynamic Mechanistic Models.

PubMed

Varma, Manthena V; El-Kattan, Ayman F

2016-07-01

A large body of evidence suggests hepatic uptake transporters, organic anion-transporting polypeptides (OATPs), are of high clinical relevance in determining the pharmacokinetics of substrate drugs, based on which recent regulatory guidances to industry recommend appropriate assessment of investigational drugs for the potential drug interactions. We recently proposed an extended clearance classification system (ECCS) framework in which the systemic clearance of class 1B and 3B drugs is likely determined by hepatic uptake. The ECCS framework therefore predicts the possibility of drug-drug interactions (DDIs) involving OATPs and the effects of genetic variants of SLCO1B1 early in the discovery and facilitates decision making in the candidate selection and progression. Although OATP-mediated uptake is often the rate-determining process in the hepatic clearance of substrate drugs, metabolic and/or biliary components also contribute to the overall hepatic disposition and, more importantly, to liver exposure. Clinical evidence suggests that alteration in biliary efflux transport or metabolic enzymes associated with genetic polymorphism leads to change in the pharmacodynamic response of statins, for which the pharmacological target resides in the liver. Perpetrator drugs may show inhibitory and/or induction effects on transporters and enzymes simultaneously. It is therefore important to adopt models that frame these multiple processes in a mechanistic sense for quantitative DDI predictions and to deconvolute the effects of individual processes on the plasma and hepatic exposure. In vitro data-informed mechanistic static and physiologically based pharmacokinetic models are proven useful in rationalizing and predicting transporter-mediated DDIs and the complex DDIs involving transporter-enzyme interplay. © 2016, The American College of Clinical Pharmacology.

Reusable glucose sensing using carbon nanotube-based self-assembly

NASA Astrophysics Data System (ADS)

Bhattacharyya, Tamoghna; Samaddar, Sarbani; Dasgupta, Anjan Kr.

2013-09-01

Lipid functionalized single walled carbon nanotube-based self assembly forms a super-micellar structure. This assemblage has been exploited to trap glucose oxidase in a molecular cargo for glucose sensing. The advantage of such a molecular trap is that all components of this unique structure (both the trapping shell and the entrapped enzyme) are reusable and rechargeable. The unique feature of this sensing method lies in the solid state functionalization of single walled carbon nanotubes that facilitates liquid state immobilization of the enzyme. The method can be used for soft-immobilization (a new paradigm in enzyme immobilization) of enzymes with better thermostability that is imparted by the strong hydrophobic environment provided through encapsulation by the nanotubes.Lipid functionalized single walled carbon nanotube-based self assembly forms a super-micellar structure. This assemblage has been exploited to trap glucose oxidase in a molecular cargo for glucose sensing. The advantage of such a molecular trap is that all components of this unique structure (both the trapping shell and the entrapped enzyme) are reusable and rechargeable. The unique feature of this sensing method lies in the solid state functionalization of single walled carbon nanotubes that facilitates liquid state immobilization of the enzyme. The method can be used for soft-immobilization (a new paradigm in enzyme immobilization) of enzymes with better thermostability that is imparted by the strong hydrophobic environment provided through encapsulation by the nanotubes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02609d

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

PubMed Central

2013-01-01

Background Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. Results Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. Conclusions The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production. PMID:24341331

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

PubMed

Kumar, Narendra; Skolnick, Jeffrey

2012-10-15

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

Inhibition of the ubiquitin-proteasome system by natural products for cancer therapy.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2010-08-01

The ubiquitin-proteasome system plays a critical role in selective protein degradation and regulates almost all cellular events such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein quality control, development, and neuronal function. The recent approval of bortezomib, a synthetic proteasome inhibitor, for the treatment of relapsed multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and ubiquitinating and deubiquitinating enzymes as well as the delivery system. To date, various synthetic and natural products have been reported to inhibit the components of the ubiquitin-proteasome system. Here, we review natural products targeting the ubiquitin-proteasome system as well as synthetic compounds with potent inhibitory effects. Georg Thieme Verlag KG Stuttgart-New York.

The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea

PubMed Central

Brand, Philipp; Lin, Wei; Johnson, Brian R.

2018-01-01

Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components. PMID:29588379

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

PubMed

Bindon, Keren A; Li, Sijing; Kassara, Stella; Smith, Paul A

2016-11-09

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication

PubMed Central

Soares, Flávia V.; de Castro, Alexandre A.; Pereira, Ander F.; Leal, Daniel H. S.; Mancini, Daiana T.; da Cunha, Elaine F. F.; Kuca, Kamil

2018-01-01

Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways. PMID:29690585

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication.

PubMed

Soares, Flávia V; de Castro, Alexandre A; Pereira, Ander F; Leal, Daniel H S; Mancini, Daiana T; Krejcar, Ondrej; Ramalho, Teodorico C; da Cunha, Elaine F F; Kuca, Kamil

2018-04-23

Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways.

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Towards an Understanding of How Protein Hydrolysates Stimulate More Efficient Biosynthesis in Cultured Cells

NASA Astrophysics Data System (ADS)

Siemensma, André; Babcock, James; Wilcox, Chris; Huttinga, Hans

In the light of the growing demand for high quality plant-derived hydrolysates (i.e., HyPep™ and UltraPep™ series), Sheffield Bio-Science has developed a new hydrolysate platform that addresses the need for animal-free cell culture medium supplements while also minimizing variability concerns. The platform is based upon a novel approach to enzymatic digestion and more refined processing. At the heart of the platform is a rationally designed animal component-free (ACF) enzyme cocktail that includes both proteases and non-proteolytic enzymes (hydrolases) whose activities can also liberate primary components of the polymerized non-protein portion of the raw material. This enzyme system is added during a highly optimized process step that targets specific enzyme-substrate reactions to expand the range of beneficial nutritional factors made available to cells in culture. Such factors are fundamental to improving the bio-performance of the culture system, as they provide not merely growth-promoting peptides and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that improve both rate and quality of protein expression, and serve to improve culture life due to osmo-protectant and anti-apoptotic properties. Also of significant note is that, compared to typical hydrolysates, the production process is greatly reduced and requires fewer steps, intrinsically yielding a better-controlled and therefore more reproducible product. Finally, the more sophisticated approach to enzymatic digestion renders hydrolysates more amenable to sterile filtration, allowing hydrolysate end users to experience streamlined media preparation and bioreactor supplementation activities. Current and future development activities will evolve from a better understanding of the complex interactions within a handful of key biochemical pathways that impact the growth and productivity of industrially relevant organisms. Presented in this chapter are some examples of the efforts that have been made so far to elucidate the mechanisms for the often dramatic benefits that hydrolysates can impart on cell culture processes. Given the variety of roles that hydrolysates likely play in each cell type, close collaboration between protein hydrolysate manufacturers and biopharmaceutical developers will continue to be critical to expanding the industry's knowledge and retaining hydrolysates as a tool for enhancing media formulations.

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

PubMed Central

Roh, Kyung-Baeg; Kim, Chan-Hee; Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Ryu, Ji-Hwan; Kurokawa, Kenji; Ha, Nam-Chul; Lee, Won-Jae; Lemaitre, Bruno; Söderhäll, Kenneth; Lee, Bok-Luel

2009-01-01

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. PMID:19473968

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

PubMed Central

Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

2016-01-01

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. PMID:27316952

Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

PubMed Central

Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S.; Storms, Reginald; Walton, Jonathan D.

2014-01-01

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5. PMID:25295862

Enhancement of synthetic Trichoderma-based enzyme mixtures for biomass conversion with an alternative family 5 glycosyl hydrolase from Sporotrichum thermophile.

PubMed

Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S; Storms, Reginald; Walton, Jonathan D

2014-01-01

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4-glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.

Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

PubMed

Sauber, K; Fröhner, C; Rosenberg, G; Eberspächer, J; Lingens, F

1977-03-15

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.

Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

PubMed

Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

2014-04-01

We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mtDNA copy number and systemic lupus erythematosus disease activity index (SLEDAI) (P < 0·05), as well as plasma 8-OHdG (P < 0·05). In particular, four complicated SLE patients with increased expression of the genes encoding the anti-oxidant enzymes, GAPDH, Tfam and PDHA1, experienced better therapeutic outcomes after rituximab therapy. In conclusion, higher oxidative damage with suboptimal increases in DNA repair, anti-oxidant capacity, mitochondrial biogenesis and glucose metabolism may be implicated in SLE deterioration, and this impairment might be improved by targeted biological therapy. © 2013 British Society for Immunology.

Influence of the reaction conditions on the enzyme catalyzed transesterification of castor oil: A possible step in biodiesel production.

PubMed

Andrade, Thalles A; Errico, Massimiliano; Christensen, Knud V

2017-11-01

The identification of the influence of the reaction parameters is of paramount importance when defining a process design. In this work, non-edible castor oil was reacted with methanol to produce a possible component for biodiesel blends, using liquid enzymes as the catalyst. Temperature, alcohol-to-oil molar ratio, enzyme and added water contents were the reaction parameters evaluated in the transesterification reactions. The optimal conditions, giving the optimal final FAME yield and FFA content in the methyl ester-phase was identified. At 35°C, 6.0 methanol-to-oil molar ratio, 5wt% of enzyme and 5wt% of water contents, 94% of FAME yield and 6.1% of FFA in the final composition were obtained. The investigation was completed with the analysis of the component profiles, showing that at least 8h are necessary to reach a satisfactory FAME yield together with a minor FFA content. Copyright © 2017 Elsevier Ltd. All rights reserved.

Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components.

PubMed

Gilliam-Davis, Shea; Gallagher, Patricia E; Payne, Valerie S; Kasper, Sherry O; Tommasi, Ellen N; Westwood, Brian M; Robbins, Michael E; Chappell, Mark C; Diz, Debra I

2011-07-14

In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT(1)) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT(1) antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT(1) receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT(1b), AT(2), and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT(1) receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1-7) and prevent age-related declines in the leptin receptor and its signaling pathway.

A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A.

PubMed

Keefe, A D; Newton, G L; Miller, S L

1995-02-23

The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

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

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A

NASA Technical Reports Server (NTRS)

Keefe, A. D.; Newton, G. L.; Miller, S. L.

1995-01-01

The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.

Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis†

PubMed Central

Bridwell-Rabb, Jennifer; Iannuzzi, Clara; Pastore, Annalisa; Barondeau, David P.

2012-01-01

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich’s ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homolog CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule. PMID:22352884

Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis.

PubMed

Bridwell-Rabb, Jennifer; Iannuzzi, Clara; Pastore, Annalisa; Barondeau, David P

2012-03-27

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2013 CFR

2013-04-01

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

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2014 CFR

2014-04-01

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

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2012 CFR

2012-04-01

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

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2011 CFR

2011-04-01

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

21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

Code of Federal Regulations, 2010 CFR

2010-04-01

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

Serum levels of angiotensin converting enzyme as a biomarker of liver fibrosis

PubMed Central

Miranda, Aline Silva; Simões e Silva, Ana Cristina

2017-01-01

The renin angiotensin system (RAS) is classically conceived as a circulating hormonal system involved in blood pressure control and hydroelectrolyte balance. The discovery that RAS components are locally expressed in a wide range of organs and tissues, including the liver, pointed to a role for this system in the pathogenesis of several conditions including hepatic fibrosis and cirrhosis. It has been widely reported that the classical RAS axis composed by the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-Ang type 1 (AT1) receptor mediates pro-inflammatory, pro-thrombotic, and pro-fibrotic processes. On the other hand, the alternative axis comprising ACE2-Ang-(1-7)-Mas receptor seems to play a protective role by frequently opposing Ang II action. Chronic hepatitis B (CHB) is one of the leading causes of liver fibrosis, accounting for the death of nearly one million people worldwide. Liver fibrosis is a key factor to determine therapeutic interventions for patients with CHB. However, the establishment of non-invasive and accurate methods to detect reversible stages of liver fibrosis is still a challenge. In an elegant study published in the 36th issue of the World Journal of Gastroenterology, Noguchi et al showed the predictive value of serum ACE levels in detecting not only advanced stages of liver fibrosis but also initial and intermediate fibrotic stages. The serum levels of ACE might represent an accurate, non-invasive, widely available, and easy method to evaluate fibrosis related to CHB. Moreover, therapies involving the inhibition of the classical RAS axis components might be promising in the control of CHB-related liver fibrosis. PMID:29358853

Serum levels of angiotensin converting enzyme as a biomarker of liver fibrosis.

PubMed

Miranda, Aline Silva; Simões E Silva, Ana Cristina

2017-12-28

The renin angiotensin system (RAS) is classically conceived as a circulating hormonal system involved in blood pressure control and hydroelectrolyte balance. The discovery that RAS components are locally expressed in a wide range of organs and tissues, including the liver, pointed to a role for this system in the pathogenesis of several conditions including hepatic fibrosis and cirrhosis. It has been widely reported that the classical RAS axis composed by the angiotensin converting enzyme (ACE)-angiotensin (Ang) II-Ang type 1 (AT1) receptor mediates pro-inflammatory, pro-thrombotic, and pro-fibrotic processes. On the other hand, the alternative axis comprising ACE2-Ang-(1-7)-Mas receptor seems to play a protective role by frequently opposing Ang II action. Chronic hepatitis B (CHB) is one of the leading causes of liver fibrosis, accounting for the death of nearly one million people worldwide. Liver fibrosis is a key factor to determine therapeutic interventions for patients with CHB. However, the establishment of non-invasive and accurate methods to detect reversible stages of liver fibrosis is still a challenge. In an elegant study published in the 36 th issue of the World Journal of Gastroenterology , Noguchi et al showed the predictive value of serum ACE levels in detecting not only advanced stages of liver fibrosis but also initial and intermediate fibrotic stages. The serum levels of ACE might represent an accurate, non-invasive, widely available, and easy method to evaluate fibrosis related to CHB. Moreover, therapies involving the inhibition of the classical RAS axis components might be promising in the control of CHB-related liver fibrosis.

Human autoantibody to topoisomerase II.

PubMed

Hoffmann, A; Heck, M M; Bordwell, B J; Rothfield, N F; Earnshaw, W C

1989-02-01

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

Enzyme Characterization of Cellulase and Hemicellulases Component Enzymes and Saccharification of Ionic Liquid Pretreated Lignocellulosic Biomass

USDA-ARS?s Scientific Manuscript database

Lignocellulosic biomass is comprised of cellulose and hemicellulose, sources of polysaccharides, and lignin, a macromolecule with extensive aromaticity. Terrestrial biomass can provide a renewable carbon based feedstock for fuel and chemical production. However, recalcitrance of biomass to deconstru...

Cloning and characterization of alpha-glucuronidase enzyme

USDA-ARS?s Scientific Manuscript database

Hemicellulose is the second largest source of biomass on Earth. Xylan, a polymer of beta-1,4-linked xylose residues, is a common component of hemicellulose. The enzymes xylanase and beta-xylosidase hydrolyze the xylan into xylose which can then be fermented into value-added products. However, the...

Evolution of thiol protective systems in prokaryotes

NASA Technical Reports Server (NTRS)

Fahey, R. C.; Newton, G. L.

1986-01-01

Biological thiols are essential elements in most aspects of cell function but undergo rapid oxidation to disulfides in the presence of oxygen. The evolution of systems to protect against such oxygen toxicity was essential to the emergence of aerobic life. The protection system used by eukaryotes is based upon glutathione (GSH) and GSH-dependent enzymes but many bacteria lack GSH and apparently use other mechanisms. The objective of this research is to elaborate the thiol protective mechanisms employed by prokaryotes of widely divergent evolutionary origin and to understand why GSH became the central thiol employed in essentially all higher organisms. Thiol-selective fluorescent labeling and HPLC analysis has been used to determine key monothiol components.

Responsive materials for self-regulated insulin delivery.

PubMed

Wu, Weitai; Zhou, Shuiqin

2013-11-01

With diabetes mellitus becoming an important public health concern, insulin-delivery systems are attracting increasing interest from both scientific and technological researchers. This feature article covers the present state-of-the-art glucose-responsive insulin-delivery system (denoted as GRIDS), based on responsive polymer materials, a promising system for self-regulated insulin delivery. Three types of GRIDS are discussed, based on different fundamental mechanisms of glucose-recognition, with: a) glucose enzyme, b) glucose binding protein, and c) synthetic boronic acid as the glucose-sensitive component. At the end, a personal perspective on the major issues yet to be worked out in future research is provided. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[The role of metalloprotease in pathogenesis of nervous system diseases].

PubMed

Mirowska, D; Członkowska, A

2001-01-01

Matrix Metalloproteases (MMPs) comprise a big family of proteolytic enzymes secreted into extracellular matrix and involved in remodelling of many tissues. The MMPs' activity is regulated on many levels. It is also determined by specific inhibitors known as tissue inhibitors of metalloproteases (TIMPs). Several studies revealed that MMPs have a role not only in physiological processes but also in pathophysiology of nervous system diseases, such as multiplex sclerosis, Guillan-Barré syndrome and strokes. Concerning demyelination MMPs are responsible for degradation of myelin components and facilitation of immune cells migration into inflammatory sites by degrading vascular basement membrane. We still investigate substances with positive clinical effect on the nervous system diseases due to MMPs inactivation.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes*

PubMed Central

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-01-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the 14C-labeled crystalline polymeric substrates 14C chitin nanowhiskers and 14C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki(obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. PMID:25767120

The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.

PubMed

Kuusk, Silja; Sørlie, Morten; Väljamäe, Priit

2015-05-01

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The effect of angiotensin-converting enzyme inhibition throughout a superovulation protocol in ewes.

PubMed

Pereira, Alécio Matos; de Souza Júnior, Antônio; Machado, Fernanda Brandão; Gonçalves, Gleisy Kelly Neves; Feitosa, Lauro César Soares; Reis, Adelina Martha; Santos, Robson Augusto Souza; Honorato-Sampaio, Kinulpe; Costa, Amilton Raposo

2015-12-01

Many studies identified new components of the renin–angiotensin system (RAS), such as Angiotensin-(1-7) [Ang-(1–7)] and Angiotensin-converting enzyme type 2 (ACE2), in mammalian ovaries.We previously showed Angiotensin-Converting Enzyme (ACE) inhibition, which increases the level of Ang-(1–7), stimulated ovarian estradiol output in ewe after estrous synchronization. Considering that Ang-(1–7) stimulates ovarian function and elevated estradiol before ovulation is associated with increased chance of achieving pregnancy, the present study investigated whether ACE inhibition throughout a superovulation protocol in ewe might improve ovulation outcome. At first, immunohistochemistry in ovaries of nonpregnant ewes revealed localization of Angiotensin II (Ang II), Ang-(1–7) and ACE2 in theca cells of antral follicles and in corpus luteum. Ang II and Ang-(1–7)were also detected in follicular fluid (FF) by Radioimmunoassay (RIA). Enalapril treatment throughout the superovulation protocol decreased 17β-estradiol (E2) output and raised progesterone:estradiol (P4:E2) ratio without a direct influence on ovulation and quality of embryos.

Digestive enzymes of human and nonhuman primates.

PubMed

Janiak, Mareike Cora

2016-09-01

All living organisms need to consume nutrients to grow, survive, and reproduce, making the successful acquisition of food resources a powerful selective pressure. However, acquiring food is only part of the challenge. While all animals spend much of their daily activity budget hunting, searching for, or otherwise procuring food, a large part of what is involved in overall nutrition occurs once the meal has been swallowed. Most nutritional components are too complex for immediate use and must be broken down into simpler compounds, which can then be absorbed by the body. This process, digestion, is catalyzed by enzymes that are either endogenous or produced by the host's microbial population. 1 Research shows that the nutritional value of food is partially constrained by the digestive abilities of the microbial community present in the host's gut and that these microbes rapidly adapt to changes in diet and other environmental pressures. 2 An accumulating body of evidence suggests that endogenously produced digestive enzymes also have been, and still are, common targets of natural selection, further cementing their crucial role in an organism's digestive system. 3-5 . © 2016 Wiley Periodicals, Inc.

[Metalloproteinases in meningoencephalitis].

PubMed

Pastuszka, Ewa; Pabin, Agata; Radkowski, Marek

2008-01-01

Meningoencephalitis remains a devastating disease with high morbidity and mortality. Despite advances in antibiotic treatment and critical care, mortality rate in bacterial meningoencephalitis is close to 25%. Moreover, neurological and neuropsychological sequelae emerge in up to 50% of survivors. Adverse outcome is significantly associated with events secondary to meningitis and damage of the blood-brain barrier. Several studies conducted on animals confirmed that matrix-metalloproteinases (MMP), a family of enzymes with major actions in the remodeling of exracellural matrix components facilitate this process which results in acute neurological complications. Gelatinases (MMP-2, MMP-9), the most complex family member, through degradation of gelatine and collagen IV play an important role in the pathogenesis of brain's inflamatory diseases (e.g. Guillian-Barre syndrom) and contribute to spreading the disease beyond the central nervous system. Infection (bacterial, viral or fungal) can lead to increased concentration and activity of metalloproteinases due to excessive enzyme's secretion or decrease in level of its natural inhibitors. A detailed analysis of those enzymes could help in developing new diagnostic and prognostic markers for meningoencephalitis and could facilitate new treatment approaches.

Enhancing enzymatic hydrolysis of sugarcane bagasse by ferric chloride catalyzed organosolv pretreatment and Tween 80.

PubMed

Zhang, Hongdan; Fan, Meishan; Li, Xin; Zhang, Aiping; Xie, Jun

2018-06-01

In this work, a FeCl 3 -catalyzed organosolv pretreatment was employed at 160 °C to remove hemicellulose and lignin in sugarcane bagasse leaving the cellulose-enriched residue for enzymatic hydrolysis to sugars. The solubilized hemicellulose fractions consisted more monomer xylose than oligomer xylose. The FeCl 3 -catalyzed organosolv pretreatment significantly improved the enzymatic hydrolysis, nearly 100% of cellulose components were converted to glucose after pretreatment with 0.05 M FeCl 3 . Structural analysis was employed to reveal how pretreatment affected the enzymatic hydrolysis. With the addition of Tween 80, the same level of glucose was obtained with 50% reduction of enzyme dosage after 24 h. Furthermore, the influence of Tween 80 on different pretreatment systems was investigated, indicating that the improvement was increased as the lignin content increased, decreased with high enzyme loading and extending hydrolysis time. This work suggested that the addition of Tween 80 could improve the enzymatic hydrolysis, reduce the hydrolysis time and enzyme dosage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Acute and prolonged complement activation in the central nervous system during herpes simplex encephalitis.

PubMed

Eriksson, Charlotta E; Studahl, Marie; Bergström, Tomas

2016-06-15

Herpes simplex encephalitis (HSE) is characterized by a pronounced inflammatory activity in the central nervous system (CNS). Here, we investigated the acute and prolonged complement system activity in HSE patients, by using enzyme-linked immunosorbent assays (ELISAs) for numerous complement components (C). We found increased cerebrospinal fluid concentrations of C3a, C3b, C5 and C5a in HSE patients compared with healthy controls. C3a and C5a concentrations remained increased also compared with patient controls. Our results conclude that the complement system is activated in CNS during HSE in the acute phase, and interestingly also in later stages supporting previous reports of prolonged inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052

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

Mitchell, W.J.; Shaw, J.E.; Andrews, L.

1991-09-01

Acetone-butanol-ethanol fermentation by Clostridium acetobutylicum has been exploited on an industrial scale in the past, but for economic reasons the process has declined. However, with an increased understanding of solvent formation and the potential for genetic manipulation of the organism, this fermentation is once again receiving attention. An economical process would be founded on the use of cheap, renewable substrates, ideally carbohydrate-based waste materials. However, little is known about the mechanism and regulation of carbohydrate accumulation by C. acetobutylicum. The glucose phosphotransferase system (PTS) of C. acetobutylicum was studied by using cell extracts. The system exhibited a K{sub m} formore » glucose of 34 {mu}M, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose. The analogs 3-O-methylglucoside and methyl {alpha}-glucoside did not inhibit glucose phosphorylation significantly. Activity showed no dependence on Mg{sup 2+} ions or on pH in the range 6.0 to 8.0. The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli. In addition to a membrane-bound enzyme II{sup Glc}, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and III{sup Glc} components. The HPr was found in the soluble fraction of C. acetobutylicum extracts, whereas enzyme I, and probably also III{sup Glc}, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell.« less

Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

PubMed

Luo, Ling; Meng, Han; Gu, Ji-Dong

2017-07-15

Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

Artificial enzymes based on supramolecular scaffolds.

PubMed

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.

Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots.

PubMed

Ruíz-Torres, Carmelo; Feriche-Linares, Rafael; Rodríguez-Ruíz, Marta; Palma, José M; Corpas, Francisco J

2017-04-01

Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200μM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Functions and substrates of NEDDylation during cell cycle in the silkworm, Bombyx mori.

PubMed

Li, Zhiqing; Cui, Qixin; Wang, Xiaoyan; Li, Bingqian; Zhao, Dongchao; Xia, Qingyou; Zhao, Ping

2017-11-01

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm. Copyright © 2017. Published by Elsevier Ltd.

Numerical Analysis of the Effect of Particle Shape and Adhesion on the Segregation of Powder Mixtures

NASA Astrophysics Data System (ADS)

Alizadeh Behjani, Mohammadreza; Hassanpour, Ali; Ghadiri, Mojtaba; Bayly, Andrew

2017-06-01

Segregation of granules is an undesired phenomenon in which particles in a mixture separate from each other based on the differences in their physical and chemical properties. It is, therefore, crucial to control the homogeneity of the system by applying appropriate techniques. This requires a fundamental understanding of the underlying mechanisms. In this study, the effect of particle shape and cohesion has been analysed. As a model system prone to segregation, a ternary mixture of particles representing the common ingredients of home washing powders, namely, spray dried detergent powders, tetraacetylethylenediamine, and enzyme placebo (as the minor ingredient) during heap formation is modelled numerically by the Discrete Element Method (DEM) with an aim to investigate the effect of cohesion/adhesion of the minor components on segregation quality. Non-spherical particle shapes are created in DEM using the clumped-sphere method based on their X-ray tomograms. Experimentally, inter particle adhesion is generated by coating the minor ingredient (enzyme placebo) with Polyethylene Glycol 400 (PEG 400). The JKR theory is used to model the cohesion/adhesion of coated enzyme placebo particles in the simulation. Tests are carried out experimentally and simulated numerically by mixing the placebo particles (uncoated and coated) with the other ingredients and pouring them in a test box. The simulation and experimental results are compared qualitatively and quantitatively. It is found that coating the minor ingredient in the mixture reduces segregation significantly while the change in flowability of the system is negligible.

Digestive stimulant action of spices: a myth or reality?

PubMed

Platel, Kalpana; Srinivasan, K

2004-05-01

Spices have long been recognized for their digestive stimulant action. Several spices are also employed in medicinal preparations against digestive disorders in traditional and Indian systems of medicine. Earlier reports on the digestive stimulant action of spices are largely empirical; only in recent years, this beneficial attribute of spices has been authenticated in exhaustive animal studies. Animal studies have shown that many spices induce higher secretion of bile acids which play a vital role in fat digestion and absorption. When consumed through the diet also spices produce significant stimulation of the activities of pancreatic lipase, amylase and proteases. A few of them also have been shown to have beneficial effect on the terminal digestive enzymes of small intestinal mucosa. Concomitant with such a stimulation of either bile secretion or activity of digestive enzymes by these spices, leading to an accelerated digestion, a reduction in the food transit time in the gastrointestinal tract has also been shown. Thus, the digestive stimulant action of spices seems to be mediated through two possible modes: (i) by stimulating the liver to secrete bile rich in bile acids, components that are vital for fat digestion and absorption, and (ii) by a stimulation of enzyme activities that are responsible for digestion. This review highlights the available information on the influence of spices on the digestive secretions and enzymes.

Metabolic gene clusters encoding the enzymes of two branches of the 3-oxoadipate pathway in the pathogenic yeast Candida albicans.

PubMed

Gérecová, Gabriela; Neboháčová, Martina; Zeman, Igor; Pryszcz, Leszek P; Tomáška, Ľubomír; Gabaldón, Toni; Nosek, Jozef

2015-05-01

The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina, which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are suitable candidates for the missing enzymes of the pathway. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Plant defense induced in in vitro propagated banana (Musa paradisiaca) plantlets by Fusarium derived elicitors.

PubMed

Patel, Miral; Kothari, I L; Mohan, J S S

2004-07-01

Perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used fungal components of Fusarium under in vitro condition and checked the rise in signal molecule, salicylic acid (SA), and marker enzymes in defense reactions against the pathogen. SA level increased by 21 folds in elicitor treated plantlets as compared to that of control plantlets and there was marked increase in phenylalanine ammonia-lyase(PAL), peroxidase(POX), polyphenol oxidase(PPO) along with higher total phenolic content. Present results indicated that use of fungal components had successfully induced systemic resistance in in vitro cultured banana plantlets.

[Characteristics of soil organic carbon and enzyme activities in soil aggregates under different vegetation zones on the Loess Plateau].

PubMed

Li, Xin; Ma, Rui-ping; An, Shao-shan; Zeng, Quan-chao; Li, Ya-yun

2015-08-01

In order to explore the distribution characteristics of organic carbon of different forms and the active enzymes in soil aggregates with different particle sizes, soil samples were chosen from forest zone, forest-grass zone and grass zone in the Yanhe watershed of Loess Plateau to study the content of organic carbon, easily oxidized carbon, and humus carbon, and the activities of cellulase, β-D-glucosidase, sucrose, urease and peroxidase, as well as the relations between the soil aggregates carbon and its components with the active soil enzymes were also analyzed. It was showed that the content of organic carbon and its components were in order of forest zone > grass zone > forest-grass zone, and the contents of three forms of organic carbon were the highest in the diameter group of 0.25-2 mm. The content of organic carbon and its components, as well as the activities of soil enzymes were higher in the soil layer of 0-10 cm than those in the 10-20 cm soil layer of different vegetation zones. The activities of cellulase, β-D-glucosidase, sucrose and urease were in order of forest zone > grass zone > forest-grass zone. The peroxidase activity was in order of forest zone > forest-grass zone > grass zone. The activities of various soil enzymes increased with the decreasing soil particle diameter in the three vegetation zones. The activities of cellulose, peroxidase, sucrose and urease had significant positive correlations with the contents of various forms of organic carbon in the soil aggregates.

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

PubMed

Bremner, P D; Blacklock, C J; Paganga, G; Mullen, W; Rice-Evans, C A; Crozier, A

2000-06-01

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

PubMed

Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

2013-01-01

NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

Wood mimetic hydrogel beads for enzyme immobilization.

PubMed

Park, Saerom; Kim, Sung Hee; Won, Keehoon; Choi, Joon Weon; Kim, Yong Hwan; Kim, Hyung Joo; Yang, Yung-Hun; Lee, Sang Hyun

2015-01-22

Wood component-based composite hydrogels have potential applications in biomedical fields owing to their low cost, biodegradability, and biocompatibility. The controllable properties of wood mimetic composites containing three major wood components are useful for enzyme immobilization. Here, lipase from Candida rugosa was entrapped in wood mimetic beads containing cellulose, xylan, and lignin by dissolving wood components with lipase in [Emim][Ac], followed by reconstitution. Lipase entrapped in cellulose/xylan/lignin beads in a 5:3:2 ratio showed the highest activity; this ratio is very similar to that in natural wood. The lipase entrapped in various wood mimetic beads showed increased thermal and pH stability. The half-life times of lipase entrapped in cellulose/alkali lignin hydrogel were 31- and 82-times higher than those of free lipase during incubation under denaturing conditions of high temperature and low pH, respectively. Owing to their biocompatibility, biodegradability, and controllable properties, wood mimetic hydrogel beads can be used to immobilize various enzymes for applications in the biomedical, bioelectronic, and biocatalytic fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

Extracellular enzymatic activity of two hydrolases in wastewater treatment for biological nutrient removal.

PubMed

Berrio-Restrepo, Jorge Mario; Saldarriaga, Julio César; Correa, Mauricio Andrés; Aguirre, Néstor Jaime

2017-10-01

Due to the complex nature of the wastewater (both domestic and non-domestic) composition, biological processes are widely used to remove nutrients, such as carbon (C), nitrogen (N), and phosphorous (P), which cause instability and hence contribute to the damage of water bodies. Systems with different configurations have been developed (including anaerobic, anoxic, and aerobic conditions) for the joint removal of carbon, nitrogen, and phosphorus. The goal of this research is to evaluate the extracellular activity of β-glucosidase and phosphatase enzymes in a University of Cape Town (UCT) system fed with two synthetic wastewaters of different molecular complexity. Both types of waters have medium strength characteristics similar to those of domestic wastewater with a mean C/N/P ratio of 100:13:1. The operation parameters were hydraulic retention time (HRT) of 10 h, solid retention time (SRT) of 12 days, mean concentration of the influent in terms of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) of 600, 80, and 6 mg/L, respectively. According to the results obtained, statistically significant differences have been found in the extracellular enzyme activities with the evaluated wastewaters and in the units comprising the treatment system in some of the cases. An analysis of principal components showed that the extracellular enzymatic activity has been correlated to nutrient concentration in wastewater, biomass concentration in the system, and metabolic conditions of treatment phases. Additionally, this research has allowed determining an inverse relationship between wastewater biodegradability and the extracellular enzyme activity of β-glucosidase and phosphatase. These results highlight the importance of including the analysis of biomass biochemical characteristics as control methods in wastewater treatment systems for the nutrient removal.

A microfluidic device for evaluating the dynamics of the metabolism-dependent antioxidant activity of nutrients.

PubMed

Lee, Jungwoo; Choi, Jong-ryul; Ha, Sang Keun; Choi, Inwook; Lee, Seung Hwan; Kim, Donghyun; Choi, Nakwon; Sung, Jong Hwan

2014-08-21

Various food components are known for their health-promoting effects. However, their biochemical effects are generally evaluated in vitro, and their actual in vivo effect can vary significantly, depending on their metabolic profiles. To evaluate the effect of the liver metabolism on the antioxidant activity, we have developed a two-compartment microfluidic system that integrates the dynamics of liver metabolism and the subsequent antioxidant activity of food components. In the first compartment of the device, human liver enzyme fractions were immobilized inside a poly(ethylene glycol) diacrylate (PEGDA) hydrogel to mimic the liver metabolism. The radical scavenging activity was evaluated by the change of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) absorbance in the second compartment. Reaction engineering and fluid mechanics principles were used to develop a simplified analytical model and a more complex finite element model, which were used to design the chip and determine the optimal flow conditions. For real-time measurements of the reaction on a chip, we developed a custom-made photospectrometer system with an LED light source. The developed microfluidic system showed a linear and dose-dependent antioxidant activity in response to increasing concentration of flavonoid. We also compared the antioxidant activity of flavonoid after various liver metabolic reactions. This microfluidic system can serve as a novel in vitro platform for predicting the antioxidant activity of various food components in a more physiologically realistic manner, as well as for studying the mechanism of action of such food components.

In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

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

Coelho, Pedro S.; Brustad, Eric M.; Arnold, Frances H.

The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cellsmore » expressing the heme enzymes are also provided by the present invention.« less

21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Angiotensin converting enzyme (A.C.E.) test system... Test Systems § 862.1090 Angiotensin converting enzyme (A.C.E.) test system. (a) Identification. An angiotensin converting enzyme (A.C.E.) test system is a device intended to measure the activity of angiotensin...

Differential Gene Expression in Explanted Human Retinal Pigment Epithelial Cells 12-Hours Post-Exposure to 532 nm, 120 ps Pulsed Laser Light

DTIC Science & Technology

2004-04-01

cycling, anaerobic enzymes and kinase enzymes as well as specific cellular channel or receptor components. However, the most striking revelation of the...degradation. Most notably up-regulated were the genes for the enzymes essential in the ubiquitin-proteoasome pathway (UPP) shown to be up-regulated in response...to oxidative stress in eye tissue (1). These were ubiquitin [2.0], 3 ubiquitin-conjugating enzyme genes E2 [2.3], E2D2 [2.3] and E2D3 [2.8]. Also up

Cations in component reactions of `malic' enzyme catalysis

PubMed Central

Tsai, C. S.; Tsai, Y. H.; Samad, R. A.

1971-01-01

The `malic' enzyme (EC 1.1.1.40) has been purified (300-fold) from wheat germ and its abilities to catalyse the decarboxylation and the hydrogenation of oxaloacetic acid and oxaloacetate esters was studied. The free 1-carboxyl group is essential for the interaction of oxaloacetates and substituted oxaloacetates with the enzyme via cations. The free 4-carboxyl group is required for the decarboxylation but is not indispensable for the hydrogenation. At high concentrations, cations inhibit the enzymic hydrogenation of oxaloacetic acid but not that of 4-ethyl oxaloacetate. A plausible inhibitory mechanism is proposed. PMID:4399519

Regulation of Toxin Production in Clostridium perfringens

PubMed Central

Ohtani, Kaori; Shimizu, Tohru

2016-01-01

The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

PubMed

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

PubMed

Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing.

PubMed

Wiseman, Alan

2003-04-01

Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).

In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

PubMed

Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

2016-01-01

In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

Miniaturized flow system based on enzyme modified PMMA microreactor for amperometric determination of glucose.

PubMed

Cerdeira Ferreira, Luís Marcos; da Costa, Eric Tavares; do Lago, Claudimir Lucio; Angnes, Lúcio

2013-09-15

This paper describes the development of a microfluidic system having as main component an enzymatic reactor constituted by a microchannel assembled in poly(methyl methacrylate) (PMMA) substrate connected to an amperometric detector. A CO2 laser engraving machine was used to make the channels, which in sequence were thermally sealed. The internal surfaces of the microchannels were chemically modified with polyethyleneimine (PEI), which showed good effectiveness for the immobilization of the glucose oxidase enzyme using glutaraldehyde as crosslinking agent, producing a very effective microreactor for the detection of glucose. The hydrogen peroxide generated by the enzymatic reaction was detected in an electrochemical flow cell localized outside of the reactor using a platinum disk as the working electrode. The proposed system was applied to the differential amperometric determination of glucose content in soft drinks showing good repeatability (DPR=1.72%, n=50), low detection limit (1.40×10(-6)molL(-1)), high sampling frequency (calculated as 345 samples h(-1)), and relatively good stability for long-term use. The results were in close agreement with those obtained by the classical spectrophotometric method utilized to quantify glucose in biological fluids. Copyright © 2013 Elsevier B.V. All rights reserved.

Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown rot wood-decay fungi.

PubMed

Goodell, Barry; Zhu, Yuan; Kim, Seong; Kafle, Kabindra; Eastwood, Daniel; Daniel, Geoffrey; Jellison, Jody; Yoshida, Makoto; Groom, Leslie; Pingali, Sai Venkatesh; O'Neill, Hugh

2017-01-01

Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a "pretreatment" used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability.

Pan-Cellulosomics of Mesophilic Clostridia: Variations on a Theme.

PubMed

Dassa, Bareket; Borovok, Ilya; Lombard, Vincent; Henrissat, Bernard; Lamed, Raphael; Bayer, Edward A; Moraïs, Sarah

2017-11-18

The bacterial cellulosome is an extracellular, multi-enzyme machinery, which efficiently depolymerizes plant biomass by degrading plant cell wall polysaccharides. Several cellulolytic bacteria have evolved various elaborate modular architectures of active cellulosomes. We present here a genome-wide analysis of a dozen mesophilic clostridia species, including both well-studied and yet-undescribed cellulosome-producing bacteria. We first report here, the presence of cellulosomal elements, thus expanding our knowledge regarding the prevalence of the cellulosomal paradigm in nature. We explored the genomic organization of key cellulosome components by comparing the cellulosomal gene clusters in each bacterial species, and the conserved sequence features of the specific cellulosomal modules (cohesins and dockerins), on the background of their phylogenetic relationship. Additionally, we performed comparative analyses of the species-specific repertoire of carbohydrate-degrading enzymes for each of the clostridial species, and classified each cellulosomal enzyme into a specific CAZy family, thus indicating their putative enzymatic activity (e.g., cellulases, hemicellulases, and pectinases). Our work provides, for this large group of bacteria, a broad overview of the blueprints of their multi-component cellulosomal complexes. The high similarity of their scaffoldin clusters and dockerin-based recognition residues suggests a common ancestor, and/or extensive horizontal gene transfer, and potential cross-species recognition. In addition, the sporadic spatial organization of the numerous dockerin-containing genes in several of the genomes, suggests the importance of the cellulosome paradigm in the given bacterial species. The information gained in this work may be utilized directly or developed further by genetically engineering and optimizing designer cellulosome systems for enhanced biotechnological biomass deconstruction and biofuel production.

The involvement of heparan sulfate proteoglycans in stem cell differentiation and in malignant glioma

NASA Astrophysics Data System (ADS)

Kundu, Soumi; Xiong, Anqi; Forsberg-Nilsson, Karin

2016-04-01

Heparan sulfate (HS) proteoglycans (HSPG) are major components of the extracellular matrix. They interact with a plethora of macromolecules that are of physiological importance. The pattern of sulfation of the HS chain determines the specificity of these interactions. The enzymes that synthesize and degrade HS are thus key regulators of processes ranging from embryonic development to tissue homeostasis and tumor development. Formation of the nervous system is also critically dependent on appropriate HSPGs as shown by several studies on the role of HS in neural induction from embryonic stem cells. High-grade glioma is the most common primary malignant brain tumor among adults, and the prognosis is poor. Neural and glioma stem cells share several traits, including sustained proliferation and highly efficient migration in the brain. There are also similarities between the neurogenic niche where adult neural stem cells reside and the tumorigenic niche, including their interactions with components of the extracellular matrix (ECM). The levels of many of these components, for example HSPGs and enzymes involved in the biosynthesis and modification of HS are attenuated in gliomas. In this paper, HS regulation of pathways involved in neural differentiation and how these may be of importance for brain development are discussed. The literature suggesting that modifications of HS could regulate glioma growth and invasion is reviewed. Targeting the invasiveness of glioma cells by modulating HS may improve upon present therapeutic options, which only marginally enhance the survival of glioma patients.

Fungal biodegradation and enzymatic modification of lignin

PubMed Central

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

Synergism between ultrasonic pretreatment and white rot fungal enzymes on biodegradation of wheat chaff.

PubMed

Sabarez, Henry; Oliver, Christine Maree; Mawson, Raymond; Dumsday, Geoff; Singh, Tanoj; Bitto, Natalie; McSweeney, Chris; Augustin, Mary Ann

2014-11-01

Lignocellulosic biomass samples (wheat chaff) were pretreated by ultrasound (US) (40kHz/0.5Wcm(-2)/10min and 400kHz/0.5Wcm(-2)/10min applied sequentially) prior to digestion by enzyme extracts obtained from fermentation of the biomass with white rot fungi (Phanerochaete chrysosporium or Trametes sp.). The accessibility of the cellulosic components in wheat chaff was increased, as demonstrated by the increased concentration of sugars produced by exposure to the ultrasound treatment prior to enzyme addition. Pretreatment with ultrasound increased the concentration of lignin degradation products (guaiacol and syringol) obtained from wheat chaff after enzyme addition. In vitro digestibility of wheat chaff was also enhanced by the ultrasonics pretreatment in combination with treatment with enzyme extracts. Degradation was enhanced with the use of a mixture of the enzyme extracts compared to that for a single enzyme extract. Copyright © 2014. Published by Elsevier B.V.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

PubMed

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.

Enzymatic degradation of cell wall and related plant polysaccharides.

PubMed

Ward, O P; Moo-Young, M

1989-01-01

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.

The role of enzyme activities in soil ecosystem services: Location, origin and connection to the phytobiome

USDA-ARS?s Scientific Manuscript database

Soil enzymes are important components of soil quality and its health because of their involvement in ecosystem services related to biogeochemical cycling, global C and organic matter dynamics, and soil detoxification. This talk will provide an overview of the field of soil enzymology, the location a...

NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOMPONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

EPA Science Inventory

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. ix open reading frames were identified, four of which are, homologous to the components of toluene dioxy...

[Specific features of digestive function development in larvae of some salmonid fish].

PubMed

Ershova, T S; Volkova, I V; Zaĭtseva, V F

2004-01-01

We studied the activities of digestive enzymes responsible for the digestion of food carbohydrate and protein components in plant-eating fish at various stages of larval development. The activities of all digestive enzymes tend to rise during larval development. Species specific features of the alimentary canal functioning have been described.

Effects of salinity and ascorbic acid on growth, water status and antioxidant system in a perennial halophyte

PubMed Central

Hameed, Abdul; Gulzar, Salman; Aziz, Irfan; Hussain, Tabassum; Gul, Bilquees; Khan, M. Ajmal

2015-01-01

Salinity causes oxidative stress in plants by enhancing production of reactive oxygen species, so that an efficient antioxidant system, of which ascorbic acid (AsA) is a key component, is an essential requirement of tolerance. However, antioxidant responses of plants to salinity vary considerably among species. Limonium stocksii is a sub-tropical halophyte found in the coastal marshes from Gujarat (India) to Karachi (Pakistan) but little information exists on its salt resistance. In order to investigate the role of AsA in tolerance, 2-month-old plants were treated with 0 (control), 300 (moderate) and 600 (high) mM NaCl for 30 days with or without exogenous application of AsA (20 mM) or distilled water. Shoot growth of unsprayed plants at moderate salinity was similar to that of controls while at high salinity growth was inhibited substantially. Sap osmolality, AsA concentrations and activities of AsA-dependant antioxidant enzymes increased with increasing salinity. Water spray resulted in some improvement in growth, indicating that the growth promotion by exogenous treatments could partly be attributed to water. However, exogenous application of AsA on plants grown under saline conditions improved growth and AsA dependent antioxidant enzymes more than the water control treatment. Our data show that AsA-dependent antioxidant enzymes play an important role in salinity tolerance of L. stocksii. PMID:25603966

Structural studies on the decameric S. typhimurium arginine decarboxylase (ADC): Pyridoxal 5'-phosphate binding induces conformational changes.

PubMed

Deka, G; Bharath, S R; Savithri, H S; Murthy, M R N

2017-09-02

Enteric pathogens such as Salmonella typhimurium colonize the human gut in spite of the lethal acidic pH environment (pH < 2.5) due to the activation of inducible acid tolerance response (ATR) systems. The pyridoxal 5'-phosphate (PLP)-dependent enzyme, biodegradative arginine decarboxylase (ADC, encoded by AdiA), is a component of an ATR system. The enzyme consumes a cytoplasmic proton in the process of arginine degradation to agmatine. Arginine-agmatine antiporter (AdiC) exchanges the product agmatine for arginine. In this manuscript, we describe the structure of Salmonella typhimurium ADC (StADC). The decameric structure assembled from five dimers related by a non crystallographic 5-fold symmetry represents the first apo-form of the enzyme. The structure suggests that PLP-binding is not a prerequisite for oligomerization. Comparison with E. coli ADC reveals that PLP-binding is accompanied by the movement and ordering of two loops (residues 150-159 and 191-197) and a few active site residues such as His256 and Lys257. A number of residues important for substrate binding are disordered in the apo-StADC structure indicating that PLP binding is important for substrate binding. Unlike the interactions between 5-fold related protomers, interactions that stabilize the dimeric structure are not pH dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

A real-scale soil phytoremediation.

PubMed

Macci, Cristina; Doni, Serena; Peruzzi, Eleonora; Bardella, Simone; Filippis, Giorgio; Ceccanti, Brunello; Masciandaro, Grazia

2013-07-01

In the present investigation, a phytoremediation process with a combination of different plant species (Populus nigra (var.italica), Paulownia tomentosa and Cytisus scoparius), and natural growing vegetation has been proposed at real-scale (10.000 m(2)) to bioremediate and functionally recover a soil historically contaminated by heavy metals and hydrocarbons. In the attempts to assess both effectiveness and evolution of the remediation system towards a natural soil ecosystem, besides the pollution parameters, also parameters describing the efficiency of the microbiological components (enzyme activities), were investigated. In 3 years, the total content of hydrocarbons and heavy metals in soil decreased with time (40 % and 20-40 %, respectively), reaching concentrations under the limit of National legislation and making the site suitable for environmental reusing. The reduction in pollutants was probably the reason of the increase in dehydrogenase (indicator of overall microbial activity), β-glucosidase and phosphatase activities, enzymes related to C and P cycles, respectively. However, this trend was obviously due also to the increase of chemical nutrients, acting as substrate of these enzymes. Moreover, a phytotest carried out with Raphanus sativus, showed, after 3 years, a significant increase in percentage of plant growth, confirming a reduction in soil toxicity and an improvement in soil nutritional state. Therefore, this phytoremediation system seems very promising to perform both decontamination and functional recovery of a polluted soil at real-scale level.

Importance of single molecular determinants in the fidelity of expanded genetic codes.

PubMed

Antonczak, Alicja K; Simova, Zuzana; Yonemoto, Isaac T; Bochtler, Matthias; Piasecka, Anna; Czapinska, Honorata; Brancale, Andrea; Tippmann, Eric M

2011-01-25

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

Importance of single molecular determinants in the fidelity of expanded genetic codes

PubMed Central

Antonczak, Alicja K.; Simova, Zuzana; Yonemoto, Isaac T.; Bochtler, Matthias; Piasecka, Anna; Czapińska, Honorata; Brancale, Andrea; Tippmann, Eric M.

2011-01-01

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented. PMID:21224416

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

PubMed

Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita

2006-06-25

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Effects of stinging nettle root extracts and their steroidal components on the Na+,K(+)-ATPase of the benign prostatic hyperplasia.

PubMed

Hirano, T; Homma, M; Oka, K

1994-02-01

The effects of organic-solvent extracts of Urtica dioica (Urticaceae) on the Na+,K(+)-ATPase of the tissue of benign prostatic hyperplasia (BPH) were investigated. The membrane Na+,K(+)-ATPase fraction was prepared from a patient with BPH by a differential centrifugation of the tissue homogenate. The enzyme activity was inhibited by 10(-4)-10(-5) M of ouabain. The hexane extract, the ether extract, the ethyl acetate extract, and the butanol extract of the roots caused 27.6-81.5% inhibition of the enzyme activity at 0.1 mg/ml. In addition, a column extraction of stinging nettle roots using benzene as an eluent afforded efficient enzyme inhibiting activity. Steroidal components in stinging nettle roots, such as stigmast-4-en-3-one, stigmasterol, and campesterol inhibited the enzyme activity by 23.0-67.0% at concentrations ranging from 10(-3)-10(-6) M. These results suggest that some hydrophobic constituents such as steroids in the stinging nettle roots inhibited the membrane Na+,K(+)-ATPase activity of the prostate, which may subsequently suppress prostate-cell metabolism and growth.

Innate immune memory in plants.

PubMed

Reimer-Michalski, Eva-Maria; Conrath, Uwe

2016-08-01

The plant innate immune system comprises local and systemic immune responses. Systemic plant immunity develops after foliar infection by microbial pathogens, upon root colonization by certain microbes, or in response to physical injury. The systemic plant immune response to localized foliar infection is associated with elevated levels of pattern-recognition receptors, accumulation of dormant signaling enzymes, and alterations in chromatin state. Together, these systemic responses provide a memory to the initial infection by priming the remote leaves for enhanced defense and immunity to reinfection. The plant innate immune system thus builds immunological memory by utilizing mechanisms and components that are similar to those employed in the trained innate immune response of jawed vertebrates. Therefore, there seems to be conservation, or convergence, in the evolution of innate immune memory in plants and vertebrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Processing and fractional utilization of lignocellulosic substrates by "pure" and "natural and defined mixed" culture C.TM1, C.SA IV and rumen mixed culture consortia CD.

PubMed

Sankar, M; Chandra, T S

2003-01-01

A detailed analysis was made of chemical fractions of common agro-residues before and after pretreatment (alkali and hydrogen peroxide), and the selective utilization of components such as WSS, EBS, TSS, lignin, cellulose and hemicellulose by pure and mixed cultures of cellulolytic and xylanolytic Clostridia was monitored and correlated with the organisms' enzyme activity. For all cultures pretreatment gave higher utilization of hemicellulose and cellulose fractions; hydrogen peroxide pretreatment was more effective than NaOH treatment. Lignin utilization was not very significant even on pretreatment. C.TM1 and C.SA IV utilized hemicellulose and cellulose better than mixed cultures in selected substrates. These results help to determine the substrate composition, pretreatment conditions and enzyme system of the organism needed when designing an inoculum for agricultural waste treatment processes such as composting or biogas generation.

Development of small-size tubular-flow continuous reactors for the analysis of operational stability of enzymes in low-water systems.

PubMed

Pirozzi, D; Halling, P J

2001-01-20

A very small-scale continuous flow reactor has been designed for use with enzymes in organic media, particularly for operational stability studies. It is constructed from fairly inexpensive components, and typically uses 5 mg of catalyst and flow rates of 1 to 5 mL/h, so only small quantities of feedstock need to be handled. The design allows control of the thermodynamic water activity of the feed, and works with temperatures up to at least 80 degrees C. The reactor has been operated with both nonpolar (octane) and polar (4-methyl-pentan-2-one) solvents, and with the more viscous solvent-free reactant mixture. It has been applied to studies of the operational stability of lipases from Chromobacterium viscosum (lyophilized powder or polypropylene-adsorbed) and Rhizomucor miehei (Lipozyme) in different experimental conditions. Transesterification of geraniol and ethylcaproate has been adopted as a model transformation.

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

PubMed

Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

1991-01-05

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

PubMed

Huang, Jinjin; Xia, Ji; Yang, Zhen; Guan, Feifei; Cui, Di; Guan, Guohua; Jiang, Wei; Li, Ying

2014-01-01

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

PubMed

Bansal, Vibha; Delgado, Yamixa; Legault, Marc; Barletta, Gabriel

2012-02-14

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Antioxidant Activity and Induction of mRNA Expressions of Antioxidant Enzymes in HEK-293 Cells of Moringa oleifera Leaf Extract.

PubMed

Vongsak, Boonyadist; Mangmool, Supachoke; Gritsanapan, Wandee

2015-08-01

The leaves of Moringa oleifera, collected in different provinces in Thailand, were determined for the contents of total phenolics, total flavonoids, major components, and antioxidant activity. The extract and its major active components were investigated for the inhibition of H2O2-induced reactive oxygen species production and the effects on antioxidant enzymes mRNA expression. The extract, crypto-chlorogenic acid, isoquercetin and astragalin, significantly reduced the reactive oxygen species production inducing by H2O2 in HEK-293 cells. Treatment with isoquercetin significantly increased the mRNA expression levels of antioxidant enzymes such as superoxide dismutase, catalase and heme oxygenase 1. These results confirm that M. oleifera leaves are good sources of natural antioxidant with isoquercetin as an active compound. Georg Thieme Verlag KG Stuttgart · New York.

Effect of phenolic compounds from pretreated sugarcane bagasse on cellulolytic and hemicellulolytic activities.

PubMed

Michelin, Michele; Ximenes, Eduardo; de Lourdes Teixeira de Moraes Polizeli, Maria; Ladisch, Michael R

2016-01-01

This work shows both cellulases and hemicellulases are inhibited and deactivated by water-soluble and acetone extracted phenolics from sugarcane bagasse pretreated at 10% (w/v) for 30 min in liquid hot water at 180 or 200°C. The dissolved phenolics in vacuum filtrate increased from 1.4 to 2.4 g/L as temperature increased from 180 to 200°C. The suppression of cellulose and hemicellulose hydrolysis by phenolics is dominated by deactivation of the β-glucosidase or β-xylosidase components of cellulase and hemicellulase enzyme by acetone extract at 0.2-0.65 mg phenolics/mg enzyme protein and deactivation of cellulases and hemicellulases by the water soluble components in vacuum filtrate at 0.05-2mg/mg. Inhibition was a function of the type of enzyme and the manner in which the phenolics were extracted from the bagasse. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigation of the donor and acceptor range for chiral carboligation catalyzed by the E1 component of the 2-oxoglutarate dehydrogenase complex

PubMed Central

Patel, Hetalben; Shim, Da Jeong; Farinas, Edgardo T.; Jordan, Frank

2013-01-01

The potential of thiamin diphosphate (ThDP)-dependent enzymes to catalyze C-C bond forming (carboligase) reactions with high enantiomeric excess has been recognized for many years. Here we report the application of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex in the synthesis of chiral compounds with multiple functional groups in good yield and high enantiomeric excess, by varying both the donor substrate (different 2-oxo acids) and the acceptor substrate (glyoxylate, ethyl glyoxylate and methyl glyoxal). Major findings include the demonstration that the enzyme can accept 2-oxovalerate and 2-oxoisovalerate in addition to its natural substrate 2-oxoglutarate, and that the tested acceptors are also acceptable in the carboligation reaction, thereby very much expanding the repertory of the enzyme in chiral synthesis. PMID:24277992

Impact of proteolytic enzymes in colorectal cancer development and progression.

PubMed

Herszényi, László; Barabás, Loránd; Hritz, István; István, Gábor; Tulassay, Zsolt

2014-10-07

Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enzymes (proteinases) are involved in the degradation of extracellular matrix, but also in cancer invasion and metastasis. The four categories of proteinases (cysteine-, serine-, aspartic-, and metalloproteinases) are named and classified according to the essential catalytic component in their active site. We and others have shown that proteolytic enzymes play a major role not only in colorectal cancer (CRC) invasion and metastasis, but also in malignant transformation of precancerous lesions into cancer. Tissue and serum-plasma antigen concentrations of proteinases might be of great value in identifying patients with poor prognosis in CRC. Our results, in concordance with others indicate the potential tumor marker impact of proteinases for the early diagnosis of CRC. In addition, proteinases may also serve as potential target molecules for therapeutic agents.

Regulation of monoamine oxidase A by circadian-clock components implies clock influence on mood.

PubMed

Hampp, Gabriele; Ripperger, Jürgen A; Houben, Thijs; Schmutz, Isabelle; Blex, Christian; Perreau-Lenz, Stéphanie; Brunk, Irene; Spanagel, Rainer; Ahnert-Hilger, Gudrun; Meijer, Johanna H; Albrecht, Urs

2008-05-06

The circadian clock has been implicated in addiction and several forms of depression [1, 2], indicating interactions between the circadian and the reward systems in the brain [3-5]. Rewards such as food, sex, and drugs influence this system in part by modulating dopamine neurotransmission in the mesolimbic dopamine reward circuit, including the ventral tegmental area (VTA) and the ventral striatum (NAc). Hence, changes in dopamine levels in these brain areas are proposed to influence mood in humans and mice [6-10]. To establish a molecular link between the circadian-clock mechanism and dopamine metabolism, we analyzed the murine promoters of genes encoding key enzymes important in dopamine metabolism. We find that transcription of the monoamine oxidase A (Maoa) promoter is regulated by the clock components BMAL1, NPAS2, and PER2. A mutation in the clock gene Per2 in mice leads to reduced expression and activity of MAOA in the mesolimbic dopaminergic system. Furthermore, we observe increased levels of dopamine and altered neuronal activity in the striatum, and these results probably lead to behavioral alterations observed in Per2 mutant mice in despair-based tests. These findings suggest a role of circadian-clock components in dopamine metabolism highlighting a role of the clock in regulating mood-related behaviors.

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

Coelho, Pedro S; Brustad, Eric M; Arnold, Frances H

The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cellsmore » expressing the heme enzymes are also provided by the present invention.« less

Therapeutic and prophylactic applications of bacteriophage components in modern medicine.

PubMed

Adhya, Sankar; Merril, Carl R; Biswas, Biswajit

2014-01-01

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage-bacterial and phage-human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress.

Therapeutic and Prophylactic Applications of Bacteriophage Components in Modern Medicine

PubMed Central

Adhya, Sankar; Merril, Carl R.; Biswas, Biswajit

2014-01-01

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage–bacterial and phage–human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress. PMID:24384811

Genetic Variation and Gene Expression in Antioxidant-Related Enzymes and Risk of Chronic Obstructive Pulmonary Disease: A Systematic Review

PubMed Central

Bentley, Amy R; Emrani, Parastu; Cassano, Patricia A

2011-01-01

Observational epidemiologic studies of dietary antioxidant intake, serum antioxidant concentration, and lung outcomes suggest that lower levels of antioxidant defenses are associated with decreased lung function. Another approach to understanding the role of oxidant/antioxidant imbalance in risk of Chronic Obstructive Pulmonary Disease (COPD) is to investigate the role of genetic variation in antioxidant enzymes, and indeed family-based studies suggest a heritable component to lung disease. Many studies of the genes encoding antioxidant enzymes have considered COPD or COPD-related outcomes, and a systematic review is needed to summarise the evidence to date, and to provide insights for further research. Genetic association studies of antioxidant enzymes and COPD/COPD-related traits, and comparative gene expression studies with disease or smoking as the exposure were systematically identified and reviewed. Antioxidant enzymes considered included enzymes involved in glutathione (GSH) metabolism, in the thioredoxin (TXN) system, superoxide dismutases (SOD), and catalase (CAT). A total of 29 genetic association and 15 comparative gene expression studies met the inclusion criteria. The strongest and most consistent effects were in the genes GCL, GSTM1, GSTP1, and SOD3. This review also highlights the lack of studies for genes of interest, particularly GSR, GGT, and those related to TXN. There were limited opportunities to evaluate a gene’s contribution to disease risk through a synthesis of results from different study designs, as the majority of studies considered either association of sequence variants with disease or effect of disease on gene expression. Network-driven approaches that consider potential interaction between genes and amoung genes, smoke exposure, and antioxidant intake are needed to fully characterise the role of oxidant/antioxidant balance in pathogenesis. PMID:18566111

Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells.

PubMed

De Spirt, Silke; Eckers, Anna; Wehrend, Carina; Micoogullari, Mustafa; Sies, Helmut; Stahl, Wilhelm; Steinbrenner, Holger

2016-02-01

Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1. Copyright © 2015 Elsevier Inc. All rights reserved.

The copper rush of the nineties.

PubMed

Solioz, Marc

2016-09-01

The nineties witnessed the discovery of the copper ATPases, enzymes which transport copper across the cytoplasmic membranes of bacteria and eukaryotes. In the same decade, several other key components of copper homeostasis have also been discovered, like copper chaperones and plasma membrane copper transporters. This has finally led to a molecular understanding of two inherited human diseases related to copper: Menkes disease, manifested by systemic copper deficiency, and Wilson disease, caused by defective secretion of excess copper. A historic perspective and untold stories of the events leading up to these discoveries are presented here.

NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

EPA Science Inventory

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

Chapter 5: Organopollutant Degradation by Wood Decay Basidiomycetes

Treesearch

Yitzhak Hadar; Daniel Cullen

2013-01-01

Wood decay fungi are obligate aerobes, deriving nutrients from the biological ‘combustion’ of wood, using molecular oxygen as terminal electron acceptor (Kirk and Farrell 1987; Blanchette 1991). Non-specific extracellular enzymes are generally viewed as key components in lignin depolymerization. The major enzymes implicated in lignin degradation are lignin peroxidase (...

Pectin: cell biology and prospects for functional analysis.

PubMed

Willats, W G; McCartney, L; Mackie, W; Knox, J P

2001-09-01

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.

Bacterial cellulose hydrolysis in anaerobic environmental subsystems--Clostridium thermocellum and Clostridium stercorarium, thermophilic plant-fiber degraders.

PubMed

Zverlov, Vladimir V; Schwarz, Wolfgang H

2008-03-01

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley beta-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.

Iron Homeostasis in Peripheral Nervous System, Still a Black Box?

PubMed Central

Taveggia, Carla

2014-01-01

Abstract Significance: Iron is the most abundant transition metal in biology and an essential cofactor for many cellular enzymes. Iron homeostasis impairment is also a component of peripheral neuropathies. Recent Advances: During the past years, much effort has been paid to understand the molecular mechanism involved in maintaining systemic iron homeostasis in mammals. This has been stimulated by the evidence that iron dyshomeostasis is an initial cause of several disorders, including genetic and sporadic neurodegenerative disorders. Critical Issues: However, very little has been done to investigate the physiological role of iron in peripheral nervous system (PNS), despite the development of suitable cellular and animal models. Future Directions: To stimulate research on iron metabolism and peripheral neuropathy, we provide a summary of the knowledge on iron homeostasis in the PNS, on its transport across the blood–nerve barrier, its involvement in myelination, and we identify unresolved questions. Furthermore, we comment on the role of iron in iron-related disorder with peripheral component, in demyelinating and metabolic peripheral neuropathies. Antioxid. Redox Signal. 21, 634–648. PMID:24409826

Effects of TiO2 nanoparticles on the aquatic plant Spirodela polyrrhiza: Evaluation of growth parameters, pigment contents and antioxidant enzyme activities.

PubMed

Movafeghi, Ali; Khataee, Alireza; Abedi, Mahboubeh; Tarrahi, Roshanak; Dadpour, Mohammadreza; Vafaei, Fatemeh

2018-02-01

Plants are essential components of all ecosystems and play a critical role in environmental fate of nanoparticles. However, the toxicological impacts of nanoparticles on plants are not well documented. Titanium dioxide nanoparticles (TiO 2 -NPs) are produced worldwide in large quantities for a wide range of purposes. In the present study, the uptake of TiO 2 -NPs by the aquatic plant Spirodela polyrrhiza and the consequent effects on the plant were evaluated. Initially, structural and morphological characteristics of the used TiO 2 -NPs were determined using XRD, SEM, TEM and BET techniques. As a result, an anatase structure with the average crystalline size of 8nm was confirmed for the synthesized TiO 2 -NPs. Subsequently, entrance of TiO 2 -NP S to plant roots was verified by fluorescence microscopic images. Activity of a number of antioxidant enzymes, as well as, changes in growth parameters and photosynthetic pigment contents as physiological indices were assessed to investigate the effects of TiO 2 -NPs on S. polyrrhiza. The increasing concentration of TiO 2 -NPs led to the significant decrease in all of the growth parameters and changes in antioxidant enzyme activities. The activity of superoxide dismutase enhanced significantly by the increasing concentration of TiO 2 -NPs. Enhancement of superoxide dismutase activity could be explained as promoting antioxidant system to scavenging the reactive oxygen species. In contrast, the activity of peroxidase was notably decreased in the treated plants. Reduced peroxidase activity could be attributed to either direct effect of these particles on the molecular structure of the enzyme or plant defense system damage due to reactive oxygen species. Copyright © 2017. Published by Elsevier B.V.

Coproduction of detergent compatible bacterial enzymes and stain removal evaluation.

PubMed

Niyonzima, Francois N; More, Sunil S

2015-10-01

Most of the detergents that are presently produced contain the detergent compatible enzymes to improve and accelerate the washing performance by removing tough stains. The process is environment friendly as the use of enzymes in the detergent formulation reduces the utilization of toxic detergent constituents. The current trend is to use the detergent compatible enzymes that are active at low and ambient temperature in order to save energy and maintain fabric quality. As the detergent compatible bacterial enzymes are used together in the detergent formulation, it is important to co-produce the detergent enzymes in a single fermentation medium as the enzyme stability is assured, and production cost gets reduced enormously. The review reports on the production, purification, characterization and application of detergent compatible amylases, lipases, and proteases are available. However, there is no specific review or minireview on the concomitant production of detergent compatible amylases, lipases, and proteases. In this minireview, the coproduction of detergent compatible enzymes by bacterial species, enzyme stability towards detergents and detergent components, and stain release analysis were discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Applications of micellar enzymology to clean coal technology

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

Walsh, C.T.

1990-10-26

This project is designed to develop methods for pre-combustion coal remediation by implementing recent advances in enzyme biochemistry. The novel approach of this study is incorporation of hydrophilic oxidative enzymes in reverse micelles in an organic solvent. Enzymes from commercial sources or microbial extracts are being investigated for their capacity to remove organic sulfur from coal by oxidation of the sulfur groups, splitting of C-S bonds and loss of sulfur as sulfuric acid Dibenzothiophene (DBT) and ethlyphenylsulfide (EPS) are serving as models of organic sulfur-containing components of coal in initial studies. A goal of this project is to define amore » reverse micelle system that optimizes the catalytic activity of enzymes toward desulfurization of model compounds and ultimately coal samples. Among the variables which will be examined are the surfactant, the solvent, the water:surfactant ration and the pH and ionic strength of the aqueous phase. Studies were carried out with HRP, Type I RZ=1.2 and Type VI RZ=3.2 and laccase from Polyporus versicolor. Substrates for HRP assays included hydrogen peroxide, DBT, DBT sulfoxide, and DBT sulfone. Buffers included sodium phosphate. For formation of reverse micelle solutions the surfactant AOT, di(2-ethyl-hexyl)sodium sulphosuccinate, was obtained from Sigma Chemical Co. Isooctant was used as organic solvent. 12 refs., 5 figs., 3 tabs.« less

A Novel Amidotransferase Required for Lipoic Acid Cofactor Assembly in Bacillus subtilis

PubMed Central

Christensen, Quin H.; Martin, Natalia; Mansilla, Maria C.; de Mendoza, Diego; Cronan, John E.

2011-01-01

SUMMARY In the companion paper (Martin et al., 2011) we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate:protein ligase, respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g., pyruvate dehydrogenase). We show that this function is provided by LipL which catalyzes the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably employs a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures. PMID:21338421

The emerging role of lysosomes in copper homeostasis.

PubMed

Polishchuk, Elena V; Polishchuk, Roman S

2016-09-01

The lysosomal system operates as a focal point where a number of important physiological processes such as endocytosis, autophagy and nutrient sensing converge. One of the key functions of lysosomes consists of regulating the metabolism/homeostasis of metals. Metal-containing components are carried to the lysosome through incoming membrane flows, while numerous transporters allow metal ions to move across the lysosome membrane. These properties enable lysosomes to direct metal fluxes to the sites where metal ions are either used by cellular components or sequestered. Copper belongs to a group of metals that are essential for the activity of vitally important enzymes, although it is toxic when in excess. Thus, copper uptake, supply and intracellular compartmentalization have to be tightly regulated. An increasing number of publications have indicated that these processes involve lysosomes. Here we review studies that reveal the expanding role of the lysosomal system as a hub for the control of Cu homeostasis and for the regulation of key Cu-dependent processes in health and disease.

The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae.

PubMed

Valente, Rita S; Xavier, Karina B

2016-01-15

Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae. Crop losses from bacterial diseases caused by pectolytic bacteria are a major problem in agriculture. By studying the regulatory pathways involved in controlling the expression of plant cell wall-degrading enzymes in Pectobacterium wasabiae, we showed that the Trk potassium transport system plays an important role in the regulation of these pathways. The data presented further identify potassium as an important environmental factor in the regulation of virulence in this plant pathogen. We showed that a reduction in virulence can be achieved by increasing the extracellular concentration of potassium. Therefore, this work highlights how elucidation of the mechanisms involved in regulating virulence can lead to the identification of environmental factors that can influence the outcome of infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

PubMed Central

Valente, Rita S.

2015-01-01

ABSTRACT Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae. IMPORTANCE Crop losses from bacterial diseases caused by pectolytic bacteria are a major problem in agriculture. By studying the regulatory pathways involved in controlling the expression of plant cell wall-degrading enzymes in Pectobacterium wasabiae, we showed that the Trk potassium transport system plays an important role in the regulation of these pathways. The data presented further identify potassium as an important environmental factor in the regulation of virulence in this plant pathogen. We showed that a reduction in virulence can be achieved by increasing the extracellular concentration of potassium. Therefore, this work highlights how elucidation of the mechanisms involved in regulating virulence can lead to the identification of environmental factors that can influence the outcome of infection. PMID:26483524

CYP450s analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development.

PubMed

Ventura, Tomer; Bose, Utpal; Fitzgibbon, Quinn P; Smith, Gregory G; Shaw, P Nicholas; Cummins, Scott F; Elizur, Abigail

2017-07-01

Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Isolation and characterization of novel chitinolytic bacteria

NASA Astrophysics Data System (ADS)

Gürkök, Sümeyra; Görmez, Arzu

2016-04-01

Chitin, a linear polymer of β-1,4-N-acetylglucosamine units, is one of the most abundant biopolymers widely distributed in the marine and terrestrial environments. It is found as a structural component of insects, crustaceans and the cell walls of fungi. Chitinases, the enzymes degrading chitin by cleaving the β-(1-4) bond, have gained increased attention due to their wide range of biotechnological applications, especially for biocontrol of harmful insects and phytopathogenic fungi in agriculture. In the present study, 200 bacterial isolates from Western Anatolia Region of Turkey were screened for chitinolytic activity on agar media amended with colloidal chitin. Based on the chitin hydrolysis zone, 13 isolates were selected for further study. Bacterial isolates with the highest chitinase activity were identified as Acinetobacter calcoaceticus, Arthrobacter oxydans, Bacillus cereus, Bacillus megaterium, Brevibacillus reuszeri, Kocuria erythromyxa, Kocuria rosea, Novosphingobium capsulatum, Rhodococcus bratislaviensis, Rhodococcus fascians and Staphylococcus cohnii by MIS and BIOLOG systems. The next aims of the study are to compare the productivity of these bacteria quantitatively, to purify the enzyme from the most potent producer and to apply the pure enzyme for the fight against the phytopathogenic fungi and harmful insects.

Truffles contain endocannabinoid metabolic enzymes and anandamide.

PubMed

Pacioni, Giovanni; Rapino, Cinzia; Zarivi, Osvaldo; Falconi, Anastasia; Leonardi, Marco; Battista, Natalia; Colafarina, Sabrina; Sergi, Manuel; Bonfigli, Antonella; Miranda, Michele; Barsacchi, Daniela; Maccarrone, Mauro

2015-02-01

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors. Copyright © 2014 Elsevier Ltd. All rights reserved.

PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

PubMed Central

Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M

2006-01-01

Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016

Mechanisms of copper homeostasis in bacteria

PubMed Central

Argüello, José M.; Raimunda, Daniel; Padilla-Benavides, Teresita

2013-01-01

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu+ transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures. PMID:24205499

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

PubMed

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Biochemical and immunochemical analyses of detergent solubilized antigens from membrane vesicles of Aspergillus fumigatus.

PubMed

Piechura, J E; Riefel, R S; Daft, L J

1987-11-01

A membrane vesicle fraction isolated from exponentially growing Aspergillus fumigatus strain Ag 507 cultures was obtained by mechanical disruption of intact Aspergillus cells under specific osmotic conditions followed by a pH fractionation technique. Electron micrographs of the membrane vesicles indicated unit membrane structures free from cell wall material. High glucose-6-phosphatase and low lactate dehydrogenase activities verified the relative purity of the membrane vesicle fraction. Allergic bronchopulmonary aspergillosis (ABPA) patient and normal human sera were incubated with the membrane vesicle fraction followed by colloidal gold tagged rabbit antiserum to human IgG or IgE. Electron micrographs indicated ABPA patient sera possessed specific IgG and IgE antibodies to membranous components. The detergent octyl-beta-D-glucopyranoside was used to extract membrane vesicle components (MC). The enzyme profile of MC compared with cell sap components (CS) showed differences in types of enzymes. Two-dimensional polyacrylamide gel electrophoretic analyses of MC and CS detected components shared as well as unique to each fraction. In crossed immunoelectrophoresis using both rabbit antisera raised to MC and ABPA patient sera, 5 peaks were detected, while analysis of CS using rabbit antisera raised to CS produced 20 major peaks. Immunoelectrophoresis and double immunodiffusion data supported the crossed immunoelectrophoretic data: MC differed from CS. Enzyme-linked immunosorbent assay indicated high specific IgG and IgE antibody levels to MC in ABPA patient sera. Crossed immuno-affinoelectrophoresis with concanavalin A partially characterized the MC, which consist of components which have glycoprotein elements (i.e., containing alpha-D-glucose or alpha-D-mannose).

[Complex enzyme combined with ultrasound extraction technology, physicochemical properties and antioxidant activity of Hedysarum polysaccharides].

PubMed

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.

Evidence of cellulose metabolism by the giant panda gut microbiome.

PubMed

Zhu, Lifeng; Wu, Qi; Dai, Jiayin; Zhang, Shanning; Wei, Fuwen

2011-10-25

The giant panda genome codes for all necessary enzymes associated with a carnivorous digestive system but lacks genes for enzymes needed to digest cellulose, the principal component of their bamboo diet. It has been posited that this iconic species must therefore possess microbial symbionts capable of metabolizing cellulose, but these symbionts have remained undetected. Here we examined 5,522 prokaryotic ribosomal RNA gene sequences in wild and captive giant panda fecal samples. We found lower species richness of the panda microbiome than of mammalian microbiomes for herbivores and nonherbivorous carnivores. We detected 13 operational taxonomic units closely related to Clostridium groups I and XIVa, both of which contain taxa known to digest cellulose. Seven of these 13 operational taxonomic units were unique to pandas compared with other mammals. Metagenomic analysis using ~37-Mbp contig sequences from gut microbes recovered putative genes coding two cellulose-digesting enzymes and one hemicellulose-digesting enzyme, cellulase, β-glucosidase, and xylan 1,4-β-xylosidase, in Clostridium group I. Comparing glycoside hydrolase profiles of pandas with those of herbivores and omnivores, we found a moderate abundance of oligosaccharide-degrading enzymes for pandas (36%), close to that for humans (37%), and the lowest abundance of cellulases and endohemicellulases (2%), which may reflect low digestibility of cellulose and hemicellulose in the panda's unique bamboo diet. The presence of putative cellulose-digesting microbes, in combination with adaptations related to feeding, physiology, and morphology, show that giant pandas have evolved a number of traits to overcome the anatomical and physiological challenge of digesting a diet high in fibrous matter.

'Unknown' proteins and 'orphan' enzymes: the missing half of the engineering parts list--and how to find it.

PubMed

Hanson, Andrew D; Pribat, Anne; Waller, Jeffrey C; de Crécy-Lagard, Valérie

2009-12-14

Like other forms of engineering, metabolic engineering requires knowledge of the components (the 'parts list') of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of 'unknown' proteins and 'orphan' enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the 'missing parts list' problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

Thermostable enzymes as biocatalysts in the biofuel industry.

PubMed

Yeoman, Carl J; Han, Yejun; Dodd, Dylan; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-01-01

Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. Copyright 2010 Elsevier Inc. All rights reserved.

Effect of solar irradiation on extracellular enzymes of Aeromonas proteolytica

NASA Technical Reports Server (NTRS)

Foster, B. G.

1973-01-01

The bacterium Aeromonas proteolytica was selected for studying the effects of solar irradiation on extracellular enzymes because it produces an endopeptidase that is capable of degrading proteins and a hemolysin that is active in lysing human erythrocytes. Possible alterations in the rate of enzyme production in response to the test conditions are currently underway and are not available for this preliminary report. Completed viability studies are indicative that little difference exists among the survival curves derived for cells exposed to various components of ultraviolet irradiation in space.

Thermostable Enzymes as Biocatalysts in the Biofuel Industry

PubMed Central

Yeoman, Carl J.; Han, Yejun; Dodd, Dylan; Schroeder, Charles M.; Mackie, Roderick I.

2015-01-01

Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. PMID:20359453

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

PubMed

Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

2017-10-01

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

A unified model for surface electrocatalysis based on observations with enzymes.

PubMed

Hexter, Suzannah V; Esterle, Thomas F; Armstrong, Fraser A

2014-06-28

Despite being so large, many enzymes are not only excellent electrocatalysts - making possible chemical transformations under almost reversible conditions - but they also facilitate our understanding of electrocatalysis by allowing complex processes to be dissected systematically. The electrocatalytic voltammograms obtained for enzymes attached to an electrode expose fundamental aspects of electrocatalysis that can be addressed in ways that are not available to conventional molecular or surface electrocatalysts. The roles of individual components, each characterisable by diffraction or spectroscopy, can be tested and optimised by genetic engineering. Importantly, unlike small-molecule electrocatalysts (RMM < 1000) that are structurally well-defined but invariably altered by being attached to a surface, the enzyme is a giant, multi-component assembly in which the active site is buried and relatively insensitive to the presence of the electrode and solvent interface. A central assertion is that for a given driving force (electrode potential) a true catalyst has no influence on the direction of the reaction; consequently, 'catalytic bias', i.e. the common observation that an enzyme or indeed any electrocatalyst operates preferentially in one direction, must arise from secondary effects beyond the elementary catalytic cycle. This Perspective highlights and extends a general model for electrocatalysis by surface-confined enzymes, and explains how two secondary effects control the bias: (i) the electrode potential at which electrons enter or leave the catalytic cycle; (ii) potential-dependent interconversions between states of the catalyst differing in catalytic activity due to changes in the composition and arrangements of atoms. The model, which is easily applied to enzymes that have been studied recently, highlights important considerations for understanding and developing surface-confined electrocatalysts.

Exploration of two-enzyme coupled catalysis system using scanning electrochemical microscopy.

PubMed

Wu, Zeng-Qiang; Jia, Wen-Zhi; Wang, Kang; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-12-18

In biological metabolism, a given metabolic process usually occurs via a group of enzymes working together in sequential pathways. To explore the metabolism mechanism requires the understanding of the multienzyme coupled catalysis systems. In this paper, an approach has been proposed to study the kinetics of a two-enzyme coupled reaction using SECM combining numerical simulations. Acetylcholine esterase and choline oxidase are immobilized on cysteamine self-assembled monolayers on tip and substrate gold electrodes of SECM via electrostatic interactions, respectively. The reaction kinetics of this two-enzyme coupled system upon various separation distance precisely regulated by SECM are measured. An overall apparent Michaelis-Menten constant of this enzyme cascade is thus measured as 2.97 mM at an optimal tip-substrate gap distance of 18 μm. Then, a kinetic model of this enzyme cascade is established for evaluating the kinetic parameters of individual enzyme by using the finite element method. The simulated results demonstrate the choline oxidase catalytic reaction is the rate determining step of this enzyme cascade. The Michaelis-Menten constant of acetylcholine esterase is evaluated as 1.8 mM. This study offers a promising approach to exploring mechanism of other two-enzyme coupled reactions in biological system and would promote the development of biosensors and enzyme-based logic systems.

Expression of the autoantigen TRIM33/TIF1γ in skin and muscle of patients with dermatomyositis is upregulated, together with markers of cellular stress.

PubMed

Scholtissek, B; Ferring-Schmitt, S; Maier, J; Wenzel, J

2017-08-01

Dermatomyositis (DM) is an autoimmune disorder associated with a dysregulation of immune homeostasis of both the innate and adaptive immune system. Earlier data suggested that these two arms of the immune system interconnect in DM. In the current study, we analysed the association of autoantigen expression [adaptive system components: Mi2, transcriptional intermediary factor (TIF)1γ, small ubiquitin-like modifier 1 activating enzyme subunit (SAE)1, melanoma differentiation-associated protein (MDA)5] with markers of cellular stress (innate system components: MxA, p53) in skin and muscle (immunohistology and gene expression data, respectively). We found that distinctive self-antigens of DM were elevated in both skin and muscle tissue. In particular, TIF1γ expression was seen in autoimmune diseases including DM, but not in other inflammatory skin disorders. This upregulation was closely associated with p53 expression and type I interferon-regulated inflammation, suggesting that upregulation of autoantigens in the skin and muscle of patients with DM might be driven by cellular stress. Better understanding of these mechanisms could pave the way for new therapeutic concepts focusing on stress reduction. © 2017 British Association of Dermatologists.

Endocannabinoid Signaling in Autism.

PubMed

Chakrabarti, Bhismadev; Persico, Antonio; Battista, Natalia; Maccarrone, Mauro

2015-10-01

Autism spectrum disorder (ASD) is a complex behavioral condition with onset during early childhood and a lifelong course in the vast majority of cases. To date, no behavioral, genetic, brain imaging, or electrophysiological test can specifically validate a clinical diagnosis of ASD. However, these medical procedures are often implemented in order to screen for syndromic forms of the disorder (i.e., autism comorbid with known medical conditions). In the last 25 years a good deal of information has been accumulated on the main components of the "endocannabinoid (eCB) system", a rather complex ensemble of lipid signals ("endocannabinoids"), their target receptors, purported transporters, and metabolic enzymes. It has been clearly documented that eCB signaling plays a key role in many human health and disease conditions of the central nervous system, thus opening the avenue to the therapeutic exploitation of eCB-oriented drugs for the treatment of psychiatric, neurodegenerative, and neuroinflammatory disorders. Here we present a modern view of the eCB system, and alterations of its main components in human patients and animal models relevant to ASD. This review will thus provide a critical perspective necessary to explore the potential exploitation of distinct elements of eCB system as targets of innovative therapeutics against ASD.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid.

PubMed

Stygar, Dominika; Michalczyk, Katarzyna; Dolezych, Bogdan; Nakonieczny, Miroslaw; Migula, Pawel; Zaak, Maria; Sawczyn, Tomasz; Karcz-Socha, Iwona; Kukla, Michal; Zwirska-Korczala, Krystyna; Buldak, Rafal

2013-01-01

In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1 year sublethal exposure to imidacloprid pesticide. Caterpillars - L4 stage (fourth instar, hyperphagic tissue-feeding phase) - were collected from chemically protected white horse chestnut trees 1 year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like β-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, β-glucosidase and β-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, β-glucosidase and β-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut tree significantly affected some of the digestive enzymes activities and - in consequence - also interrelationships between enzymes, what may affect the food digestion. Copyright © 2012 Elsevier Inc. All rights reserved.

Supramolecular catalysis beyond enzyme mimics.

PubMed

Meeuwissen, Jurjen; Reek, Joost N H

2010-08-01

Supramolecular catalysis - the assembly of catalyst species by harnessing multiple weak intramolecular interactions - has, until recently, been dominated by enzyme-inspired approaches. Such approaches often attempt to create an enzyme-like 'active site' and have concentrated on reactions similar to those catalysed by enzymes themselves. Here, we discuss the application of supramolecular assembly to the more traditional transition metal catalysis and to small-molecule organocatalysis. The modularity of self-assembled multicomponent catalysts means that a relatively small pool of catalyst components can provide rapid access to a large number of catalysts that can be evaluated for industrially relevant reactions. In addition, we discuss how catalyst-substrate interactions can be tailored to direct substrates along particular reaction paths and selectivities.

Nonclassical Kinetics of Clonal yet Heterogeneous Enzymes.

PubMed

Park, Seong Jun; Song, Sanggeun; Jeong, In-Chun; Koh, Hye Ran; Kim, Ji-Hyun; Sung, Jaeyoung

2017-07-06

Enzyme-to-enzyme variation in the catalytic rate is ubiquitous among single enzymes created from the same genetic information, which persists over the lifetimes of living cells. Despite advances in single-enzyme technologies, the lack of an enzyme reaction model accounting for the heterogeneous activity of single enzymes has hindered a quantitative understanding of the nonclassical stochastic outcome of single enzyme systems. Here we present a new statistical kinetics and exactly solvable models for clonal yet heterogeneous enzymes with possibly nonergodic state dynamics and state-dependent reactivity, which enable a quantitative understanding of modern single-enzyme experimental results for the mean and fluctuation in the number of product molecules created by single enzymes. We also propose a new experimental measure of the heterogeneity and nonergodicity for a system of enzymes.

Intracellular Distribution of Enzymes of the Cytidine Diphosphate Choline Pathway in Castor Bean Endosperm

PubMed Central

Lord, J. M.; Kagawa, T.; Beevers, Harry

1972-01-01

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 × g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 × g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm3 contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm2. Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm3, and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm3. Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components. Images PMID:4506764

Air Pollution Exposures and Circulating Biomarkers of Effect in a Susceptible Population: Clues to Potential Causal Component mixtures and mechanisms

PubMed Central

Delfino, Ralph J.; Staimer, Norbert; Tjoa, Thomas; Gillen, Daniel L.; Polidori, Andrea; Arhami, Mohammad; Kleinman, Micheal T.; Vaziri, Nosratola D.; Longhurst, John; Sioutas, Constantinos

2009-01-01

Background Mechanisms involving oxidative stress and inflammation have been proposed to explain associations of ambient air pollution with cardiovascular morbidity and mortality. Experimental evidence suggests that organic components and ultrafine particles (UFP) are important. Methods We conducted a panel study of 60 elderly subjects with coronary artery disease living in retirement communities within the Los Angeles, California, air basin. Weekly biomarkers of inflammation included plasma interleukin-6, tumor necrosis factor-α soluble receptor II (sTNF-RII), soluble platelet selectin (sP-selectin), and C-reactive protein (CRP). Biomarkers of erythrocyte antioxidant activity included glutathione peroxidase-1 and superoxide dismutase. Exposures included outdoor home daily particle mass [particulate matter < 0.25, 0.25–2.5, and 2.5–10 μm in aerodynamic diameter (PM0.25, PM0.25–2.5, PM2.5–10)], and hourly elemental and black carbon (EC–BC), estimated primary and secondary organic carbon (OCpri, SOC), particle number (PN), carbon monoxide (CO), and nitrogen oxides–nitrogen dioxide (NOx–NO2). We analyzed the relation of biomarkers to exposures with mixed effects models adjusted for potential confounders. Results Primary combustion markers (EC–BC, OCpri, CO, NOx–NO2), but not SOC, were positively associated with inflammatory biomarkers and inversely associated with erythrocyte anti-oxidant enzymes (n = 578). PN and PM0.25 were more strongly associated with biomarkers than PM0.25–2.5. Associations for all exposures were stronger during cooler periods when only OCpri, PN, and NOx were higher. We found weaker associations with statin (sTNF-RII, CRP) and clopidogrel use (sP-selectin). Conclusions Traffic-related air pollutants are associated with increased systemic inflammation, increased platelet activation, and decreased erythrocyte antioxidant enzyme activity, which may be partly behind air pollutant–related increases in systemic inflammation. Differences in association by particle size, OC fraction, and seasonal period suggest components carried by UFP are important. PMID:19672402

Light-Induced Activation of a Molybdenum Oxotransferase Model within a Ru(II)-Mo(VI) Dyad.

PubMed

Ducrot, Aurélien B; Coulson, Ben A; Perutz, Robin N; Duhme-Klair, Anne-Kathrin

2016-12-19

Nature uses molybdenum-containing enzymes to catalyze oxygen atom transfer (OAT) from water to organic substrates. In these enzymes, the two electrons that are released during the reaction are rapidly removed, one at a time, by spatially separated electron transfer units. Inspired by this design, a Ru(II)-Mo(VI) dyad was synthesized and characterized, with the aim of accelerating the rate-determining step in the cis-dioxo molybdenum-catalyzed OAT cycle, the transfer of an oxo ligand to triphenyl phosphine, via a photo-oxidation process. The dyad consists of a photoactive bis(bipyridyl)-phenanthroline ruthenium moiety that is covalently linked to a bioinspired cis-dioxo molybdenum thiosemicarbazone complex. The quantum yield and luminescence lifetimes of the dyad [Ru(bpy) 2 (L 2 )MoO 2 (solv)] 2+ were determined. The major component of the luminescence decay in MeCN solution (τ = 1149 ± 2 ns, 67%) corresponds closely to the lifetime of excited [Ru(bpy) 2 (phen-NH 2 )] 2+ , while the minor component (τ = 320 ± 1 ns, 31%) matches that of [Ru(bpy) 2 (H 2 -L 2 )] 2+ . In addition, the (spectro)electrochemical properties of the system were investigated. Catalytic tests showed that the dyad-catalyzed OAT from dimethyl sulfoxide to triphenyl phosphine proceeds significantly faster upon irradiation with visible light than in the dark. Methylviologen acts as a mediator in the photoredox cycle, but it is regenerated and hence only required in stoichiometric amounts with respect to the catalyst rather than sacrificial amounts. It is proposed that oxidative quenching of the photoexcited Ru unit, followed by intramolecular electron transfer, leads to the production of a reactive one-electron oxidized catalyst, which is not accessible by electrochemical methods. A significant, but less pronounced, rate enhancement was observed when an analogous bimolecular system was tested, indicating that intramolecular electron transfer between the photosensitizer and the catalytic center is more efficient than intermolecular electron transfer between the separate components.

Accurate Determination of Soluble Axl by Enzyme-Linked Immunosorbent Assay.

PubMed

Dengler, Mirko; Huber, Heidemarie; Müller, Christian J; Zellmer, Angela; Rauch, Peter; Mikulits, Wolfgang

2016-11-01

Levels of soluble Axl (sAxl) are routinely assessed in human sera by sandwich enzyme-linked immunosorbent assay (ELISA). Although sAxl values are suggested to diagnose different types of disorders, no uniform ELISA method is available, allowing the reliable interassay comparison between results. Furthermore, little is known about the stability of sAxl under storage conditions, which is a relevant parameter for biomedical trials. The evaluation of sAxl stability under various stress conditions and the determination of proper conditions to use the sAxl ELISA for routine clinical applications are of great interest. In this study, serum samples were subjected to freeze-thaw cycles and incubation at different temperatures to analyze the stability of sAxl by ELISA. Dilution and spike-in experiments were carried out to examine the impact of serum and diluent components on the ELISA performance. Various diluents and media were employed to resolve masking effects of the serum. The assay components were further optimized for long-term usability by treatment with stabilizers and validation under temperature stress. Indeed, sAxl showed long-term stability in serum during freeze-thaw cycles and incubation under temperature stress conditions. The dilution experiments revealed that unknown components in the serum caused masking effects that can be reduced by proper dilutions. The assay performance was further increased by using a standardized buffer system to dilute serum samples. Stabilization of coated plates and of streptavidin-horseradish peroxidase allowed long-term storage for up to 6 months. In sum, our data demonstrate proper ELISA conditions, allowing the accurate analysis of sAxl levels in human serum.

Reactor concepts for bioelectrochemical syntheses and energy conversion.

PubMed

Krieg, Thomas; Sydow, Anne; Schröder, Uwe; Schrader, Jens; Holtmann, Dirk

2014-12-01

In bioelectrochemical systems (BESs) at least one electrode reaction is catalyzed by microorganisms or isolated enzymes. One of the existing challenges for BESs is shifting the technology towards industrial use and engineering reactor systems at adequate scales. Due to the fact that most BESs are usually deployed in the production of large-volume but low-value products (e.g., energy, fuels, and bulk chemicals), investment and operating costs must be minimized. Recent advances in reactor concepts for different BESs, in particular biofuel cells and electrosynthesis, are summarized in this review including electrode development and first applications on a technical scale. A better understanding of the impact of reactor components on the performance of the reaction system is an important step towards commercialization of BESs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

PubMed

Barig, Susann; Funke, Andreas; Merseburg, Andrea; Schnitzlein, Klaus; Stahmann, K-Peter

2014-06-10

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values. Copyright © 2014 Elsevier Inc. All rights reserved.

Liposomal Encapsulation Enzymes: From Medical Applications to Kinetic Characteristics.

PubMed

Jahadi, M; Khosravi-Darani, K

2017-01-01

Liposomes and nanoliposomes as small vesicles composed of phospholipid bilayer (entrapping one or more hydrophilic or lipophilic components) have recently found several potential applications in medicine and food industry. These vesicles may protect the core materials from moisture, heat and other extreme conditions. They may also provide controlled release of various bioactive agents, including food ingredients at the right place and time. Potential applications of enzyme-loaded liposomes are in the medical or biomedical field, particularly for the enzymereplacement therapy, as well as cheese industry for production of functional foods with improved health beneficial impacts on the consumer. Encapsulation process has a recondite impact on enzymes. In fact, liposome preparation techniques may alter the pH and temperature optima, affinity of the enzyme to substrate (Km), and maximum rate of reaction (Vmax). In addition, in this paper, the impact of process variables on the kinetic characteristics of enzymes encapsulated in liposomes was investigated. Also, the effects of enzyme entrapment in liposomes, prepared by different methods, on the catalytic efficiency of enzyme, as well as its kinetic properties and stability compared to native (free) enzymes has been reviewed.

Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

PubMed

Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

2016-10-15

Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR This study improves our knowledge of the carbohydrate degradation systems of B. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Role of endogenous cortistatin in the regulation of ghrelin system expression at pancreatic level under normal and obese conditions.

PubMed

Chanclón, Belén; Luque, Raúl M; Córdoba-Chacón, José; Gahete, Manuel D; Pozo-Salas, Ana I; Castaño, Justo P; Gracia-Navarro, Francisco; Martínez-Fuentes, Antonio J

2013-01-01

Ghrelin-system components [native ghrelin, In1-ghrelin, Ghrelin-O-acyltransferase enzyme (GOAT) and receptors (GHS-Rs)] are expressed in a wide variety of tissues, including the pancreas, where they exert different biological actions including regulation of neuroendocrine secretions, food intake and pancreatic function. The expression of ghrelin system is regulated by metabolic conditions (fasting/obesity) and is associated with the progression of obesity and insulin resistance. Cortistatin (CORT), a neuropeptide able to activate GHS-R, has emerged as an additional link in gut-brain interplay. Indeed, we recently reported that male CORT deficient mice (cort-/-) are insulin-resistant and present a clear dysregulation in the stomach ghrelin-system. The present work was focused at analyzing the expression pattern of ghrelin-system components at pancreas level in cort-/- mice and their control littermates (cort +/+) under low- or high-fat diet. Our data reveal that all the ghrelin-system components are expressed at the mouse pancreatic level, where, interestingly, In1-ghrelin was expressed at higher levels than native-ghrelin. Thus, GOAT mRNA levels were significantly lower in cort-/- mice compared with controls while native ghrelin, In1-ghrelin and GHS-R transcript levels remained unaltered under normal metabolic conditions. Moreover, under obese condition, a significant increase in pancreatic expression of native-ghrelin, In1-ghrelin and GHS-R was observed in obese cort+/+ but not in cort-/- mice. Interestingly, insulin expression and release was elevated in obese cort+/+, while these changes were not observed in obese cort-/- mice. Altogether, our results indicate that the ghrelin-system expression is clearly regulated in the pancreas of cort+/+ and cort -/- under normal and/or obesity conditions suggesting that this system may play relevant roles in the endocrine pancreas. Most importantly, our data demonstrate, for the first time, that endogenous CORT is essential for the obesity-induced changes in insulin expression/secretion observed in mice, suggesting that CORT is a key regulatory component of the pancreatic function.

Modified kinetics of enzymes interacting with nanoparticles

NASA Astrophysics Data System (ADS)

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

2015-08-01

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

Candidate Chemosensory Genes in the Stemborer Sesamia nonagrioides

PubMed Central

Glaser, Nicolas; Gallot, Aurore; Legeai, Fabrice; Montagné, Nicolas; Poivet, Erwan; Harry, Myriam; Calatayud, Paul-André; Jacquin-Joly, Emmanuelle

2013-01-01

The stemborer Sesamia nonagrioides is an important pest of maize in the Mediterranean Basin. Like other moths, this noctuid uses its chemosensory system to efficiently interact with its environment. However, very little is known on the molecular mechanisms that underlie chemosensation in this species. Here, we used next-generation sequencing (454 and Illumina) on different tissues from adult and larvae, including chemosensory organs and female ovipositors, to describe the chemosensory transcriptome of S. nonagrioides and identify key molecular components of the pheromone production and detection systems. We identified a total of 68 candidate chemosensory genes in this species, including 31 candidate binding-proteins and 23 chemosensory receptors. In particular, we retrieved the three co-receptors Orco, IR25a and IR8a necessary for chemosensory receptor functioning. Focusing on the pheromonal communication system, we identified a new pheromone-binding protein in this species, four candidate pheromone receptors and 12 carboxylesterases as candidate acetate degrading enzymes. In addition, we identified enzymes putatively involved in S. nonagrioides pheromone biosynthesis, including a ∆11-desaturase and different acetyltransferases and reductases. RNAseq analyses and RT-PCR were combined to profile gene expression in different tissues. This study constitutes the first large scale description of chemosensory genes in S. nonagrioides. PMID:23781142

A novel enzyme-based antimicrobial system comprising iodide and a multicopper oxidase isolated from Alphaproteobacterium strain Q-1.

PubMed

Yuliana, Tri; Ebihara, Kyota; Suzuki, Mio; Shimonaka, Chie; Amachi, Seigo

2015-12-01

Alphaproteobacterium strain Q-1 produces an extracellular multicopper oxidase (IOX), which catalyzes iodide (I-) oxidation to form molecular iodine (I2). In this study, the antimicrobial activity of the IOX/iodide system was determined. Both Gram-positive and Gram-negative bacteria tested were killed completely within 5 min by 50 mU mL(-1) of IOX and 10 mM iodide. The sporicidal activity of the system was also tested and compared with a common iodophor, povidone-iodine (PVP-I). IOX (300 mU mL(-1)) killed Bacillus cereus, B. subtilis, and Geobacillus stearothermophilus spores with decimal reduction times of 2.58, 7.62, and 40.9 min, respectively. However, 0.1% PVP-I killed these spores with much longer decimal reduction times of 5.46, 38.0, and 260 min, respectively. To evaluate the more superior sporicidal activity of the IOX system over PVP-I, the amount of free iodine (non-complexed I2) was determined by an equilibrium dialysis technique. The IOX system included more than 40 mg L(-1) of free iodine, while PVP-I included at most 25 mg L(-1) free iodine. Our results suggest that the new enzyme-based antimicrobial system is effective against a wide variety of microorganisms and bacterial spores, and that its strong biocidal activity is due to its high free iodine content, which is probably maintained by re-oxidation of iodide released after oxidation of cell components by I2.

C1q complement component and -antibodies reflect SLE activity and kidney involvement.

PubMed

Horák, P; Hermanová, Z; Zadrazil, J; Ciferská, H; Ordeltová, M; Kusá, L; Zurek, M; Tichý, T

2006-07-01

The role of the complement system in the pathogenesis of systemic diseases is very ambivalent. In systemic lupus erythematosus (SLE), many abnormalities in the activation of the complement system have been reported. The most important antibodies formed against the complement system in SLE are the ones associated with the C1q component. The aim of this study was to assess separately the anti-C1q antibodies and C1q component in the serum from 65 patients with SLE, then in individuals with (n=33) and without (n=32) lupus nephritis and with active (n=36) and nonactive (n=29) form of the disease (European Consensus Lupus Activity Measurement, ECLAM>3, ECLAM

Long-term systemic angiotensin II type 1 receptor blockade regulates mRNA expression of dorsomedial medulla renin-angiotensin system components

PubMed Central

Gilliam-Davis, Shea; Gallagher, Patricia E.; Payne, Valerie S.; Kasper, Sherry O.; Tommasi, Ellen N.; Westwood, Brian M.; Robbins, Michael E.; Chappell, Mark C.

2011-01-01

In Fischer 344 (F344) rats, renin-angiotensin system (RAS) blockade for 1 yr with the angiotensin II type 1 (AT1) receptor blocker L-158,809 prevents age-related impairments in metabolic function, similar to transgenic rats with low glial angiotensinogen (Aogen). Brain RAS regulation may contribute to the benefits of long-term systemic AT1 antagonism. We assessed the mRNA of RAS components in the dorsomedial medulla of F344 rats at 3 (young; n = 8) or 15 mo of age (old; n = 7) and in rats treated from 3 to 15 mo of age with 20 mg/l of the AT1 receptor antagonist L-158,809 (Old+L; n = 6). Aogen and renin mRNA were lower in the young compared with old group. Angiotensin-converting enzyme (ACE) mRNA was lower in the old and Old+L compared with the young group. ACE2 and neprilysin expression were significantly higher in Old+L compared with young or old rats. AT1b, AT2, and Mas receptor mRNA were higher with treatment. Leptin receptor mRNA was lower in the old rats and this was prevented by L-158,809 treatment. Dual-specificity phosphatase 1 (DUSP1) mRNA was highest in the Old+L group. Aggregate correlate summation revealed a positive relationship for Mas receptor mRNA with food intake. The findings provide evidence for regulation of dorsomedial medullary renin and Aogen mRNA during aging. Long-term AT1 receptor blockade increases the mRNA of the enzymes ACE2 and neprilysin and the MAS receptor, which could potentially shift the balance from ANG II to ANG-(1–7) and prevent age-related declines in the leptin receptor and its signaling pathway. PMID:21540301

The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

ERIC Educational Resources Information Center

Splittgerber, A. G.; And Others

1975-01-01

Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

The digestive tract as the origin of systemic inflammation.

PubMed

de Jong, Petrus R; González-Navajas, José M; Jansen, Nicolaas J G

2016-10-18

Failure of gut homeostasis is an important factor in the pathogenesis and progression of systemic inflammation, which can culminate in multiple organ failure and fatality. Pathogenic events in critically ill patients include mesenteric hypoperfusion, dysregulation of gut motility, and failure of the gut barrier with resultant translocation of luminal substrates. This is followed by the exacerbation of local and systemic immune responses. All these events can contribute to pathogenic crosstalk between the gut, circulating cells, and other organs like the liver, pancreas, and lungs. Here we review recent insights into the identity of the cellular and biochemical players from the gut that have key roles in the pathogenic turn of events in these organ systems that derange the systemic inflammatory homeostasis. In particular, we discuss the dangers from within the gastrointestinal tract, including metabolic products from the liver (bile acids), digestive enzymes produced by the pancreas, and inflammatory components of the mesenteric lymph.

The influence of detergents and active components of detergent on bioproduction of organic matters and enzymatic activity of some species of fungi.

PubMed

Stojanović, Jelica; Stojanović, Marina; Iles, Deana; Mijusković, Zoran

2004-01-01

Detergent (Merix, "Merima " Krusevac) applied in concentration of 1% vol. showed specific influence on the bioproduction of some 15 different amino acids and on the enzyme activity of the species of fungi A. niger, A. alternata and T. roseum. Detergent has significantly stimulated the production of 15 analyzed amino acids of the fungi species A. niger. The same applied concentration of detergent has decreased or considerably decreased the production of some 14 of totally 15 analyzed amino acids of investigated fungi species A. alternata and T. roseum. The enzyme activity of the fungi A. niger was more intensive in relation to the species A. alternata and T. roseum during the experimental period or in some phases of the experimental period. The detergent component, ethoxyled oleyl-cetyl alcohol, in concentration of 0.01%, 0.1% and 1% showed an inhibitory effect, or significant inhibitory effect on the enzyme activity of the examined species of fungi (A. niger, A. alternata and T. roseum).

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

PubMed

Chandra, Richard P; Arantes, Valdeir; Saddler, Jack

2015-06-01

The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis. However, the amount of hemicellulose that was solubilised during pretreatment correlated (r(2)=0.98) with the effectiveness of enzyme hydrolysis of each pretreated substrate. Simons's staining, to measure the cellulose accessibility, showed good correlation (r(2)=0.83) with hemicellulose removal and the extent of enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

The immunological capacity in the larvae of Pacific oyster Crassostrea gigas.

PubMed

Song, Xiaorui; Wang, Hao; Xin, Lusheng; Xu, Jiachao; Jia, Zhihao; Wang, Lingling; Song, Linsheng

2016-02-01

As the immune system has not fully developed during early developmental stages, bivalve larvae are more susceptible for pathogens, which frequently leads to the significant mortality in hatcheries. In the present study, the development of immune system and its response against bacteria challenge were investigated in order to characterize the repertoire of immunological capacity of Pacific oyster Crassostrea gigas during the ontogenesis. The phagocytosis was firstly observed in the early D-veliger larvae (17 hpf), especially in their velum site, which indicated the appearance of functional hemocytes during early D-veliger larvae stage. The whole-mount immunofluorescence assay of three pattern recognition receptors (integrin β-1, caspase-3 and C-type lectin 3) and one immune effector gene (IL17-5) was performed in blastula, early D-veliger and umbo larvae, suggested that velum and digestive gland were the potential sites of immune system in the larvae. The lowest activities of antioxidant enzymes (superoxide dismutase and catalase) and hydrolytic enzyme (lysozyme), as well as descended expression levels of 12 immune genes at the transition between embryogenesis and planktonic, indicated that the larvae at hatching (9 hpf) were in hypo-immunity. While the ascending activities of enzymes and expression levels of seven immune genes during the trochophore stage (15 hpf) suggested the initiation of immune system. The steadily increasing trend of all the 12 candidate genes at the early umbo larvae (120 h) hinted that the immune system was well developed at this stage. After bacterial challenge, some immune recognition (TLR4) and immune effector (IL17-5 and defh2) genes were activated in blastula stage (4 hpf), and other immune genes were up regulated in D-veliger larvae, indicating that the zygotic immune system could respond earlier against the bacterial challenge during its development. These results indicated that the cellular and humoral immune components appeared at trochophore stage, and the cellular immune system was activated with its occurrence, while the humoral immune system executed until the early umbo larval stage. The immune system emerged earlier to aid larvae in defending bacterial challenge during the early stages of oyster development. Copyright © 2016 Elsevier Ltd. All rights reserved.

RNA damage in biological conflicts and the diversity of responding RNA repair systems

PubMed Central

Burroughs, A. Maxwell; Aravind, L.

2016-01-01

RNA is targeted in biological conflicts by enzymatic toxins or effectors. A vast diversity of systems which repair or ‘heal’ this damage has only recently become apparent. Here, we summarize the known effectors, their modes of action, and RNA targets before surveying the diverse systems which counter this damage from a comparative genomics viewpoint. RNA-repair systems show a modular organization with extensive shuffling and displacement of the constituent domains; however, a general ‘syntax’ is strongly maintained whereby systems typically contain: a RNA ligase (either ATP-grasp or RtcB superfamilies), nucleotidyltransferases, enzymes modifying RNA-termini for ligation (phosphatases and kinases) or protection (methylases), and scaffold or cofactor proteins. We highlight poorly-understood or previously-uncharacterized repair systems and components, e.g. potential scaffolding cofactors (Rot/TROVE and SPFH/Band-7 modules) with their respective cognate non-coding RNAs (YRNAs and a novel tRNA-like molecule) and a novel nucleotidyltransferase associating with diverse ligases. These systems have been extensively disseminated by lateral transfer between distant prokaryotic and microbial eukaryotic lineages consistent with intense inter-organismal conflict. Components have also often been ‘institutionalized’ for non-conflict roles, e.g. in RNA-splicing and in RNAi systems (e.g. in kinetoplastids) which combine a distinct family of RNA-acting prim-pol domains with DICER-like proteins. PMID:27536007

Mechanisms for kappa reduction and color removal by xylanases

Treesearch

Thomas W. Jeffries; Mark Davis; Brian Rosin; Larry L. Landucci

1998-01-01

Xylanases reduce kappa and release UV- and visibly absorptive materials from kraft pulps. The extents of these actions depend on the origin and processing of the pulp, access of enzymes to the substrate, and the natures of the enzymes. Hexeneuronic acid (HexA) is a component of kraft pulp xylans that accounts for a fraction of the kappa content. It absorbs strongly in...

Designing polymers with sugar-based advantages for bioactive delivery applications.

PubMed

Zhang, Yingyue; Chan, Jennifer W; Moretti, Alysha; Uhrich, Kathryn E

2015-12-10

Sugar-based polymers have been extensively explored as a means to increase drug delivery systems' biocompatibility and biodegradation. Here,we review he use of sugar-based polymers for drug delivery applications, with a particular focus on the utility of the sugar component(s) to provide benefits for drug targeting and stimuli responsive systems. Specifically, numerous synthetic methods have been developed to reliably modify naturally-occurring polysaccharides, conjugate sugar moieties to synthetic polymer scaffolds to generate glycopolymers, and utilize sugars as a multifunctional building block to develop sugar-linked polymers. The design of sugar-based polymer systems has tremendous implications on both the physiological and biological properties imparted by the saccharide units and are unique from synthetic polymers. These features include the ability of glycopolymers to preferentially target various cell types and tissues through receptor interactions, exhibit bioadhesion for prolonged residence time, and be rapidly recognized and internalized by cancer cells. Also discussed are the distinct stimuli-sensitive properties of saccharide-modified polymers to mediate drug release under desired conditions. Saccharide-based systems with inherent pH- and temperature-sensitive properties, as well as enzyme-cleavable polysaccharides for targeted bioactive delivery, are covered. Overall, this work emphasizes inherent benefits of sugar-containing polymer systems for bioactive delivery.

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production.

PubMed

Rahnama, Nooshin; Foo, Hooi Ling; Abdul Rahman, Nor Aini; Ariff, Arbakariya; Md Shah, Umi Kalsom

2014-12-12

Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme. Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.

Maternal hyperthyroidism alters the pattern of expression of cardiac renin-angiotensin system components in rat offspring.

PubMed

Lino, Caroline A; Shibata, Caroline E R; Barreto-Chaves, Maria Luiza M

2014-03-01

Changes in perinatal environment can lead to physiological, morphological, or metabolic alterations in adult life. It is well known that thyroid hormones (TH) are critical for the development, growth, and maturation of organs and systems. In addition, TH interact with the renin-angiotensin system (RAS), and both play a critical role in adult cardiovascular function. The objective of this study was to evaluate the effect of maternal hyperthyroidism on cardiac RAS components in pups during development. From gestational day nine (GD9), pregnant Wistar rats received thyroxine (T4, 12 mg/l in tap water; Hyper group) or vehicle (control group). Dams and pups were killed on GD18 and GD20. Serum concentrations of triiodothyronine (T3) and T4 were higher in the Hyper group than in the control group dams. Cardiac hypertrophy was observed in Hyper pups on GD20. Cardiac angiotensin-converting enzyme (ACE) activity was significantly lower in Hyper pups on both GD18 and GD20, but there was no difference in Ang I/Ang II levels. Ang II receptors expression was higher in the Hyper pup heart on GD18. Maternal hyperthyroidism is associated with alterations in fetal development and altered pattern of expression in RAS components, which in addition to cardiac hypertrophy observed on GD20 may represent an important predisposing factor to cardiovascular diseases in adult life.

Comparison of in vitro systems of protein digestion using either mammal or fish proteolytic enzymes.

PubMed

Moyano, F J; Savoie, L

2001-02-01

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.

Digestive proteolysis in the Colorado potato beetle, Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network.

PubMed

Srp, Jaroslav; Nussbaumerová, Martina; Horn, Martin; Mareš, Michael

2016-11-01

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB. Copyright © 2016 Elsevier Ltd. All rights reserved.

Insulin-egg yolk dispersions in self microemulsifying system.

PubMed

Singnurkar, P S; Gidwani, S K

2008-11-01

Formulation of insulin into a microemulsion very often presents a physicochemical instability during their preparation and storage. In order to overcome this lack of stability and facilitate the handling of these colloidal systems, stabilization of insulin in presence of hydrophobic components of a microemulsion appears as the most promising strategy. The present paper reports the use of egg yolk for stabilization of insulin in self microemulsifying dispersions. Insulin loaded egg yolk self microemulsifying dispersions were prepared by lyophilization followed by dispersion into self microemulsifying vehicle. The physicochemical characterization of selfmicroemulsifying dispersions includes such as insulin encapsulation efficiency, in vitro stability of insulin in presence of proteolytic enzymes and in vitro release. The biological activity of insulin from the dispersion was estimated by enzyme-linked immunosorbant assay and in vivo using Wistar diabetic rats. The particle size ranged 1.023±0.316 μm in diameter and insulin encapsulation efficiency was 98.2±0.9 %. Insulin hydrophobic self microemulsifying dispersions suppressed insulin release in pH 7.4 phosphate buffer and shown to protect insulin from enzymatic degradation in vitro in presence of chymotripsin. Egg yolk encapsulated insulin was bioactive, demonstrated through both in vivo and in vitro.

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

PubMed Central

Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

2011-01-01

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

Cocaine-induced behavioral sensitization decreases the expression of endocannabinoid signaling-related proteins in the mouse hippocampus.

PubMed

Blanco, Eduardo; Galeano, Pablo; Palomino, Ana; Pavón, Francisco J; Rivera, Patricia; Serrano, Antonia; Alen, Francisco; Rubio, Leticia; Vargas, Antonio; Castilla-Ortega, Estela; Decara, Juan; Bilbao, Ainhoa; de Fonseca, Fernando Rodríguez; Suárez, Juan

2016-03-01

In the reward mesocorticolimbic circuits, the glutamatergic and endocannabinoid systems are implicated in neurobiological mechanisms underlying cocaine addiction. However, the involvement of both systems in the hippocampus, a critical region to process relational information relevant for encoding drug-associated memories, in cocaine-related behaviors remains unknown. In the present work, we studied whether the hippocampal gene/protein expression of relevant glutamate signaling components, including glutamate-synthesizing enzymes and metabotropic and ionotropic receptors, and the hippocampal gene/protein expression of cannabinoid type 1 (CB1) receptor and endocannabinoid metabolic enzymes were altered following acute and/or repeated cocaine administration resulting in conditioned locomotion and locomotor sensitization. Results showed that acute cocaine administration induced an overall down-regulation of glutamate-related gene expression and, specifically, a low phosphorylation level of GluA1. In contrast, locomotor sensitization to cocaine produced an up-regulation of several glutamate receptor-related genes and, specifically, an increased protein expression of the GluN1 receptor subunit. Regarding the endocannabinoid system, acute and repeated cocaine administration were associated with an increased gene/protein expression of CB1 receptors and a decreased gene/protein expression of the endocannabinoid-synthesis enzymes N-acyl phosphatidylethanolamine D (NAPE-PLD) and diacylglycerol lipase alpha (DAGLα). These changes resulted in an overall decrease in endocannabinoid synthesis/degradation ratios, especially NAPE-PLD/fatty acid amide hydrolase and DAGLα/monoacylglycerol lipase, suggesting a reduced endocannabinoid production associated with a compensatory up-regulation of CB1 receptor. Overall, these findings suggest that repeated cocaine administration resulting in locomotor sensitization induces a down-regulation of the endocannabinoid signaling that could contribute to the specifically increased GluN1 expression observed in the hippocampus of cocaine-sensitized mice. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Microbial P450 Enzymes in Bioremediation and Drug Discovery: Emerging Potentials and Challenges.

PubMed

Bhattacharya, Sukanta S; Yadav, Jagjit S

2018-01-01

Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and demonstration of their potential in bioremediation of these chemicals. This review is an attempt to summarize the postgenomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Polyamine formation by arginine decarboxylase as a transducer of hormonal, environmental and stress stimuli in higher plants

NASA Technical Reports Server (NTRS)

Galston, A. W.; Flores, H. E.; Kaur-Sawhney, R.

1982-01-01

Recent evidence implicates polyamines including putrescine in the regulation of such diverse plant processes as cell division, embryogenesis and senescence. We find that the enzyme arginine decarboxylase, which controls the rate of putrescine formation in some plant systems, is activated by light acting through P(r) phytochrome as a receptor, by the plant hormone gibberellic acid, by osmotic shock and by other stress stimuli. We therefore propose arginine decarboxylase as a possible transducer of the various initially received tropistic stimuli in plants. The putrescine formed could act by affecting cytoskeletal components.

Applications of micellar enzymology to clean coal technology. [Laccase from Polyporus versicolor

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

Walsh, C.T.

1990-07-24

This project is designed to develop methods for pre-combustion coal remediation by implementing recent advances in enzyme biochemistry. The novel approach of this study is incorporation of hydrophilic oxidative enzymes in reverse micelles in an organic solvent. Enzymes from commercial sources or microbial extracts are being investigated for their capacity to remove organic sulfur from coal by oxidation of the sulfur groups, splitting of C-S bonds and loss of sulfur as sulfuric acid. Dibenzothiophen (DBT) and ethylphenylsulfide (EPS) are serving as models of organic sulfur-containing components of coal in initial studies.

Determining the safety of enzymes used in animal feed.

PubMed

Pariza, Michael W; Cook, Mark

2010-04-01

The purpose of this paper is to provide guidance for evaluating the safety of enzyme preparations used in animal feed. Feed enzymes are typically added to animal feed to increase nutrient bioavailability by acting on feed components prior to or after consumption, i.e., within the gastrointestinal tract. In contrast, food processing enzymes are generally used during processing and then inactivated or removed prior to consumption. The enzymes used in both applications are almost always impure mixtures of active enzyme and other metabolites from the production strain, hence similar safety evaluation procedures for both are warranted. We propose that the primary consideration should be the safety of the production strain and that the decision tree mechanism developed previously for food processing enzymes (Pariza and Johnson, 2001) is appropriate for determining the safety of feed enzymes. Thoroughly characterized non-pathogenic, non-toxigenic microbial strains with a history of safe use in enzyme manufacture are also logical candidates for generating safe strain lineages, from which additional strains may be derived via genetic modification by traditional and non-traditional strategies. For new feed enzyme products derived from a safe strain lineage, it is important to ensure a sufficiently high safety margin for the intended use, and that the product complies with appropriate specifications for chemical and microbial contamination. Copyright 2009 Elsevier Inc. All rights reserved.

Functional interrelationships between the kallikrein-related peptidases family and the classical kinin system in the human neutrophil.

PubMed

Ehrenfeld, Pamela; Bhoola, Kanti D; Matus, Carola E; Figueroa, Carlos D

2018-06-19

In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.

Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

PubMed

Selvaraj, Brinda; Buckel, Wolfgang; Golding, Bernard T; Ullmann, G Matthias; Martins, Berta M

2016-01-01

4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs. © 2016 S. Karger AG, Basel.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

NADPH-generating systems in bacteria and archaea

PubMed Central

Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

2015-01-01

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

Discovery and characterization of heme enzymes from unsequenced bacteria: application to microbial lignin degradation.

PubMed

Brown, Margaret E; Walker, Mark C; Nakashige, Toshiki G; Iavarone, Anthony T; Chang, Michelle C Y

2011-11-16

Bacteria and other living organisms offer a potentially unlimited resource for the discovery of new chemical catalysts, but many interesting reaction phenotypes observed at the whole organism level remain difficult to elucidate down to the molecular level. A key challenge in the discovery process is the identification of discrete molecular players involved in complex biological transformations because multiple cryptic genetic components often work in concert to elicit an overall chemical phenotype. We now report a rapid pipeline for the discovery of new enzymes of interest from unsequenced bacterial hosts based on laboratory-scale methods for the de novo assembly of bacterial genome sequences using short reads. We have applied this approach to the biomass-degrading soil bacterium Amycolatopsis sp. 75iv2 ATCC 39116 (formerly Streptomyces setonii and S. griseus 75vi2) to discover and biochemically characterize two new heme proteins comprising the most abundant members of the extracellular oxidative system under lignin-reactive growth conditions.

Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

NASA Astrophysics Data System (ADS)

Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

1994-03-01

Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone*

PubMed Central

Bartoszewska, Magdalena; Williams, Chris; Kikhney, Alexey; Opaliński, Łukasz; van Roermund, Carlo W. T.; de Boer, Rinse; Veenhuis, Marten; van der Klei, Ida J.

2012-01-01

Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities. PMID:22733816

Application of nitrogen metabolism in autotrophic bacteria to chemosynthetic bioregeneration in space missions, supplement

NASA Technical Reports Server (NTRS)

Wixom, R. L.

1974-01-01

The chemolithotroph, Hydrogenomonas eutropha, was considered as a life support, bioregenerative system. This project focuses on several metabolic functions that are related to the proposed nitrogen cycle between man and this microbe. Specifically this organism has the capability to utilize as the sole nitrogen source such urine components as urea and fifteen individual amino acids, but not nine other amino acids. The effectiveness of utilization was high for many amino acids. Several specific growth inhibitions were also observed. The enzyme that catalyzes the incorporation of ammonia in the medium into amino acids was identified as a NADP-specific, L-glutamate dehydrogenase. This enzyme has a constitutive nature. This organism can synthesize all of its amino acids from carbon dioxide and ammonia. Therefore with the background literature of multiple pathways of individual amino acid biosyntheses, our evidence to date is consistent with the Hydrogeneomonas group having the same pathway of valine-isoleucine formation as the classical E. coli.

Development of a Multi-Point Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the Clostridium thermocellum Cellulosome Protein Complex

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

Dykstra, Andrew B; St. Brice, Lois; Rodriguez, Jr., Miguel

2014-01-01

Clostridium thermocellum has emerged as a leading bioenergy-relevant microbe due to its ability to solubilize cellulose into carbohydrates, mediated by multi-component membrane-attached complexes termed cellulosomes. To probe microbial cellulose utilization rates, it is desirable to be able to measure the concentrations of saccharolytic enzymes and estimate the total amount of cellulosome present on a mass basis. Current cellulase determination methodologies involve labor-intensive purification procedures and only allow for indirect determination of abundance. We have developed a method using multiple reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic and structural components of the cellulosome protein complex in samples ranging in complexitymore » from purified cellulosomes to whole cell lysates, as an alternative to a previously-developed enzyme-linked immunosorbent assay (ELISA) method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision for determination of the mass concentration of cellulosome components.« less

Effect of Valsartan on Cerebellar Adrenomedullin System Dysregulation During Hypertension.

PubMed

Figueira, Leticia; Israel, Anita

2017-02-01

Adrenomedullin (AM) and its receptors components, calcitonin-receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3) are expressed in cerebellum. Cerebellar AM, AM binding sites and receptor components are altered during hypertension, suggesting a role for cerebellar AM in blood pressure regulation. Thus, we assessed the effect of valsartan, on AM and its receptor components expression in the cerebellar vermis of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Additionally, we evaluated AM action on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS) production in cerebellar vermis. Animals were treated with valsartan or vehicle for 11 days. Rats were sacrificed by decapitation; cerebellar vermis was dissected; and AM, CRLR, RAMP1, RAMP2, and RAMP3 expression was quantified by Western blot analysis. CAT, SOD, and GPx activity was determined spectrophotometrically and blood pressure by non-invasive plethysmography. We demonstrate that AM and RAMP2 expression was lower in cerebellum of SHR rats, while CRLR, RAMP1, and RAMP3 expression was higher than those of WKY rats. AM reduced cerebellar CAT, SOD, GPx activities, and TBARS production in WKY rats, but not in SHR rats. Valsartan reduced blood pressure and reversed the altered expression of AM and its receptors components, as well the loss of AM capacity to reduce antioxidant enzyme activity and TBARS production in SHR rats. These findings demonstrate that valsartan is able to reverse the dysregulation of cerebellar adrenomedullinergic system; and they suggest that altered AM system in the cerebellum could represent the primary abnormality leading to hypertension.

Recent advances in material science for developing enzyme electrodes.

PubMed

Sarma, Anil Kumar; Vatsyayan, Preety; Goswami, Pranab; Minteer, Shelley D

2009-04-15

The enzyme-modified electrode is the fundamental component of amperometric biosensors and biofuel cells. The selection of appropriate combinations of materials, such as: enzyme, electron transport mediator, binding and encapsulation materials, conductive support matrix and solid support, for construction of enzyme-modified electrodes governs the efficiency of the electrodes in terms of electron transfer kinetics, mass transport, stability, and reproducibility. This review investigates the varieties of materials that can be used for these purposes. Recent innovation in conductive electro-active polymers, functionalized polymers, biocompatible composite materials, composites of transition metal-based complexes and organometallic compounds, sol-gel and hydro-gel materials, nanomaterials, other nano-metal composites, and nano-metal oxides are reviewed and discussed here. In addition, the critical issues related to the construction of enzyme electrodes and their application for biosensor and biofuel cell applications are also highlighted in this article. Effort has been made to cover the recent literature on the advancement of materials sciences to develop enzyme electrodes and their potential applications for the construction of biosensors and biofuel cells.

Effect of surface modifiers on an ectoenzyme: granulocyte 5'-nucleotidase.

PubMed

Smolen, J E; Karnovsky, M L

1980-05-01

Several agents that react with plasma membranes, namely the native lectins concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin, the modified lectin succinyl concanavalin A, and sodium meta-periodate, inhibited the ecto-5'-nucleotidase of intact guinea pig granulocytes. Stimulation of the enzyme was not observed at any lectin concentration. Inhibition by native lectins could be blocked or reversed by appropriate competing hapten sugars. In the case of concanavalin A, reversal could be achieved at 37 degrees C, but not at 5 degrees C. When lectins were used in combination with each other, the effects were found to be largely independent. However, when concanavalin A and R. communis agglutinin were applied together, complications arose because the former lectin binds to the latter as well as to the cell surface. To avoid some of the complexities inherent in studying intact cell 5'-nucleotidase and to gain additional information about the system, two broken cell enzyme preparations were also examined. The enzyme of plasma membrane-enriched fractions was inhibited by all five agents mentioned above. 5'-Nucleotidase solubilized in sodium deoxycholate was inhibited by the four lectins but stimulated by periodate. The effects of the surface modifiers on kinetic data for all three enzyme preparations are consistent with the hypothesis that direct interactions with the enzyme molecule give rise to changes in Vmax; interactions at membrane sites other than 5'-nucleotidase itself could cause increases in apparent Km values. Effects of interactions of ectoenzymes with plant lectins may serve as models for phenomena that result from cell-cell interactions or from interactions of animal cells with lectin-like components of the cellular environment.

Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides.

PubMed Central

Devonshire, A L

1975-01-01

Acetylcholinesterase from the heads of insecticide-resistant and -susceptible houseflies (Musca domestica L.) was studied in vitro. The enzymes could not be distinguished electrophoretically, and their behaviour on polyacrylamide-disc-gel electrophoresis was influenced by the presence of Triton X-100 in both the homogenate and the gels. In the absence of detergent, the acetylcholinesterase was heterogeneous, but behaved as a single enzyme when it was present. By analogy with studies of acetylcholinesterase from other sources, these observations were attributed to aggregation of the enzyme when not bound by membranes. The enzyme from resistant flies was more slowly inhibited than the susceptible enzyme, bimolecular rate constants (ki) differing by approx. 4-20-fold for a range of organophosphorus compounds. The kinetics of inhibition of acetylcholinesterase were consistent with the results of electrophoresis, i.e. they corresponded to those of a single enzyme in the presence of Triton X-100, but a mixture of enzymes in its absence. The susceptibility of the more sensitive components in these mixtures was determined. Images PLATE 1 PMID:1180906

Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings.

PubMed

Kumar, Linoj; Chandra, Richard; Saddler, Jack

2011-10-01

It is recognized that some form of post-treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post-treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post-treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post-treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post-treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post-treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post-treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post-treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Copyright © 2011 Wiley Periodicals, Inc.

Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry.

PubMed

Wu, Fei; Minteer, Shelley

2015-02-02

It has been hypothesized that the high metabolic flux in the mitochondria is due to the self-assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross-linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low-resolution structure for the three-enzyme complex was achieved, as well as the two-fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein-protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods to identify enzymes that degrade the main extracellular polysaccharide component of Staphylococcus epidermidis biofilms

PubMed Central

Gökçen, Anke; Vilcinskas, Andreas; Wiesner, Jochen

2013-01-01

The production of extracellular poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by Staphylococcus epidermidis is the principal determinant of biofilm formation on indwelling medical devices. Enzymes that degrade PNAG therefore provide an attractive strategy for biofilm removal and for the manufacture of biofilm-resistant coatings. Here we present methods that allow the identification of PNAG-degrading enzymes with the ability to detach biofilms. Our protocol includes the preparation of soluble PNAG from S. epidermidis cultures, the incubation of soluble PNAG with candidate enzymes and assays that detect the release of N-acetyl-d-glucosamine using high-pH anion-exchange chromatography (HPAEC) followed in parallel by pulsed amperometric detection (PAD) and online electrospray ionization mass spectrometry (ESI-MS). We validated our procedures using dispersin B, which is currently the only known PNAG-degrading enzyme. PMID:23357872

[THE SYSTEM OF XENOBIOTICS BIOTRANSFORMATION OF HELMINTHS. RESEMBLANCE AND DIFFERENSES FROM SIMILAR HOST SYSTEMS (REWEW)].

PubMed

Smirnov, L P; Borvinskaya, E V; Suhovskaya, I V

2016-01-01

The three phases system xenobiotic biotransformation in cells as prokaryotes as eukaryotes was formed during the process of evolution. Clear and managed function of all three links of this system guarantee the survival of living organisms at alteration of chemical component of environment. Oxidation, reduction or hydrolysis of xenobiotics realize in phase I by insertion or opening reactive and hydrophilic groups in structure of drug molecule. In phase II xenobiotics or their metabolites from phase I conjugate with endogenic compounds, main of there are glutathione, glucuronic acid, amino acids and sulphates. Active transport of substrata, metabolites and conjugates through cell lipid membranes special transport proteins carry out (phase III). The system of xenobiotics biotransformation of helminths has essential differences from the same of vertebrate hosts. In particular, parasites do not reveal the activity of prime oxidases of phase I, such as CYP or FMO, in spite of the genes of these enzymes in DNA. As this phenomenon displays mainly in adult helminths, living in guts of vertebrates, then the hypothesis was formulated that this effect is related with adaptation to conditions of strong deficiency of oxygen, arise in a process of evolution (Kotze et al., 2006). Literature data testify the existence in helminths of unique forms of enzymes of phase II, the investigation of which present doubtless interest in relation with possible role in adaptation to parasitic mode of life. Notwithstanding that many of helminths GST in greater or lesser degree similar with enzymes of M, P, S and О classes of other organisms, nevertheless they have essential structural differences as compared with enzymes of hosts that makes perspective the search of specific anthelminthics vaccines. Transport of xenobiotics is now considered phase III of biotransformation. It was shown that proteins of this phase (ATP binding cassette transporters (ABC ) of parasites) play a key role in efflux of lipophilic xenobiotics, hydrophilic metabolites and conjugates and take part in forming of anthelminthics resistance. Some of these transporters, such as P-glycoprotein (Pgp), are important for drug resistance of helminths. In particular, a correlation between the level of expression of Pgp and resistance of S. mansoni and F. hepatica to widely used anthelminthics as praziquantel and triclabendazol exist.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

PubMed

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

NASA Astrophysics Data System (ADS)

Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

2012-10-01

We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions.

Exploration of cellular reaction systems.

PubMed

Kirkilionis, Markus

2010-01-01

We discuss and review different ways to map cellular components and their temporal interaction with other such components to different non-spatially explicit mathematical models. The essential choices made in the literature are between discrete and continuous state spaces, between rule and event-based state updates and between deterministic and stochastic series of such updates. The temporal modelling of cellular regulatory networks (dynamic network theory) is compared with static network approaches in two first introductory sections on general network modelling. We concentrate next on deterministic rate-based dynamic regulatory networks and their derivation. In the derivation, we include methods from multiscale analysis and also look at structured large particles, here called macromolecular machines. It is clear that mass-action systems and their derivatives, i.e. networks based on enzyme kinetics, play the most dominant role in the literature. The tools to analyse cellular reaction networks are without doubt most complete for mass-action systems. We devote a long section at the end of the review to make a comprehensive review of related tools and mathematical methods. The emphasis is to show how cellular reaction networks can be analysed with the help of different associated graphs and the dissection into modules, i.e. sub-networks.

Cannabinoid receptor 1 signaling in embryo neurodevelopment

PubMed Central

Psychoyos, Delphine; Vinod, K. Yaragudri; Cao, Jin; Hyson, Richard L.; Wlodarczyk, Bogdan; He, Weimin; Cooper, Thomas B.; Hungund, Basalingappa L.; Finnell, Richard H.

2014-01-01

In utero exposure to THC, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time it is not clearly known whether the eCB system is present prior to neurogenesis. Using an array of biochemical techniques we analyzed the levels of CB1 receptors, eCBs (AEA and 2-AG), and the enzymes (NAPE-PLD, DAGLα, DAGLβ MAGL and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo, prior to neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period. PMID:22311661

A novel method for fabrication of continuous-relief optical elements

NASA Astrophysics Data System (ADS)

Guo, Xiaowei; Du, Jinglei; Chen, Mingyong; Ma, Yanqin; Zhu, Jianhua; Peng, Qinjun; Guo, Yongkang; Du, Chunlei

2005-08-01

A novel method for the fabrication of continuous micro-optical components is presented in this paper. It employs a computer controlled spatial-light-modulator (SLM) as a switchable projection mask and silver-halide sensitized gelatin (SHSG) as recording material. By etching SHSG with enzyme solution, the micro-optical components with relief modulation can be generated through special processing procedures. The principles of digital SLM-based lithography and enzyme etching SHSG are discussed in detail, and microlens arrays, micro axicon-lens arrays and gratings with good profile were achieved. This method is simple, cheap and the aberration in processing procedures can be in-situ corrected in the step of designing mask, so it is a practical method to fabricate continuous profile for low-volume production.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

PubMed Central

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

Functional diversity for biomass deconstruction in family 5 subfamily 5 (GH5_5) of fungal endo-β1,4-glucanases.

PubMed

Li, Bingyao; Walton, Jonathan D

2017-05-01

Endo-β1,4-glucanases in glycosyl hydrolase family 5 (GH5) are ubiquitous enzymes in the multicellular fungi and are common components of enzyme cocktails for biomass conversion. We recently showed that an endo-glucanase of subfamily 5 of GH5 (GH5_5) from Sporotrichum thermophile (StCel5A) was more effective at releasing glucose from pretreated corn stover, when part of an eight-component synthetic enzyme mixture, compared to its closely related counterpart from Trichoderma reesei, TrCel5A. StCel5A and TrCel5A belong to different clades of GH5_5 (GH5_5_1 and GH5_5_2, respectively). To test whether the superior activity of StCel5A was a general property of all enzymes in the GH5_5_2 clade, StCel5A, TrCel5A, and two additional members of each subfamily were expressed in a common host that had been engineered to suppress its native cellulases (T. reesei Δxyr1) and compared against each other alone on pure substrates, in synthetic mixtures on pure substrates, and against each other in synthetic mixtures on real biomass. The results indicated that superiority is a unique property of StCel5A and not of GH5_5_2 generally. The six Cel5A enzymes had significant differences in relative activities on different substrates, in specific activities, and in sensitivities to mannan inhibition. Importantly, the behavior of the six endo-glucanases on pure cellulose substrates did not predict their behavior in combination with other cellulolytic enzymes on a real lignocellulosic biomass substrate.

Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges.

PubMed

Jensen, Michael A; Davis, Ronald W

2018-03-27

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

Gene expression analysis of a critical enzyme in intermediary metabolism in oyster pathogen Perkinsus marinus .

NASA Astrophysics Data System (ADS)

Noell, K.

2016-02-01

A key regulatory component in the Krebs cycle pathway is the mitochondrial aconitase enzyme which has been posited to balance energy needs and oxidative growth total storage via citrate utilization. The presence of a cytosolic aconitase (cAcon) activity which serves as a competitor for citrate substrate has been recognized for years. cAcon is a dual function protein with mutually exclusive roles as a post transcriptional regulator of animal cell iron metabolism or as the cytosolic isoform of the iron sulfur enzyme aconitase. We are interested in establishing the role of this orthologue in Perkinsus marnius metabolism through demonstrating its function as aconitase, by looking at gene expression under certain environmental conditions. P. marinus is a close evolutionary relative of the dinoflagellates and is the causative agent of Dermo disease, which has significantly impacted oyster populations along the eastern seaboard. An understanding of intermediary metabolism will yield important insights into how c-aconitase may be involved in stress response systems such as oxidative tension and metabolite deficiency, which could be used to help aquaculturists alleviate the severe impact of "dermo" on the on the oyster population. This study will present data regarding our preliminary analysis of the gene aconitase and its role in intermediary metabolism.

Effect of Chitosan Coating on the Postharvest Quality and Antioxidant Enzyme System Response of Strawberry Fruit during Cold Storage

PubMed Central

Petriccione, Milena; Mastrobuoni, Francesco; Pasquariello, Maria Silvia; Zampella, Luigi; Nobis, Elvira; Capriolo, Giuseppe; Scortichini, Marco

2015-01-01

The effectiveness of chitosan fruit coating to delay the qualitative and nutraceutical traits of three strawberry cultivars, namely “Candonga”, “Jonica” and “Sabrina”, as well as the effects of chitosan on antioxidant enzymes were evaluated. The fruits were coated with 1% and 2% chitosan solution and stored at 2 °C for nine days. Samples were taken every three days. Physico-chemical (weight loss, soluble solid content and titratable acidity) and nutraceutical (total polyphenol, anthocyanin, flavonoid, ascorbic acid content and antioxidant capacity) properties along with the enzymatic activity (catalase (CAT), ascorbate peroxidase (APX), polyphenol oxidase (PPO), guaiacol peroxidase (GPX) and lipoxygenase (LOX)) were evaluated. Chitosan treatment significantly reduced water loss and delayed the qualitative changes in color, titratable acidity and ascorbic acid content in dose- and cultivar-dependent manners. Additionally, changes in the total polyphenol, anthocyanin and flavonoid contents and the antioxidant capacity of chitosan-coated strawberry fruits were delayed. Chitosan coating enhanced the activity of some antioxidant enzymes, preventing flesh browning and reducing membrane damage. A global view of the responses of the three strawberry cultivars to chitosan coating and storage temperature was obtained using principal component analysis. Chitosan-coated fruit exhibited a slower rate of deterioration, compared to uncoated fruit in all tested cultivars. PMID:28231220

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

PubMed Central

Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

2014-01-01

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals.

PubMed

Fritz, R R; Hodgins, D S; Abell, C W

1976-08-10

Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.

Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo.

PubMed

Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

2016-01-01

Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015. Published by Elsevier B.V.

Biofouling of groundwater systems by Thiothrix spp.

PubMed

Brigmon, R L; Martin, H W; Aldrich, H C

1997-09-01

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.

The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

NASA Technical Reports Server (NTRS)

Huppe, H. C.; de Lamotte-Guery, F.; Buchanan, B. B.

1990-01-01

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

DOEpatents

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.

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

PubMed

Suwannarangsee, Surisa; Bunterngsook, Benjarat; Arnthong, Jantima; Paemanee, Atchara; Thamchaipenet, Arinthip; Eurwilaichitr, Lily; Laosiripojana, Navadol; Champreda, Verawat

2012-09-01

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

PubMed Central

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

Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

PubMed

Francki, Michael G; Hayton, Sarah; Gummer, Joel P A; Rawlinson, Catherine; Trengove, Robert D

2016-02-01

Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

PubMed

Li, Caixia; Culver, Silas A; Quadri, Syed; Ledford, Kelly L; Al-Share, Qusai Y; Ghadieh, Hilda E; Najjar, Sonia M; Siragy, Helmy M

2015-11-01

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis. Copyright © 2015 the American Physiological Society.

TLR and NLRP3 inflammasome expression deregulation in macrophages of adult rats subjected to neonatal malnutrition and infected with methicillin-resistant Staphylococcus aureus.

PubMed

Gomes de Morais, Natália; Barreto da Costa, Thacianna; Bezerra de Lira, Joana Maria; da Cunha Gonçalves de Albuquerque, Suênia; Alves Pereira, Valéria Rêgo; de Paiva Cavalcanti, Milena; Machado Barbosa de Castro, Célia Maria

2017-01-01

Nutritional aggression in critical periods may lead to epigenetic changes that affect gene expression. The aim of this study was to assess the effect of neonatal malnutrition on the expression of toll-like receptor (TLR)-2, TLR-4, and NLRP3 receptors, caspase-1 enzyme, and interleukin (IL)-1 β production in macrophages infected with methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) Staphylococcus aureus. Wistar rats (N = 24) were divided in two distinct groups: nourished (17% casein) and malnourished (8% casein). Four systems were established after the isolation of mononuclear cells: negative control, positive control, MRSA, and MSSA. The plates were incubated at 37°C for 24 h in humidified atmosphere and 5% carbon dioxide. Tests were performed after this period to analyze the expression of standard recognition receptors, caspase-1 enzyme, and the production of IL-1 β. Student's t test and analysis of variance were used in the statistical analysis; P < 0.05 was statistically significant. Malnutrition reduced animal growth and the expression of TLR-2, TLR-4, and NLRP3 receptors, the caspase-1 enzyme, and the IL-1 β levels in macrophages infected with lipopolysaccharides in the present study. However, the interaction between the S. aureus and the macrophages promoted greater gene expression of receptors and enzymes. The neonatal malnutrition model compromised the expression of standard recognition receptors, of the caspase-1 enzyme as well as the production of IL-1 β. However, the S. aureus and neonatal malnutrition combination led to intense transcription of such innate immunity components. Therefore, the deregulation in the expression of TLR and NLRP3 receptors and of the caspase-1 enzyme may induce extensive tissue injury and favor the permanence and spread of these bacteria, especially those that are methicillin resistant. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

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

Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle

2010-07-23

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. Atmore » the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.« less

Pathogenesis of aortic dilatation in mucopolysaccharidosis VII mice may involve complement activation

PubMed Central

Baldo, Guilherme; Wu, Susan; Howe, Ruth A.; Ramamoothy, Meera; Knutsen, Russell H.; Fang, Jiali; Mecham, Robert P.; Liu, Yuli; Wu, Xiaobo; Atkinson, John P.; Ponder, Katherine P.

2012-01-01

Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme β-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding β-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases. PMID:21944884

Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

NASA Astrophysics Data System (ADS)

Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

2017-02-01

The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems.

PubMed

Blandino, A; Macías, M; Cantero, D

2003-07-01

Encapsulation within calcium alginate gel capsules was used to produce a coimmobilized enzyme system. Glucose oxidase (GOD) and catalase (CAT) were chosen as model enzymes. The same values of Vmax and Km app for the GOD encapsulated system and for the GOD-CAT coencapsulated system were calculated. When gel beads and capsules were compared, the same catalyst deactivation sequence for the two enzymes was observed. However, when capsules were employed as immobilization support, GOD efficiencies were higher than for the gel beads. These results were explained in terms of the structure of the capsules.

Macrophage Biochemistry, Activation and Function

DTIC Science & Technology

1981-01-01

vacuolar apparatus become more abundant. Functional capabilities, including phagocytic activity, protein synthesis and surface receptors, also increase...properties of cell components of other tissues has led to the following assignment of marker enzymes to specific macrophage components. This assessment is...subfractions. The surface area of each histogram bar then gives the frac- tional amount of constituent present within each normalized fraction. Distribution

Bone sialoprotein binding to matrix metalloproteinase-2 alters enzyme inhibition kinetics.

PubMed

Jain, Alka; Fisher, Larry W; Fedarko, Neal S

2008-06-03

Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl- N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive K I values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression.

Functional Zonation of the Adult Mammalian Adrenal Cortex

PubMed Central

Vinson, Gavin P.

2016-01-01

The standard model of adrenocortical zonation holds that the three main zones, glomerulosa, fasciculata, and reticularis each have a distinct function, producing mineralocorticoids (in fact just aldosterone), glucocorticoids, and androgens respectively. Moreover, each zone has its specific mechanism of regulation, though ACTH has actions throughout. Finally, the cells of the cortex originate from a stem cell population in the outer cortex or capsule, and migrate centripetally, changing their phenotype as they progress through the zones. Recent progress in understanding the development of the gland and the distribution of steroidogenic enzymes, trophic hormone receptors, and other factors suggests that this model needs refinement. Firstly, proliferation can take place throughout the gland, and although the stem cells are certainly located in the periphery, zonal replenishment can take place within zones. Perhaps more importantly, neither the distribution of enzymes nor receptors suggest that the individual zones are necessarily autonomous in their production of steroid. This is particularly true of the glomerulosa, which does not seem to have the full suite of enzymes required for aldosterone biosynthesis. Nor, in the rat anyway, does it express MC2R to account for the response of aldosterone to ACTH. It is known that in development, recruitment of stem cells is stimulated by signals from within the glomerulosa. Furthermore, throughout the cortex local regulatory factors, including cytokines, catecholamines and the tissue renin-angiotensin system, modify and refine the effects of the systemic trophic factors. In these and other ways it more and more appears that the functions of the gland should be viewed as an integrated whole, greater than the sum of its component parts. PMID:27378832

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor.

PubMed

Strillinger, Eva; Grötzinger, Stefan Wolfgang; Allers, Thorsten; Eppinger, Jörg; Weuster-Botz, Dirk

2016-02-01

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.

PubMed

Matosevic, S; Lye, G J; Baganz, F

2011-09-20

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation. Copyright © 2011 Elsevier B.V. All rights reserved.

Red cell metabolism studies on Skylab

NASA Technical Reports Server (NTRS)

Mengel, C. E.

1977-01-01

Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.

PubMed

Grimaldi, Joseph; Collins, Cynthia H; Belfort, Georges

2016-01-01

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system. © 2015 American Institute of Chemical Engineers.

Structural studies of the molybdenum center of mitochondrial amidoxime reducing component (mARC) by pulsed EPR spectroscopy and 17O-labeling

PubMed Central

Rajapakshe, Asha; Astashkin, Andrei V.; Klein, Eric L.; Reichmann, Debora; Mendel, Ralf R.; Bittner, Florian; Enemark, John H.

2011-01-01

Mitochondrial amidoxime reducing components (mARC-1 and mARC-2) represent a novel group of Mo containing enzymes in eukaryotes. These proteins form the catalytic part of a three-component enzyme complex known to be responsible for the reductive activation of several N-hydroxylated prodrugs. No X-ray crystal structures are available for these enzymes as yet. Previous biochemical investigation by B. Wahl et al. (J. Biol. Chem. 285 (2010) 37847–37859) has revealed that two of the Mo coordination positions are occupied by sulfur atoms from a pyranopterindithiolate (molybdopterin, MPT) cofactor. In this work, we have used continuous wave and pulsed electron paramagnetic resonance (EPR) and density functional theoretical (DFT) calculations to determine the nature of remaining ligands in the Mo(V) state of the active site of mARC-2. The experiments with samples in D2O have identified the exchangeable equatorial ligand as a hydroxyl group. The experiments on samples in H217O-enriched buffer have shown the presence of a slowly exchangeable axial oxo ligand. The comparison of the experimental 1H and 17O hyperfine interactions with those calculated using DFT has shown that the remaining non-exchangeable equatorial ligand is, most likely, protein-derived, and that the possibility of an equatorial oxo ligand can be excluded. PMID:21916412

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

PubMed

Bernal, Claudia; Rodríguez, Karen; Martínez, Ronny

2018-06-09

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

Regulation of hazardous exposure by protective exposure: modulation of phase II detoxification and lipid peroxidation by Camellia sinensis and Swertia chirata.

PubMed

Saha, Prosenjit; Das, Sukta

2003-01-01

Many natural compounds are now known to have a modulatory role on physiological functions and biotransformation reactions involved in the detoxification process, thereby affording protection from cytotoxic, genotoxic, and metabolic actions of environmental toxicants. As part of a programme on evaluation of food, beverage, and traditional medicinal plants for their anticarcinogenic activity, their effects on detoxification enzymes were also studied. The present report deals with Camellia sinensis and Swertia chirata. The effect of water infusions as well as crude and purified components of these plants on glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) was analyzed in mice that were exposed to the chemical carcinogen DMBA. All the four enzymes were found to be activated in different degrees following treatment. The effect of Theaflavin, a component of black tea, was highly significant. The activation of the enzymes was accompanied by significant reduction in lipid peroxidation. The observation suggest the chemopreventive potential of both Camellia sinensis and Swertia chirata. Copyright 2003 Wiley-Liss, Inc.

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

PubMed Central

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

2015-01-01

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component. PMID:26463274

Refractive microlensarray made of silver-halide sensitized gelatin (SHSG) etched by enzyme with SLM-based lithography

NASA Astrophysics Data System (ADS)

Guo, Xiaowei; Chen, Mingyong; Zhu, Jianhua; Ma, Yanqin; Du, Jinglei; Guo, Yongkang; Du, Chunlei

2006-01-01

A novel method for the fabrication of continuous micro-optical components is presented in this paper. It employs a computer controlled digital-micromirror-device(DMD TM) as a switchable projection mask and silver-halide sensitized gelatin (SHSG) as recording material. By etching SHSG with enzyme solution, the micro-optical components with relief modulation can be generated through special processing procedures. The principles of etching SHSG with enzyme and theoretical analysis for deep etching are also discussed in detail, and the detailed quantitative experiments on the processing procedures are conducted to determine optimum technique parameters. A good linear relationship within a depth range of 4μm was experimentally obtained between exposure dose and relief depth. At last, the microlensarray with 256.8μm radius and 2.572μm depth was achieved. This method is simple, cheap and the aberration in processing procedures can be corrected in the step of designing mask, so it is a practical method to fabricate good continuous profile for low-volume production.

Linen Most Useful: Perspectives on Structure, Chemistry, and Enzymes for Retting Flax

PubMed Central

Akin, Danny E.

2013-01-01

The components of flax (Linum usitatissimum) stems are described and illustrated, with reference to the anatomy and chemical makeup and to applications in processing and products. Bast fiber, which is a major economic product of flax along with linseed and linseed oil, is described with particular reference to its application in textiles, composites, and specialty papers. A short history of retting methods, which is the separation of bast fiber from nonfiber components, is presented with emphasis on water retting, field retting (dew retting), and experimental methods. Past research on enzyme retting, particularly by the use of pectinases as a potential replacement for the current commercial practice of field retting, is reviewed. The importance and mechanism of Ca2+ chelators with pectinases in retting are described. Protocols are provided for retting of both fiber-type and linseed-type flax stems with different types of pectinases. Current and future applications are listed for use of a wide array of enzymes to improve processed fibers and blended yarns. Finally, potential lipid and aromatic coproducts derived from the dust and shive waste streams of fiber processing are indicated. PMID:25969769

Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

PubMed

Han, Lei; Liang, Bo; Song, Jianxia

2018-02-01

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

1995-11-28

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

1995-01-01

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Broad spectrum detoxification: the major longevity assurance process regulated by insulin/IGF-1 signaling?

PubMed

Gems, David; McElwee, Joshua J

2005-03-01

Our recent survey of genes regulated by insulin/IGF-1 signaling (IIS) in Caenorhabditis elegans suggests a role for a number of gene classes in longevity assurance. Based on these findings, we propose a model for the biochemistry of longevity assurance and ageing, which is as follows. Ageing results from molecular damage from highly diverse endobiotic toxins. These are stochastic by-products of diverse metabolic processes, of which reactive oxygen species (ROS) are likely to be only one component. Our microarray analysis suggests a major role in longevity assurance of the phase 1, phase 2 detoxification system involving cytochrome P450 (CYP), short-chain dehydrogenase/reductase (SDR) and UDP-glucuronosyltransferase (UGT) enzymes. Unlike superoxide and hydrogen peroxide detoxification, this system is energetically costly, and requires the excretion from the cell of its products. Given such costs, its activity may be selected against, as predicted by the disposable soma theory. CYP and UGT enzymes target lipophilic molecular species; insufficient activity of this system is consistent with age-pigment (lipofuscin) accumulation during ageing. We suggest that IIS-regulated longevity assurance involves: (a) energetically costly detoxification and excretion of molecular rubbish, and (b) conservation of existing proteins via molecular chaperones. Given the emphasis in this theory on investment in cellular waste disposal, and on protein conservation, we have dubbed it the green theory.

DNA-Based Enzyme Reactors and Systems

PubMed Central

Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.

2016-01-01

During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. PMID:28335267

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

PubMed

Songsiriritthigul, Chomphunuch; Buranabanyat, Bancha; Haltrich, Dietmar; Yamabhai, Montarop

2010-04-11

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

A synthetic system for expression of components of a bacterial microcompartment.

PubMed

Sargent, Frank; Davidson, Fordyce A; Kelly, Ciarán L; Binny, Rachelle; Christodoulides, Natasha; Gibson, David; Johansson, Emelie; Kozyrska, Katarzyna; Lado, Lucia Licandro; Maccallum, Jane; Montague, Rachel; Ortmann, Brian; Owen, Richard; Coulthurst, Sarah J; Dupuy, Lionel; Prescott, Alan R; Palmer, Tracy

2013-11-01

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV restriction enzymes.

PubMed

Machnicka, Magdalena A; Kaminska, Katarzyna H; Dunin-Horkawicz, Stanislaw; Bujnicki, Janusz M

2015-10-23

GmrSD is a modification-dependent restriction endonuclease that specifically targets and cleaves glucosylated hydroxymethylcytosine (glc-HMC) modified DNA. It is encoded either as two separate single-domain GmrS and GmrD proteins or as a single protein carrying both domains. Previous studies suggested that GmrS acts as endonuclease and NTPase whereas GmrD binds DNA. In this work we applied homology detection, sequence conservation analysis, fold recognition and homology modeling methods to study sequence-structure-function relationships in the GmrSD restriction endonucleases family. We also analyzed the phylogeny and genomic context of the family members. Results of our comparative genomics study show that GmrS exhibits similarity to proteins from the ParB/Srx fold which can have both NTPase and nuclease activity. In contrast to the previous studies though, we attribute the nuclease activity also to GmrD as we found it to contain the HNH endonuclease motif. We revealed residues potentially important for structure and function in both domains. Moreover, we found that GmrSD systems exist predominantly as a fused, double-domain form rather than as a heterodimer and that their homologs are often encoded in regions enriched in defense and gene mobility-related elements. Finally, phylogenetic reconstructions of GmrS and GmrD domains revealed that they coevolved and only few GmrSD systems appear to be assembled from distantly related GmrS and GmrD components. Our study provides insight into sequence-structure-function relationships in the yet poorly characterized family of Type IV restriction enzymes. Comparative genomics allowed to propose possible role of GmrD domain in the function of the GmrSD enzyme and possible active sites of both GmrS and GmrD domains. Presented results can guide further experimental characterization of these enzymes.

ENZYMATIC PROCESSES USED BY PLANTS TO DEGRADE ORGANIC COMPOUNDS

EPA Science Inventory

This is a review of recent plant enzyme systems that have been studied in uptake and transformation of organic contaminants. General procedures of plant preparation and enzyme isolation are covered. Six plant enzyme systems have been investigated for activity with selected pollut...

Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

PubMed

Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

2016-11-01

The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D. Copyright © 2016 Elsevier Inc. All rights reserved.

Salicylic acid improves root antioxidant defense system and total antioxidant capacities of flax subjected to cadmium.

PubMed

Belkadhi, Aïcha; De Haro, Antonio; Soengas, Pilar; Obregon, Sara; Cartea, Maria Elena; Djebali, Wahbi; Chaïbi, Wided

2013-07-01

Cadmium (Cd) disrupts the normal growth and development of plants, depending on their tolerance to this toxic element. The present study was focused on the impacts of exogenous salicylic acid (SA) on the response and regulation of the antioxidant defense system and membrane lipids to 16-day-old flax plantlets under Cd stress. Exposure of flax to high Cd concentrations led to strong inhibition of root growth and enhanced lipid peroxides, membrane permeability, protein oxidation, and hydrogen peroxide (H2O2) production to varying degrees. Concomitantly, activities of the antioxidant enzymes catalase (CAT, EC 1.11.1.6), guaïcol peroxydase (GPX, EC 1.11.1.7), ascorbate peroxydase (APX, EC 1.11.1.11), and superoxide dismutase (SOD, EC 1.15.1.1), and the total antioxidant capacities (2,2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and ferric reducing antioxidant power (FRAP)) were significantly altered by Cd. In contrast, exogenous SA greatly reduced the toxic effects of Cd on the root growth, antioxidant system, and membrane lipid content. The Cd-treated plantlets pre-soaked with SA exhibited less lipid and protein oxidation and membrane alteration, as well as a high level of total antioxidant capacities and increased activities of antioxidant enzymes except of CAT. These results may suggest that SA plays an important role in triggering the root antioxidant system, thereby preventing membrane damage as well as the denaturation of its components.

Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

PubMed

Wang, Weixue; Liang, Alexandria D; Lippard, Stephen J

2015-09-15

A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of "Fe2O2" intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.

Effects of frying oil and Houttuynia cordata thunb on xenobiotic-metabolizing enzyme system of rodents

PubMed Central

Chen, Ya-Yen; Chen, Chiao-Ming; Chao, Pi-Yu; Chang, Tsan-Ju; Liu, Jen-Fang

2005-01-01

AIM: To evaluate the effects of frying oil and Houttuynia cordata Thunb (H. cordata), a vegetable traditionally consumed in Taiwan, on the xenobiotic-metabolizing enzyme system of rodents. METHODS: Forty-eight Sprague-Dawley rats were fed with a diet containing 0%, 2% or 5% H. cordata powder and 15% fresh soybean oil or 24-h oxidized frying oil (OFO) for 28 d respectively. The level of microsomal protein, total cytochrome 450 content (CYP450) and enzyme activities including NADPH reductase, ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-dealkylase (PROD), aniline hydroxylase (ANH), aminopyrine demethylase (AMD), and quinone reductase (QR) were determined. QR represented phase II enzymes, the rest of the enzymes tested represented phase I enzymes. RESULTS: The oxidized frying oil feeding produced a significant increase in phase I and II enzyme systems, including the content of CYP450 and microsomal protein, and the activities of NADPH reductase, EROD, PROD, ANH, AMD and QR in rats (P<0.05). In addition, the activities of EROD, ANH and AMD decreased and QR increased after feeding with H. cordata in OFO-fed group (P<0.05). The feeding with 2% H. cordata diet showed the most significant effect. CONCLUSION: The OFO diet induces phases I and II enzyme activity, and the 2% H. cordata diet resulted in a better regulation of the xenobiotic-metabolizing enzyme system. PMID:15637750

Simultaneously and separately immobilizing incompatible dual-enzymes on polymer substrate via visible light induced graft polymerization

NASA Astrophysics Data System (ADS)

Zhu, Xing; He, Bin; Zhao, Changwen; Ma, Yuhong; Yang, Wantai

2018-04-01

Developing facile and mild strategy to construct multi-enzymes immobilization system has attracted considerable attentions in recent years. Here a simple immobilization strategy called visible light induced graft polymerization that can simultaneously and separately encapsulate two kinds of enzymes on one polymer film was proposed. Two incompatible enzymes, trypsin and transglutaminase (TGase) were selected as model dual-enzymes system and simultaneously immobilized on two sides of low-density polyethylene (LDPE) film. After immobilization, it was found that more than 90% of the enzymes can be embedded into dual-enzymes loaded film without leakage. And the activities of both separately immobilized enzymes were higher than the activities of mixed co-immobilized enzymes or the sequential immobilized ones. This dual-enzymes loaded film (DEL film) showed excellent recyclability and can retain >87% activities of both enzymes after 4 cycles of utilization. As an example, this DEL film was used to conjugate a prodrug of cytarabine with a target peptide. The successful preparation of expected product demonstrated that the separately immobilized two enzymes can worked well together to catalyze a two-step reaction.

A homolog of Drosophila grainy head is essential for epidermal integrity in mice.

PubMed

Ting, Stephen B; Caddy, Jacinta; Hislop, Nikki; Wilanowski, Tomasz; Auden, Alana; Zhao, Lin-Lin; Ellis, Sarah; Kaur, Pritinder; Uchida, Yoshikazu; Holleran, Walter M; Elias, Peter M; Cunningham, John M; Jane, Stephen M

2005-04-15

The Drosophila cuticle is essential for maintaining the surface barrier defenses of the fly. Integral to cuticle resilience is the transcription factor grainy head, which regulates production of the enzyme required for covalent cross-linking of the cuticular structural components. We report that formation and maintenance of the epidermal barrier in mice are dependent on a mammalian homolog of grainy head, Grainy head-like 3. Mice lacking this factor display defective skin barrier function and deficient wound repair, accompanied by reduced expression of transglutaminase 1, the key enzyme involved in cross-linking the structural components of the superficial epidermis. These findings suggest that the functional mechanisms involving protein cross-linking that maintain the epidermal barrier and induce tissue repair are conserved across 700 million years of evolution.

Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.

1973-01-01

After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased. PMID:4570780

Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase

PubMed Central

Jazayeri, Fatemeh S.; Hosseinkhani, Saman

2017-01-01

Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase. PMID:28672033

Importance of prebiotics in aquaculture as immunostimulants. Effects on immune system of Sparus aurata and Dicentrarchus labrax.

PubMed

Carbone, Donatella; Faggio, Caterina

2016-07-01

Infectious diseases in fish represent a major problem for the aquaculture field as they produce extensive damages and loss. Over the last few years, with increased development of the aquaculture industry, different methods have been used to contrast these pathologies. Common interest has led to the use of components (as additives in diets) that could contrast diseases without causing any negative impact on the environment. These components are represented by prebiotics, probiotics, and plant extracts. In this review, the effects of prebiotics are described. Prebiotics are indigestible fibres fermented by gut enzymes and commensal bacteria, whose beneficial effects are due to the by-products generated from fermentation. The influence of pre-biotics on the immune system of fish is called immunosaccharides. Mannanoligosaccharides (MOS), Fructooligosaccharides (FOS) and Inulin act at different levels in the innate immune response. For example, through phagocytosis, lysozyme activity, and the complement system activity, an increase in fish growth and an amelioration of their health status is brought about. In this review, the use of prebiotics in aquaculture, such as immunostimulants, has been highlighted: particularly in two teleost fish species, Sparus aurata and Dicentrarchus labrax. The results demonstrate that the road is still long and further studies are required, but the use of prebiotics, individually or coupled together, can open the doors to pioneering a new model of alternative components to antimicrobial agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microbial expression of alkaloid biosynthetic enzymes for characterization of their properties.

PubMed

Minami, Hiromichi; Ikezawa, Nobuhiro; Sato, Fumihiko

2010-01-01

A wide variety of secondary metabolites are produced in higher plants. These metabolites are synthesized in specific organs/cells at certain developmental stages and/or under specific environmental conditions. Since these biosynthetic activities are rather restricted and difficult to detect, the biochemical characterization of biosynthetic enzymes involved in secondary metabolism has been limited compared to those involved in primary metabolism. Recently, however, progress in tissue culture and molecular biology has made it easier to study biosynthetic enzymes. Here we describe protocols for expressing some biosynthetic enzymes in Escherichia coli expression systems, since this system is both efficient and cost-effective. First, we describe a standard system for expressing biosynthetic enzymes as a soluble protein under the T7 promoter of the pET expression system in E. coli. In addition, the successful expression of cytochrome P450 in E. coli in an active soluble form with N-terminal modification is discussed, since P450 is the critical enzyme in secondary metabolite biosynthesis.

Combination Approaches to Combat Multi-Drug Resistant Bacteria

PubMed Central

Worthington, Roberta J.; Melander, Christian

2013-01-01

The increasing prevalence of infections caused by multi-drug resistant bacteria is a global health problem that is exacerbated by the dearth of novel classes of antibiotics entering the clinic over the past 40 years. Herein we describe recent developments toward combination therapies for the treatment of multi-drug resistant bacterial infections. These efforts include antibiotic-antibiotic combinations, and the development of adjuvants that either directly target resistance mechanisms such as the inhibition of β-lactamase enzymes, or indirectly target resistance by interfering with bacterial signaling pathways such as two-component systems. We also discuss screening of libraries of previously approved drugs to identify non-obvious antimicrobial adjuvants. PMID:23333434

Transition States and transition state analogue interactions with enzymes.

PubMed

Schramm, Vern L

2015-04-21

Enzymatic transition states have lifetimes of a few femtoseconds (fs). Computational analysis of enzyme motions leading to transition state formation suggests that local catalytic site motions on the fs time scale provide the mechanism to locate transition states. An experimental test of protein fs motion and its relation to transition state formation can be provided by isotopically heavy proteins. Heavy enzymes have predictable mass-altered bond vibration states without altered electrostatic properties, according to the Born-Oppenheimer approximation. On-enzyme chemistry is slowed in most heavy proteins, consistent with altered protein bond frequencies slowing the search for the transition state. In other heavy enzymes, structural changes involved in reactant binding and release are also influenced. Slow protein motions associated with substrate binding and catalytic site preorganization are essential to allow the subsequent fs motions to locate the transition state and to facilitate the efficient release of products. In the catalytically competent geometry, local groups move in stochastic atomic motion on the fs time scale, within transition state-accessible conformations created by slower protein motions. The fs time scale for the transition state motions does not permit thermodynamic equilibrium between the transition state and stable enzyme states. Isotopically heavy enzymes provide a diagnostic tool for fast coupled protein motions to transition state formation and mass-dependent conformational changes. The binding of transition state analogue inhibitors is the opposite in catalytic time scale to formation of the transition state but is related by similar geometries of the enzyme-transition state and enzyme-inhibitor interactions. While enzymatic transition states have lifetimes as short as 10(-15) s, transition state analogues can bind tightly to enzymes with release rates greater than 10(3) s. Tight-binding transition state analogues stabilize the rare but evolved enzymatic geometry to form the transition state. Evolution to efficient catalysis optimized this geometry and its stabilization by a transition state mimic results in tight binding. Release rates of transition state analogues are orders of magnitude slower than product release in normal catalytic function. During catalysis, product release is facilitated by altered chemistry. Compared to the weak associations found in Michaelis complexes, transition state analogues involve strong interactions related to those in the transition state. Optimum binding of transition state analogues occurs when the complex retains the system motions intrinsic to transition state formation. Conserved dynamic motion retains the entropic components of inhibitor complexes, improving the thermodynamics of analogue binding.

Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

NASA Astrophysics Data System (ADS)

Kabulski, Jarod L.

The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein-protein interactions on drug metabolism.

Stability of Ensemble Models Predicts Productivity of Enzymatic Systems

DOE PAGES

Theisen, Matthew K.; Lafontaine Rivera, Jimmy G.; Liao, James C.

2016-03-10

Stability in a metabolic system may not be obtained if incorrect amounts of enzymes are used. Without stability, some metabolites may accumulate or deplete leading to the irreversible loss of the desired operating point. Even if initial enzyme amounts achieve a stable steady state, changes in enzyme amount due to stochastic variations or environmental changes may move the system to the unstable region and lose the steady-state or quasi-steady-state flux. This situation is distinct from the phenomenon characterized by typical sensitivity analysis, which focuses on the smooth change before loss of stability. Here we show that metabolic networks differ significantlymore » in their intrinsic ability to attain stability due to the network structure and kinetic forms, and that after achieving stability, some enzymes are prone to cause instability upon changes in enzyme amounts. We use Ensemble Modelling for Robustness Analysis (EMRA) to analyze stability in four cell-free enzymatic systems when enzyme amounts are changed. Loss of stability in continuous systems can lead to lower production even when the system is tested experimentally in batch experiments. The predictions of instability by EMRA are supported by the lower productivity in batch experimental tests. Finally, the EMRA method incorporates properties of network structure, including stoichiometry and kinetic form, but does not require specific parameter values of the enzymes.« less

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

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

Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.

PubMed

Parry, R J; Hoyt, J C

1997-02-01

Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase.

Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves.

PubMed

Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang

2016-01-15

A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. Copyright © 2015 Elsevier B.V. All rights reserved.

A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

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

Tasayco, M.L.; Prestwich, G.D.

1990-02-25

Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor ofmore » this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.« less

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito.

PubMed

Lai, Yiling; Chen, Huan; Wei, Ge; Wang, Guandong; Li, Fang; Wang, Sibao

2017-08-01

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission. To better understand infection stratagems of insect pathogenic fungi, we analyzed the global gene expression profiling of Beauveria bassiana at 36, 60, 84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing (RNA-Seq). A total of 5,354 differentially expressed genes (DEGs) are identified over the course of fungal infection. When the fungus grows on the mosquito cuticle, up-regulated DEGs include adhesion-related genes involved in cuticle attachment, Pth11-like GPCRs hypothesized to be involved in host recognition, and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle. Once in the mosquito hemocoel, the fungus evades mosquito immune system probably through up-regulating expression of β-1,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls. Moreover, six previous unknown SSCP (small secreted cysteine-rich proteins) are significantly up-regulated, which may serve as "effectors" to suppress host defense responses. B. bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress. At late stage of infection, B. bassiana activates a broad spectrum of genes including nutrient degrading enzymes, some transporters and metabolism pathway components, to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth. These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosquito interactions.

A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells

NASA Astrophysics Data System (ADS)

Kasbawati; Kalondeng, A.; Aris, N.; Erawaty, N.; Azis, M. I.

2018-03-01

Metabolic regulation plays an important role in the metabolic engineering of a cellular process. It is conducted to improve the productivity of a microbial process by identifying the important regulatory nodes of a metabolic pathway such as fermentation pathway. Regulation of enzymes involved in a particular pathway can be held to improve the productivity of the system. In the central metabolism of yeast cell, some enzymes are known as regulating enzymes that can be inhibited to increase the production of ethanol. In this research we study the kinetic modelling of the enzymes in the central pathway of yeast metabolism by taking into consideration the enzyme inhibition effects to the ethanol production. The existence of positive steady state solution and the stability of the system are also analysed to study the property and dynamical behaviour of the system. One stable steady state of the system is produced if some conditions are fulfilled. The conditions concern to the restriction of the maximum reactions of the enzymes in the pyruvate and acetaldehyde branch points. There exists a certain time of fermentation reaction at which a maximum and a minimum ethanol productions are attained after regulating the system. Optimal ethanol concentration is also produced for a certain initial concentration of inhibitor.

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

Theisen, Matthew K.; Lafontaine Rivera, Jimmy G.; Liao, James C.

Stability in a metabolic system may not be obtained if incorrect amounts of enzymes are used. Without stability, some metabolites may accumulate or deplete leading to the irreversible loss of the desired operating point. Even if initial enzyme amounts achieve a stable steady state, changes in enzyme amount due to stochastic variations or environmental changes may move the system to the unstable region and lose the steady-state or quasi-steady-state flux. This situation is distinct from the phenomenon characterized by typical sensitivity analysis, which focuses on the smooth change before loss of stability. Here we show that metabolic networks differ significantlymore » in their intrinsic ability to attain stability due to the network structure and kinetic forms, and that after achieving stability, some enzymes are prone to cause instability upon changes in enzyme amounts. We use Ensemble Modelling for Robustness Analysis (EMRA) to analyze stability in four cell-free enzymatic systems when enzyme amounts are changed. Loss of stability in continuous systems can lead to lower production even when the system is tested experimentally in batch experiments. The predictions of instability by EMRA are supported by the lower productivity in batch experimental tests. Finally, the EMRA method incorporates properties of network structure, including stoichiometry and kinetic form, but does not require specific parameter values of the enzymes.« less

Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

NASA Astrophysics Data System (ADS)

Steen, A. D.

2015-12-01

Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

PubMed

Sornlake, Warasirin; Rattanaphanjak, Phatcharamon; Champreda, Verawat; Eurwilaichitr, Lily; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Fujii, Tatsuya; Inoue, Hiroyuki

2017-07-01

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.

Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

PubMed

Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

2016-12-15

Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. Copyright © 2016 Elsevier Ltd. All rights reserved.

tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase

PubMed Central

Mathevon, Carole; Pierrel, Fabien; Oddou, Jean-Louis; Garcia-Serres, Ricardo; Blondin, Geneviève; Latour, Jean-Marc; Ménage, Stéphane; Gambarelli, Serge; Fontecave, Marc; Atta, Mohamed

2007-01-01

MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification. PMID:17679698

Sequential Reactions of Surface-Tethered Glycolytic Enzymes

PubMed Central

Mukai, Chinatsu; Bergkvist, Magnus; Nelson, Jacquelyn L.; Travis, Alexander J.

2014-01-01

SUMMARY The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices. PMID:19778729

Medium Optimization for the Production of Fibrinolytic Enzyme by Paenibacillus sp. IND8 Using Response Surface Methodology

PubMed Central

Prakash Vincent, Samuel Gnana

2014-01-01

Production of fibrinolytic enzyme by a newly isolated Paenibacillus sp. IND8 was optimized using wheat bran in solid state fermentation. A 25 full factorial design (first-order model) was applied to elucidate the key factors as moisture, pH, sucrose, yeast extract, and sodium dihydrogen phosphate. Statistical analysis of the results has shown that moisture, sucrose, and sodium dihydrogen phosphate have the most significant effects on fibrinolytic enzymes production (P < 0.05). Central composite design (CCD) was used to determine the optimal concentrations of these three components and the experimental results were fitted with a second-order polynomial model at 95% level (P < 0.05). Overall, 4.5-fold increase in fibrinolytic enzyme production was achieved in the optimized medium as compared with the unoptimized medium. PMID:24523635

Isolation of an N-acetyl-DL-phenylalanine beta-naphthyl esterase from rabbit peritoneal polymorphonuclear leukocytes.

PubMed

Tsung, P; Kegeles, S W; Showell, H J; Becker, E L

1975-09-22

An N-acetyl-DL-phenylalanine beta-naphthyl esterase has been purified 26-fold from rabbit peritoneal polymorphonuclear leukocytes. The purified enzyme was inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate. The apparent Km for hydrolysis of N-acetyl-DL-phenylalanine beta-naphthyl ester is 71 muM. Optimal reaction rates were observed at pH 6-8. No divalent cation requirement for the activation of the enzyme activity was observed. The esterase activity was neither inhibited nor stimulated by bacterial factor, complement component C5a, guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) which are attractants or repellents for polymorphonuclear leukocytes. High chemotactic activity was observed in the partially purified fraction of the enzyme. The chemotactic activity, like the enzyme activity, was completely inhibited by 10(-7) M phosphonate.

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

Novel RAAS agonists and antagonists: clinical applications and controversies.

PubMed

Romero, Cesar A; Orias, Marcelo; Weir, Matthew R

2015-04-01

The renin-angiotensin-aldosterone system (RAAS) regulates blood pressure homeostasis and vascular injury and repair responses. The RAAS was originally thought to be an endocrine system critically important in regulating blood pressure homeostasis. Yet, important local forms of the RAAS have been described in many tissues, which are mostly independent of the systemic RAAS. These systems have been associated with diverse physiological functions, but also with inflammation, fibrosis and target-organ damage. Pharmacological modulation of the RAAS has brought about important advances in preventing morbidity and mortality associated with cardiovascular disease. Yet, traditional RAAS blockers such as angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) only reduce the risk of disease progression in patients with established cardiovascular or renal disease by ∼20% compared with other therapies. As more components of the RAAS are described, other potential therapeutic targets emerge, which could provide improved cardiovascular and renal protection beyond that provided by an ACE inhibitor or ARB. This Review summarizes the present and future pharmacological manipulation of this important system.

Load-induced modulation of signal transduction networks.

PubMed

Jiang, Peng; Ventura, Alejandra C; Sontag, Eduardo D; Merajver, Sofia D; Ninfa, Alexander J; Del Vecchio, Domitilla

2011-10-11

Biological signal transduction networks are commonly viewed as circuits that pass along information--in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks--to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets.