Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-β-d-xylanase 10B in complex with xylohexaose

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Najmudin, Shabir, E-mail: shabir@dq.fct.unl.pt [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Pinheiro, Benedita A. [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); Romão, Maria J. [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Prates, José A. M.; Fontes, Carlos M. G. A., E-mail: shabir@dq.fct.unl.pt [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal)

2008-08-01

The N-terminal moiety of C. thermocellum endo-1,4-β-d-xylanase 10B, comprising a carbohydrate-binding module (CBM22-1) and a GH10 E337A mutant domain, has been crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21, contain a dimer in the asymmetric unit and diffract to beyond 2.0 Å resolution. The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32–551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3{sub 2}21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 Å resolution.

Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.

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Beth A Rasala

Full Text Available Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly 'cleaved' at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (~100-fold increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.

Comparison of kinetic characteristics of xylanases from Aspergillus ...

African Journals Online (AJOL)

Arabinoxylans are the predominant non-starch polysaccharides of the cell walls of wheat grain, and can contribute up to 3% of the total polysaccharide content of the flour. Endo-(1-4)-β-xylanase is able to hydrolyze the glycosidic bonds between two xylose units in the xylan backbone during baking process. The use of ...

Heterologous Expression of Xylanase II from Aspergillus usamii in Pichia pastoris

OpenAIRE

Zhou, Chenyan; Wang, Yongtao; Wu, Minchen; Wang, Wu; Li, Dongfeng

2009-01-01

To efficiently produce xylanase for food processing industry, a gene encoding xylanase II (XynII) from Aspergillus usamii has been cloned into the vector pPIC9K and integrated into the genome of Pichia pastoris KM71 by electroporation. By means of minimal dextrose (MD) plates and PCR, the recombinant P. pastoris strains (His+Muts) have been obtained. Activity assay and SDS-PAGE demonstrate that XynII was extracellularly expressed in P. pastoris with the induction of methanol. In shake flask c...

Purification and characterization of endo-xylanases from Aspergillus Niger. III. An enzyme of PL 365

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Fournier, R.A.; Frederick, M.M.; Frederick, J.R.; Reilly, P.J.

1985-04-01

An endo-xylanase (1,4-..beta..-D-xylan xylanohydrolase, EC 3.2.1.8) from Aspergillus niger was purified to homogeneity by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and DEAE-Sephadex A-25 at pH 5.15. The enzyme was active on soluble xylan, on insoluble xylan only after arabinosyl-initiated branch points were removed, and on xylooligosaccharides longer than xylotetraose. There was slight activity on carboxymethyl-cellulose, arabinogalactan, glucomannan, and p-nitrophenyl-..beta..-D- glucopyranoside. The main products of the hydrolysis of soluble and insoluble xylan were oligosaccharides of intermediate length, especially the tri- and pentasaccharides. The isolectric point of the enzyme was 3.65. It had a molecular weight of 2.8 x 10/sup 4/ by SDS-gel electrophoresis, and was high in acidic amino acids but low in those containing sulfur. Highest activity in a 20-min assay at pH 5 was between 40 and 45 degrees C, with an activation energy up to 40 degrees C of 11.1 kJ/mol. The optimum pH for activity was at 5.0. The enzyme was strongly activated by Ca/sup 2 +/. 15 references.

Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

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Néstor García

2017-12-01

Full Text Available The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.

Xylanases, nucleic acids encoding them and methods for making and using them

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Gray, Kevin A.; Dirmeier, Richard

2018-02-27

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

Xylanases, nucleic acids encoding them and methods for making and using them

Science.gov (United States)

Gray, Kevin A; Dirmeier, Reinhard

2013-07-16

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

The Use of Xylanases from Different Microbial Origin in Bread Baking and Their Effects on Bread Qualities

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Al-Widyan, Omar; Khataibeh, Moayad H.; Abu-Alruz, Khaled

Effects of xylanases on bread quality were examined. Enzymes used were endo-xylanase (EC 3.2.1.8) from different sources of microorganisms. Baked loaves were assessed for Loaves volume, colour and staling rate. Xylanases produced from rumen microorganisms M6 had clearly positive effects on loaf volume of bread as well as anti-firming potential. M3 (produced from Trichoderma longibrachiatum) improved crumb softness. The use of xylanase for breadmaking lowered firmness of bread crumb effectively compared with control loaf. It can be summarized that xylanases had significant positive effects on bread characteristics. In particular, they had advantage in retarding the staling rate of bread. It is recommended that the optimum dosage of enzymes, method of application in industrial scale especially with xylanase should be studied further in order to gain the great advantages of enzyme addition in breadmaking.

Homologue expression of a fungal endo-1,4-β-D- xylanase using ...

African Journals Online (AJOL)

Jane

2011-03-07

Mar 7, 2011 ... Hemicellulose is the second source of renewable organic carbon on earth, with a high potential for the recovery of ... SSF, solid-state fermentation; XE, xylanase extract. .... Active fractions were pooled, concentrated and.

Heterologous expression of chaetomium thermophilum xylanase 11-a (ctx 11-a) gene

International Nuclear Information System (INIS)

Wajid, S.; Shahid, S.; Mukhtar, Z.; Mansoor, S.

2009-01-01

Chaetomium has a potential source of xylanase and cellulase enzymes, both of which are required in the treatment of fibre in the poultry feed. The titre of the enzymes needs to be enhanced by using recombinant DNA technology for fulfilling the requirement of the industries. Efforts are made to construct prokaryotic and eukaryotic expression cassettes that can be cloned under specific strong promoters i.e., T7 and AOX1, respectively, and the enhancer elements to get the maximum gene expression. In the present study BL21 E. coli and GS115 Pichia pastoris strains are used as model organisms to express the CtX 11-A gene in the presence of 1 mM IPTG and 100% methanol upto final concentration of 0.5. In case of BL21 expression, the maximum xylanase activity was observed after 1.5 h in the presence of 1% xylose, which was 2.302 U/ml and after 7 h in the presence of 0.5% lactose, was 1.708 U/ml. However, in Pichia pastoris the maximum production of xylanase was 2.904 and 0.006 U/ml as compared to control 0.484 and 0.06 U/ml, respectively. (author)

Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII.

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Takahashi, Yui; Kawabata, Hiroaki; Murakami, Shuichiro

2013-01-01

Xylanases produced by Aspergillus niger are industrially important and many types of xylanases have been reported. Individual xylanases have been well studied for their enzymatic properties, gene cloning, and heterologous expression. However, less attention has been paid to the relationship between xylanase genes carried on the A. niger genome and xylanases produced by A. niger strains. Therefore, we examined xylanase genes encoded on the genome of A. niger E-1 and xylanases produced in culture. Seven putative xylanase genes, xynI-VII (named in ascending order of the molecular masses of the deduced amino acid sequences), were amplified from the strain E-1 genome using primers designed from the genome sequence of A. niger CBS 513.88 by PCR and phylogenetically classified into three clusters. Additionally, culture supernatant analysis by DE52 anion-exchange column chromatography revealed that this strain produced three xylanases, XynII, XynIII, and XynVII, which were identified by N-terminal amino acid sequencing and MALDI-TOF-MS analyses, in culture when gown in 0.5% xylan medium supplemented with 50 mM succinate. Furthermore, XynVII, the only GH family 10 xylanase in A. niger E-1, was purified and characterized. The purified enzyme showed a single band with a molecular mass of 35 kDa by SDS-PAGE. The highest activity of purified XynVII was observed at 55°C and pH 5.5. The enzyme was stable in the broad pH range of 3-10 and up to 60°C and was resistant to most metal ions and modifying regents. XynVII showed high specificity against beechwood xylan with K m and V max values of 2.8 mg mL(-1) and 127 μmol min(-1)mg(-1), respectively. TLC and MALDI-TOF-MS analyses showed that the final hydrolyzed products of the enzyme from beechwood xylan were xylose, xylobiose, and xylotriose substituted with a 4-o-metylglucuronic acid residue.

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

Science.gov (United States)

William R. Kenealy; Thomas W. Jeffries

2003-01-01

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

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Ventorino, Valeria; Ionata, Elena; Birolo, Leila; Montella, Salvatore; Marcolongo, Loredana; de Chiaro, Addolorata; Espresso, Francesco; Faraco, Vincenza; Pepe, Olimpia

2016-01-01

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and

Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis.

Science.gov (United States)

Liu, Xin; Liu, Tengfei; Zhang, Yuebin; Xin, Fengjiao; Mi, Shuofu; Wen, Boting; Gu, Tianyi; Shi, Xinyuan; Wang, Fengzhong; Sun, Lichao

2018-01-10

Xylanases (EC 3.2.1.8) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90 °C with a half-life of about 1 h at 80 °C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.

Characterization of Two Endo-β-1, 4-Xylanases from Myceliophthora thermophila and Their Saccharification Efficiencies, Synergistic with Commercial Cellulase

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Abdul Basit

2018-02-01

Full Text Available The xylanases with high specific activity and resistance to harsh conditions are of high practical value for biomass utilization. In the present study, two new GH11 xylanase genes, MYCTH_56237 and MYCTH_49824, have been cloned from thermophilic fungus Myceliophthora thermophila and expressed in Pichia pastoris. The specific activities of purified xylanases reach approximately 1,533.7 and 1,412.5 U/mg, respectively. Based on multiple template-based homology modeling, the structures of their catalytic domains are predicted. Enzyme activity was more effective in 7.5 L fermentor, yielding 2,010.4 and 2,004.2 U/mL, respectively. Both enzymes exhibit optimal activity at 60°C with pH of 6.0 and 7.0, respectively. Their activities are not affected by EDTA and an array of metal ions. The kinetic constants have been determined for MYCTH_56237 (Km = 8.80 mg/mL, Vmax = 2,380 U/mg and MYCTH_49824 (Km = 5.67 mg/mL, Vmax = 1,750 U/mg. More importantly, both xylanases significantly cooperate with the commercial cellulase Celluclast 1.5 L in terms of the saccharification efficiency. All these biochemical properties of the xylanases offer practical potential for future applications.

Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

Energy Technology Data Exchange (ETDEWEB)

Wang, Wei; Wei, Hui; Alahuhta, Markus; Zhang, Min; Himmel, Michael E.

2016-07-08

To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of

The xylanase inhibitor TAXI-III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants.

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Moscetti, Ilaria; Faoro, Franco; Moro, Stefano; Sabbadin, Davide; Sella, Luca; Favaron, Francesco; D'Ovidio, Renato

2015-08-01

The xylanase inhibitor TAXI-III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI-III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre-incubation of xylanase FGSG_03624 with TAXI-III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non-transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI-III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI-III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI-III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI-III to avoid host cell death. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Molecular Cloning and Sequencing of AlkalophilicCellulosimicrobium cellulans CKMX1 Xylanase Gene Isolated from Mushroom Compost and Characterization of the Gene Product

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Abhishek Walia

2015-12-01

Full Text Available ABSTRACT A xylanolytic bacterium was isolated from mushroom compost by using enrichment technique. Results from the metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16S rDNA sequencing suggested the bacterium to be Cellulosimicrobium cellulans CKMX1. Due to the xylanolytic activity of this bacterium, isolation and characterization of the xylanase gene were attempted. A distinct fragment of about 1671 bp was successfully amplified using PCR and cloned into Escherichia coli DH5α. A BLAST search confirmed that the DNA sequence from the amplified fragment was endo-1, 4-beta-xylanase, which was a member of glycoside hydrolase family 11. It showed 98% homology withCellulosimicrobium sp. xylanase gene (Accession no. FJ859907.1 reported from the gut of Eisenia fetida in Korea. In silicophysico-chemical characterization of amino acid sequence of xylanase showed an open reading frame encoding a 556 amino acid sequence with a molecular weight of 58 kDa and theoretical isolectric point (pI of 4.46 was computed using Expasy's ProtParam server. Secondary and homology based 3D structure of xylanase was analysed using SOPMA and Swiss-Prot software.

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

International Nuclear Information System (INIS)

Santos, Camila Ramos; Meza, Andreia Navarro; Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto; Giesel, Guilherme Menegon; Verli, Hugo; Squina, Fabio Marcio; Prade, Rolf Alexander; Murakami, Mario Tyago

2010-01-01

Research highlights: → The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. → Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. → Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 o C, and exclusively xylobiose at 90 o C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

Energy Technology Data Exchange (ETDEWEB)

Santos, Camila Ramos; Meza, Andreia Navarro [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Giesel, Guilherme Menegon; Verli, Hugo [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Squina, Fabio Marcio [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Prade, Rolf Alexander [Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK (United States); Murakami, Mario Tyago, E-mail: mario.murakami@lnbio.org.br [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil)

2010-12-10

Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

Optimization of moistening solution concentration on xylanase activity in solid state fermentation from oil palm empty fruit bunches

Science.gov (United States)

Mardawati, Efri; Parlan; Rialita, Tita; Nurhadi, Bambang

2018-03-01

Xylanase is an enzyme used in the industrial world, including food industry. Xylanase can be utilized as a 1,4-β-xylosidic endo-hydrolysis catalyst of xylanase, a hemicellulose component for obtaining a xylose monomer. This study aims to determine the optimum concentration of the fermentation medium using Response Surface Method (RSM) in the production of xylanase enzyme from oil palm empty fruit bunches (OPEFB) through solid state fermentation process. The variables varied in this study used factor A (ammonium sulphate concentration 1.0-2.0 g/L), B (concentration of potassium dihydrogen phosphate 1.5-2.5 g/L) and C (urea concentration 0.2 – 0.5 g/L). The data was analysed by using Design Expert version 10.0.1.0 especially CCD with total 17 running including 3 times replicated of canter point. Trichoderma viride was used for the process production of xylanase enzyme. The ratio between substrate and moistening solution used was 0.63 g / mL with temperature of 32.80C, 60 h incubation time. The analysis of enzyme activity was done by DNS method with 1% xylan as substrate. Analysis of protein content in enzyme was done by Bradford method. The optimum of moistening solution concentration in this fermentation was obtained. They are, the ammonium sulphate concentration of 1.5 g/L, potassium dihydrogen phosphate 2.0 g/L and urea 0.35 g/L with activity of 684.70 U/mL, specific activity enzyme xylanase 6261.58 U/mg, protein content 0.1093 U/mg, the model was validated using experiment design with perfect reliability value 0.96.

Role in pathogenesis of two endo-beta-1,4-xylanase genes from the vascular wilt fungus Fusarium oxysporum.

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Gómez-Gómez, E; Ruíz-Roldán, M C; Di Pietro, A; Roncero, M I G; Hera, C

2002-04-01

A gene, xyl4, whose predicted amino acid sequence shows significant homology with family 11 xylanases, was identified from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Expression of xyl4 is induced on oat spelt xylan as the carbon source, subject to carbon catabolite repression and preferentially expressed at alkaline ambient pH. Transcript levels of xyl4 on an inducing carbon source are differentially regulated by the nature and concentration of the nitrogen source. As shown by RT-PCR, xyl4 is expressed by F. oxysporum during the entire cycle of infection on tomato plants. Targeted inactivation of xyl4 and of xyl3, a previously identified gene of F. oxysporum f. sp. lycopersici encoding a family 10 xylanase, had no detectable effect on virulence on tomato plants, demonstrating that both genes are not essential for pathogenicity.

Characterization of a novel xylanase gene from rumen content of Hu sheep.

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Wang, Qian; Luo, Yang; He, Bo; Jiang, Lin-Shu; Liu, Jian-Xin; Wang, Jia-Kun

2015-12-01

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

Structural Analysis of Xylanase from Marine Thermophilic Geobacillus stearothermophilus in Tanjung Api, Poso, Indonesia

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BUDI SAKSONO

2010-12-01

Full Text Available A xylanase gene, xynA, has been cloned from thermophilic strain Geobacillus stearothermophilus, which was isolated from marine Tanjung Api, Indonesia. The polymerase chain reaction product of 1266 bp of xynA gene consisted of 1221 bp open reading frame and encoded 407 amino acids including 30 residues of signal peptide. The sequence exhibited highest identity of 98.7% in the level of amino acid, with an extracellular endo-1,4-â-xylanase from G stearothermophilus T-6 (E-GSX T-6 of the glycoside hydrolase family 10 (GH10. A comparative study between the local strain G. stearothermophilus (GSX L and E-GSX T-6 on homology of amino acid sequence indicated five differents amino acids in the gene. They were Threonine/Alanine (T/A, Asparagine/Aspartate (N/D, Lysine/Asparagine (K/N, Isoleucine/Methionine (I/M, Serine/Threonine (S/T at the position 220, 227, 228, 233, and 245, respectively. Protein structural analysis of those differences suggested that those amino acids may play role in biochemical properties such as enzyme stability, in particular its thermostability.

Cloning of a novel xylanase gene from a newly isolated Fusarium sp. Q7-31 and its expression in Escherichia coli

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Zhan-Ling Xie

2012-03-01

Full Text Available A strain of Q7-31 was isolated from Qinghai-Tibet Plateau and was identified as Fusarium sp. based on its morphological characteristics and ITS rDNA gene sequence analysis. It has the highest capacity of degrading cell wall activity compared with other 11 strains. To do research on its xylanase activity of Fusarium sp. Q7-31 while the degrading the rice cell walls, the complete gene xyn8 that encodes endo-1, 4-β-xylanase secreted by Fusarium sp. Q7-31 was cloned and sequenced. The coding region of the gene is separated by two introns of 56bp and 55bp. It encodes 230 amino acid residues of a protein with a calculated molecular weight of 25.7 kDa. The animo acids sequence of xyn8 gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The nature peptide encodeing cDNA was subcloned into pGEX5x-1 expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3-RIL, and xylanase activity was measured. The expression fusion protein was identified by SDS-PAGE and Western blotting, a new specific band of about 52kDa was identified when induced by IPTG. Enzyme activity assay verified the recombinants proteins as a xylanase. A maxium activity of 2.34U/ mg, the xylanase had optimal activity at pH 6.0 and temperature 40ºC .

Effect of combined xylanase and phytase on growth performance, apparent total tract digestibility, and carcass characteristics in growing pigs fed corn-based diets containing high-fiber coproducts.

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Jang, Y D; Wilcock, P; Boyd, R D; Lindemann, M D

2017-09-01

Phytate has been shown to be an antinutrient, and the feeding of high levels of phytase can break down phytate to improve nutrient utilization and pig performance. Dietary xylanase targets arabinoxylan breakdown, thereby improving energy utilization in pigs. However, the effects of simultaneous supplementation have not been clearly determined. Crossbred pigs ( = 45; mean initial weight, 26.4 ± 0.2 kg) were allotted to 1 of 9 treatments to evaluate the effects of both xylanase (endo-1,4-β xylanase [EC 3.2.1.8]) and phytase (6-phytase [EC 3.1.3.26]) supplementation as follows: 1) positive control (PC), a corn-soybean meal-based diet with 15% corn distillers dried grains with solubles, 15% wheat middlings, and 13% corn germ meal; 2) negative control (NC), ME was reduced by 103 kcal/kg from the PC diet by replacement of fat with corn starch; 3) NC + phytase (500 phytase units (FTU)/kg diet); 4) NC + phytase (1,000 FTU/kg diet); 5) NC + phytase (2,000 FTU/kg diet); 6) NC + xylanase (24,000 xylanase units [BXU]/kg diet); 7) NC + phytase (500 FTU/kg diet) + xylanase (24,000 BXU/kg diet); 8) NC + phytase (1,000 FTU/kg diet) + xylanase (24,000 BXU/kg diet); and 9) NC + phytase (2,000 FTU/kg diet) + xylanase (24,000 BXU/kg diet). All diets were formulated to meet nutrient requirements before phytase and xylanase addition to the diets. There were no significant interactions between xylanase and phytase supplementation on growth performance, carcass characteristics, and apparent total tract digestibility (ATTD). The ADG ( phytase level increased. The ATTD of P increased as phytase supplementation level increased ( phytase level increased. Estimated carcass lean percentage and lean gain increased ( phytase level increased. Xylanase supplementation had no effect on growth performance, ATTD, and carcass characteristics. The results demonstrated an improved nutrient digestibility, performance, and carcass response to phytase supplementation beyond P provision because all diets

Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

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Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh

2018-03-01

Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

Xylanase production by Trichoderma harzianum E58

Energy Technology Data Exchange (ETDEWEB)

Senior, D.J.; Mayers, P.R.; Saddler, J.N. (Fortintek Canada Corp., Ottawa, ON (Canada). Dept. of Biotechnology and Chemistry)

1989-12-01

Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles of the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, watersoluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose. (orig.).

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

NARCIS (Netherlands)

Rohman, Ali; van Oosterwijk, Niels; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

2007-01-01

The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of

Xylanases of thermophilic bacteria from Icelandic hot springs

Energy Technology Data Exchange (ETDEWEB)

Pertulla, M; Raettoe, M; Viikari, L [VTT, Biotechnical Lab., Espoo (Finland); Kondradsdottir, M [Dept. of Biotechnology, Technological Inst. of Iceland, Reykjavik (Iceland); Kristjansson, J K [Dept. of Biotechnology, Technological Inst. of Iceland, Reykjavik (Iceland) Inst. of Biotechnology, Iceland Univ., Reykjavik (Iceland)

1993-02-01

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80deg C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. [beta]-Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70deg C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70deg C, although xylan depolymerization was detected even up to 90deg C. (orig.).

Thermophilic xylanases: from bench to bottle.

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Basit, Abdul; Liu, Junquan; Rahim, Kashif; Jiang, Wei; Lou, Huiqiang

2018-01-17

Lignocellulosic biomass is a valuable raw material. As technology has evolved, industrial interest in new ways to take advantage of this raw material has grown. Biomass is treated with different microbial cells or enzymes under ideal industrial conditions to produce the desired products. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are excellent sources of industrially relevant thermostable enzymes that can withstand the harsh conditions of industrial processing. Thermostable xylanases display high-specific activity at elevated temperatures and distinguish themselves in biochemical properties, structures, and modes of action from their mesophilic counterparts. Natural xylanases can be further improved through genetic engineering. Rapid progress with genome editing, writing, and synthetic biological techniques have provided unlimited potential to produce thermophilic xylanases in their natural hosts or cell factories including bacteria, yeasts, and filamentous fungi. This review will discuss the biotechnological potential of xylanases from thermophilic microorganisms and the ways they are being optimized and produced for various industrial applications.

Characterization of Xylanase Streptomyces spp. SKK1-8

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ANJA MERYANDINI

2006-12-01

Full Text Available Streptomyces spp. SKK1-8 producing xylanase was isolated from soil sample from Sukabumi West Java. The xylanase have an optimum condition at pH 6 and 50 °C. Addition of 5 mM Cu2+ decreased the xylanase activity up to about 77%, whereas not by other cations. The xylanase was stable at 3 °C for 48 hours, and the enzyme half lifetime was 1 hour 45 minute at 50 °C. This xylanase showed the highest activity on oatspelt xylan, and their molecular masses were estimated approximately 16.80, 15.21, and 13.86 kDa. HPLC analysis showed that xylosa and arabinosa were the main hydrolytic product of birchwood xylan.

Xylanases and Their Applications in Baking Industry

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Masood Sadiq Butt

2008-01-01

Full Text Available Xylan is the second most abundant polysaccharide and a major component of plant cell wall. Cereal xylans contain large quantities of L-arabinose and are therefore, often referred to as arabinoxylans. Xylanases are hydrolytic enzymes, which randomly cleave the β-1,4 backbone of this complex plant cell wall polysaccharide. Different species of Aspergillus and Trichoderma produce these enzymes. Xylanases are of great value in baking as they have been found to improve the bread volume, crumb structure and reduce stickiness. When xylanases are used at optimum levels, they play a significant role in increasing shelf life of bread and reduce bread staling. There is an increasing trend in baking industry towards the application of xylanases in bread production. This review discusses the application of xylanase in the bakery industry, alone and in combination with other enzymes when it shows synergism in the action with them.

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production

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Giardina Thierry

2011-04-01

Full Text Available Abstract Background The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH. The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. Results High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX was maximal at pH 5.0 with Km(app and kcat/Km(app of 3.7 ± 0.2 (mg.mL-1 and 132 (s-1mg-1.mL, respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS. The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. Conclusion Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.

Characterization of endo-β-mannanase from Enterobacter ludwigii MY271 and application in pulp industry.

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Yang, Miao; Cai, Jun; Wang, Changgao; Du, Xin; Lin, Jianguo

2017-01-01

β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses. An endo-β-mannanase from Enterobacter ludwigii MY271 was purified at 11.7 ± 0.2-fold to homogeneity with a final recovery of 15.2 ± 0.2 %. Using purified β-mannanase protein and SDS-PAGE, the molecular mass was found to be 43.16 kDa. The optimal pH and temperature of the enzyme was found to be 7.0 and 55 °C, respectively. The β-mannanase activity was stable over a broad pH range of pH 2.0-10.0. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents, such as Mg 2+ , L-cysteine, glutathione (GSH) and β-mercaptoethanol. Whereas the enzyme was strongly inhibited by Hg 2+ , Cu 2+ , N-bromosuccinimide (NBS), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide (EDC), phenylmethanesulfonyl fluoride (PMSF), and sodium dodecyl sulfate (SDS). The β-mannanase was highly active towards glucomannan, and showed endo-activity by producing a mixture of oligosaccharides. Moreover, the enzyme displayed a classical endo-type mode on mannooligosaccharides. The β-mannanase coupled with xylanase significantly improved the brightness of kraft pulp, whereas it has no remarkable effect on the tensile strength of the pulp. Our functional studies of the purified β-mannanase indicate that the enzyme is beneficial to industrial applications, in particular, biotechnological processes, such as food, feed and pulp industry.

Production and characterization of xylanase from Thielaviopsis basicola

Energy Technology Data Exchange (ETDEWEB)

Ghosh, V K; Deb, J K

1988-08-01

The black rot fungus Thielaviopsis basicola has the ability to grow on cellulosic biomass, producing xylanase. Of the four cellulosic substrates tested, rice straw was found to be the best for production of xylanase. A xylanase activity of 34 U/ml was obtained with rice straw which was more than three times that obtained with larchwood xylan. The ..beta..-xylosidase activities obtained with these two substrates were 0.05 U/ml and 0.016 U/ml respectively. Both enzymes are active at pH 5 but the temperature optima of xylanase and ..beta..-xylosidase activities are 60/sup 0/C and 40/sup 0/C respectively. The xylanase activity is stable over a pH range of 4-8 but the stability towards temperature falls sharply above 50/sup 0/C.

Heterologous Acidothermus cellulolyticus 1,4-β-Endoglucanase E1 Produced Within the Corn Biomass Converts Corn Stover Into Glucose

Science.gov (United States)

Ransom, Callista; Balan, Venkatesh; Biswas, Gadab; Dale, Bruce; Crockett, Elaine; Sticklen, Mariam

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced, heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active from.

The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants.

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Immaculada Llop-Tous

Full Text Available BACKGROUND: Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs. METHODOLOGY/PRINCIPAL FINDINGS: Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. CONCLUSION/SIGNIFICANCE: In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low

Leishmania infantum EndoG is an endo/exo-nuclease essential for parasite survival.

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Eva Rico

Full Text Available EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

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Moscetti, Ilaria; Tundo, Silvio; Janni, Michela; Sella, Luca; Gazzetti, Katia; Tauzin, Alexandra; Giardina, Thierry; Masci, Stefania; Favaron, Francesco; D'Ovidio, Renato

2013-12-01

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

Comparison of kinetic characteristics of xylanases from Aspergillus ...

African Journals Online (AJOL)

LAB

2013-05-08

May 8, 2013 ... convert the substrate into products. The xylanase from A. ... be evidence that this enzyme is less active than the xylanase from A. niger. Moreover, the ..... lignocellulosic biomass structural polysaccharides. Biotechnol. Agron.

Purification and characterization of xylanase from Aspergillus ...

African Journals Online (AJOL)

MIET

2013-05-15

May 15, 2013 ... processing (Collins et al., 2005). Frequent ... xylanases (Stricker et al., 2008). The use of cheaper lignocellulosic residues viz. wheat bran, wheat straw, corn cob and sugarcane bagasse can be used as growth ..... Table 3. Effect of different temperature on xylanase activity from A. fumigatus (enzyme reaction.

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

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Leda Maria Fortes Gottschalk

2013-01-01

Full Text Available The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 ºC and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L, which was not detected in the T. reesei culture.

Thermophilic and alkalophilic xylanases from several Dictyoglomus isolates

Energy Technology Data Exchange (ETDEWEB)

Mathrani, I M; Ahring, B K [Technical Univ. of Denmark, Lyngby (Denmark). Anaerobic Microbiology/Biotechnology Group

1992-10-01

Supernatant xylanases from three thermophilic and strictly anaerobic Dictyoglomus strains isolated from very different environments were examined: The type species, D. thermophilum[sup T], from a hot-spring in Japan; strain B1, a recently described strictly xylanutilizing Dictyoglomus from a paper-pulp factory in Finland; and strain B4a, isolated from a thermal pool on Iceland. The highest enzymatic activity observed from batch-culture supernatant with 4 g l[sup -1] of beech xylan as growth substrate was 3.8x10[sup -6] kat l[sup -1]. The K[sub m] for the xylanases of strain B1 was 4.7 g beech xylan l[sup -1]. The xylanases of all the isolates had a broad range of activity with respect to pH, showing good activity from pH 5.5 to near 9.0. The xylanases from the three isolates had a very high temperature optimum of 80deg C, maximum temperature for extended activity between 80 and 90deg C, and a thermal half-life of over 1 h at 90deg C for strain B1. The application of thermophilic alkalophilic xylanases to paper pulping was discussed. (orig.).

Purification and Characterization of Streptomyces sp. SKK1-8 xylanase

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Nunuk Widhyastuti

2008-11-01

Full Text Available Streptomyces sp. SKK1-8 is a xylanase produced bacteria. Purified xylanase has an optimum condition at pH 4.5 and 50oC. The molecular mass of purified xylanase were determined to be 14.4 kDa and 13.4 kDa. The xylanase was capable of hydrolysing p-NP-α-L-arabinofuranoside, p-NP-β-D-xylanopiranoside, p-NP-β-D-glucopiranoside, p-NP-α-D-galactopiranoside. The Km and Vmax values at 50oC measured on Birchwood xylan were 0.101 mg/ml and 0.1796 μmoles/minute/ml.

Cloning and expression of chaetomium thermophilum xylanase 11-A

International Nuclear Information System (INIS)

Andleeb, S.; Latif, F.; Afzal, S.; Mukhtar, Z.; Mansoor, S.; Rajoka, I.

2008-01-01

The various thermophilic fungi like Chaetomium thermophile has potential to secrete xylanase and cellulase enzymes. In the present study eukaryotic expression system of Pichia pastoris (yeast) was used to express xylanase gene. The xylanase (Xyn 11-A) gene was isolated from C. thermophile strain NIBGE-1. Primers were designed to amplify the gene, ligated into P. pastoris pPIC3.5K vector, the resultant recombinant clone pSSZ810 was transformed into the genome of P. pastoris GS115 strain through electroporation. Transformants were selected on yeast peptone dextrose medium (YPD) plates containing antibiotic geneticin (100 mg/ml) upto final concentration of 0.75 mg/ml. The maximum activity of xylanase 2.04 U/ml after incubation of 2 hours at 50 degree C was observed in the presence of 100% methanol inducer upto final concentration of 30 macro L (0.5%) as compared to control. HPLC analysis represented high peak of xylose as compared to control. SDS-PAGE indicated approx. 28 kDa protein of expressed xylanase gene. (author)

Extractive fermentation of xylanase from Aspergillus tamarii URM 4634 in a bioreactor.

Science.gov (United States)

da Silva, Anna Carolina; Soares de França Queiroz, Alana Emília; Evaristo dos Santos Nascimento, Talita Camila; Rodrigues, Cristine; Gomes, José Erick Galindo; Souza-Motta, Cristina Maria; Porto de Souza Vandenberghe, Luciana; Valente de Medeiros, Erika; Moreira, Keila Aparecida; Herculano, Polyanna Nunes

2014-08-01

Of the many reported applications for xylanase, its use as a food supplement has played an important role for monogastric animals, because it can improve the utilisation of nutrients. The aim of this work was to produce xylanase by extractive fermentation in an aqueous two-phase system using Aspergillus tamarii URM 4634, increasing the scale of production in a bioreactor, partially characterising the xylanase and evaluating its influence on monogastric digestion in vitro. Through extractive fermentation in a bioreactor, xylanase was obtained with an activity of 331.4 U mL(-1) and 72% yield. The xylanase was stable under variable pH and temperature conditions, and it was optimally active at pH 3.6 and 90 °C. Xylanase activity potentiated the simulation of complete monogastric digestion by 6%, and only Mg2+ inhibited its activity. This process provides a system for efficient xylanase production by A. tamarii URM 4634 that has great potential for industrial use.

Screening and production study of microbial xylanase producers from Brazilian Cerrado.

Science.gov (United States)

Alves-Prado, Heloiza Ferreira; Pavezzi, Fabiana Carina; Leite, Rodrigo Simões Ribeiro; de Oliveira, Valéria Maia; Sette, Lara Durães; Dasilva, Roberto

2010-05-01

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by beta-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 degrees C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0-5.5. They were

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Digital Repository Service at National Institute of Oceanography (India)

Raghukumar, C.; Muraleedharan, U.; Gaud, V.R.; Mishra, R.

isolates obtained from marine habitat showed alkaline xylanase activity. The crude enzyme from NIOCC isolate # 3 (Aspergillus niger) with high xylanase activity, cellulase-free and unique properties containing 580 U L-1 of xylanase, could bring about...

Improvement of Xylanase Production by Cochliobolus sativus in Submerged Culture

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Yasser Bakri

2008-01-01

Full Text Available The xylanase production by a new Cochliobolus sativus Cs5 strain was improved under submerged fermentation. The xylanase was induced by xylan and repressed by glucose, sucrose, maltose, xylose, starch and cellulose. Highest enzyme production (98.25 IU/mL was recorded when wheat straw (4 % by mass per volume was used as a carbon source after 120 h of incubation. NaNO3 increased xylanase production 5.4-fold as compared to the control. Optimum initial pH was found to be 4.5 to 5. The C. sativus Cs5 strain grown under submerged culture in a simple medium proved to be a promising microorganism for xylanase production.

Xylanase production by a newly isolated Aspergillus niger SS7 in submerged culture.

Science.gov (United States)

Bakri, Yasser; Al-Jazairi, Manal; Al-Kayat, Ghassan

2008-01-01

Xylanase production by a newly isolated Aspergillus niger SS7 was studied in submerged culture. The optimum initial pH for xylanase production was found to be 7.0. Different agricultural and industrial wastes were evaluated for their ability to induce xylanase production by this isolate. The best xylanase production (293.82 IU/ml) was recorded at 3% (w/v) corn cob hulls after 120 h of incubation. The Aspergillus niger SS7 isolate grown in a simple medium, proved to be a promising microorganism for xylanase production.

Molecular characterization of a Xylanase-producing fungus isolated from fouled soil

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Punniavan Sakthiselvan

2014-12-01

Full Text Available Xylanase (EC 3. 2. 1. 8, hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa using SDS-PAGE.

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus

Energy Technology Data Exchange (ETDEWEB)

Gilbert, M [Ottawa Univ., Dept. of Biology, ON (Canada); Yaguchi, M [Inst. for Biological Sciences, National Research Council of Canada, Ottawa, ON (Canada); Watson, D C [Inst. for Biological Sciences, National Research Council of Canada, Ottawa, ON (Canada); Wong, K K.Y. [Chair of Forest Products Biotechnology, Faculty of Forestry, British Columbia Univ., Vancouver, BC (Canada); Breuil, C [Chair of Forest Products Biotechnology, Faculty of Forestry, British Columbia Univ., Vancouver, BC (Canada); Saddler, J N [Chair of Forest Products Biotechnology, Faculty of Forestry, British Columbia Univ., Vancouver, BC (Canada)

1993-12-01

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of [beta]-1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylooligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xyalanses were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps. (orig.)

Biotechnology of microbial xylanases: enzymology, molecular biology, and application.

Science.gov (United States)

Subramaniyan, S; Prema, P

2002-01-01

Xylanases are hydrolases depolymerizing the plant cell wall component xylan, the second most abundant polysaccharide. The molecular structure and hydrolytic pattern of xylanases have been reported extensively and the mechanism of hydrolysis has also been proposed. There are several models for the gene regulation of which this article could add to the wealth of knowledge. Future work on the application of these enzymes in the paper and pulp, food industry, in environmental science, that is, bio-fueling, effluent treatment, and agro-waste treatment, etc. require a complete understanding of the functional and genetic significance of the xylanases. However, the thrust area has been identified as the paper and pulp industry. The major problem in the field of paper bleaching is the removal of lignin and its derivatives, which are linked to cellulose and xylan. Xylanases are more suitable in the paper and pulp industry than lignin-degrading systems.

Production of xylanases by an Aspergillus niger strain in wastes grain

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Simone Cristine Izidoro

2014-08-01

Full Text Available Many fungi are used in order to extract products from their metabolism through bioprocesses capable of minimizing adverse effects caused by agro-industrial wastes in the environment. The aim of this study was to evaluate the xylanase production by an Aspergillus niger strain, using agro-industrial wastes as substrate. Brewer’s spent grain was the best inducer of xylanase activity. Higher levels of xylanase were obtained when the fungus was grown in liquid Vogel medium, pH 5.0, at 30ºC, during 5 days. The temperature for optimum activity was 50ºC and optimum pH 5.0. The enzyme was stable at 50ºC, with a half-life of 240 min. High pH stability was verified from pH 4.5 to 7.0. These characteristics exhibited by A. niger xylanase turn this enzyme attractive for some industrial applications, such as in feed and food industries. Additionally, the use of brewer’s spent grain, an abundantly available and low-cost residue, as substrate for xylanase production can not only add value and decrease the amount of this waste, but also reduce xylanase production cost.

Thermo-mechanical and micro-structural properties of xylanase containing whole wheat bread

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G. Ghoshal

2016-12-01

Full Text Available Xylanase is a hemicellulase that can hydrolyses the complex polysaccharides. Hemicelluloses are main components of cell walls of cereal grains. Moreover, hemicelluloses are considered as potential sources of mono- and oligosaccharides. In this study, influence of xylanase on the physicochemical properties and sensory qualities of the whole wheat bread during storage was investigated. Studies of whole wheat bread on microstructure, texture, thermotics, Scanning Electron Microscopic (SEM, X-Ray Diffraction (XRD were conducted at ambient temperature of 25 and 4 °C respectively. During storage at different temperatures, bread containing xylanase exhibited less firmness but larger volume with whiter crumb color and longer shelf life as compared to control bread. Results of firmness, enthalpy, Fourier Transformation Infra Red (FTIR and X-Ray Diffraction (XRD studies suggested a lower staling rate of bread containing xylanase as compared to control one. Bread containing xylanase showed a smoother surface and more uniform pore size than the control. Significant differences in microstructure of control and bread containing xylanase were observed which might be attributed due to the change in water starch gluten interaction. These differences were also found to be interrelated to the textural properties of bread. Better sensory features were achieved in bread containing xylanase.

Effect of corn cobs concentration on xylanase biosynthesis by ...

African Journals Online (AJOL)

Corn cobs, an indigenous carbon source, were tested as substrate by Aspergillus niger for optimum synthesis of xylanase using the submerged fermentation technique. The trials for xylanase production were conducted at three concentration levels (2.5, 3.0 and 3.5%) of corn cobs, four different fermentation temperatures ...

Production of cellulase-free xylanase by Aspergillus flavus: Effect of ...

African Journals Online (AJOL)

Nelciele

Aspergillus flavus produced high levels of xylanase on agricultural residues with ... addition of 5% glycerol, mannitol or xylitol protected the xylanase from thermal inactivation at 50°C. The .... most often included in nutrient media for microbial.

Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates.

Science.gov (United States)

Song, Hui-Ting; Gao, Yuan; Yang, Yi-Min; Xiao, Wen-Jing; Liu, Shi-Hui; Xia, Wu-Cheng; Liu, Zi-Lu; Yi, Li; Jiang, Zheng-Bing

2016-11-01

Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum

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D Tagu

2009-12-01

Full Text Available Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Statistical optimization of activity and stability of β-xylanase ...

African Journals Online (AJOL)

STORAGESEVER

2008-10-20

Oct 20, 2008 ... A factorial design was performed to find the best conditions of pH and temperature for β-xylanase activity and to maintain its activity for prolonged periods of time of pure xylanase produced by newly isolated Thermomyces lanuginosus THKU-49. The central composite design (CCD) used for the analysis of ...

Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

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Hui Xu

2015-01-01

Full Text Available The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA and Csac_1078 (celB from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA and TM0070 (xynB, resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

Cloning and expression of chaetomium thermophilum xylanase 11-A gene in prokaryote

International Nuclear Information System (INIS)

Wajid, S.; Latif, F.; Afzal, S.; Rajoka, I.

2008-01-01

The xylanase gene was cloned into pET32a(+) and expressed in E. coli BL21 under T7 promotor alongwith fusion protein. The SDS-PAGE and western blot analysis showed a protein of 42 kDa. The best expression of xylanase enzyme was found by using xylose as carbon source and lactose as an inducer. The maximum activity of xylanase expressed in E. coli was 6.02 U/mL in the presence of 2% xylose in DS medium. The activity of recombinant xylanase was observed on 1% xylan LB agar plates, showed halos of xylan clearance when lactose was used as an inducer. (author)

A xylanase-aided enzymatic pretreatment facilitates cellulose nanofibrillation.

Science.gov (United States)

Long, Lingfeng; Tian, Dong; Hu, Jinguang; Wang, Fei; Saddler, Jack

2017-11-01

Although biological pretreatment of cellulosic fiber based on endoglucanases has shown some promise to facilitate cellulose nanofibrillation, its efficacy is still limited. In this study, a xylanase-aided endoglucanase pretreatment was assessed on the bleached hardwood and softwood Kraft pulps to facilitate the downstream cellulose nanofibrillation. Four commercial xylanase preparations were compared and the changes of major fiber physicochemical characteristics such as cellulose/hemicellulose content, gross fiber properties, fiber morphologies, cellulose accessibility/degree of polymerization (DP)/crystallinity were systematically evaluated before and after enzymatic pretreatment. It showed that the synergistic cooperation between endoglucanase and certain xylanase (Biobrite) could efficiently "open up" the hardwood Kraft pulp with limited carbohydrates degradation (cellulose nanofibrillation during mild sonication process (90Wh) with more uniform disintegrated nanofibril products (50-150nm, as assessed by scanning electron microscopy and UV-vis spectroscopy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Hou, Jin; Österlund, Tobias; Liu, Zihe

2013-01-01

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

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Aditya eBhalla

2015-06-01

Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

Xylanase-Aided Chlorine Dioxide Bleaching of Bagasse Pulp to Reduce AOX Formation

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Yi Dai

2016-02-01

Full Text Available Xylanase pretreatment was used to improve the chlorine dioxide bleaching of bagasse pulp. The pulp was pretreated with xylanase, which was followed by a chlorine dioxide bleaching stage. The HexA content of the pulp and the AOX content of the bleaching effluent were measured using UV-Vis and GC-MS methods, respectively. The results showed that a good correlation occurred between HexA and kappa number. HexA content of the pulp decreased significantly after the xylanase pretreatment. The AOX content of the bleaching effluent decreased as HexA was removed from the pulp. It was found that AOX could be reduced by up to 29.8%, comparing XD0 with a D0 stage. Fourier transform infrared spectroscopy (FTIR was employed to determine the breakage of chemical bonds in the pulp. It revealed that some lignin and hemicellulose were removed after xylanase treatment. The GC-MS results showed that some toxic chloride such as 2,4,6-trichlorophenol could be completely removed after xylanase pretreatment.

Agro-residues as Alternative for Xylanase Production by Filamentous Fungi

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Adriana Knob

2014-07-01

Full Text Available Agro-industrial wastes are the most abundant renewable resource on earth and are available in large quantities. However, the disposal of these wastes presents an increasing environmental problem. Recently, there has been a great interest in the exploitation of these wastes as low-cost raw materials for the production of value-added compounds as microbial enzymes by submerged or solid-state fermentation systems. This review focuses on alternatives for xylanase production using agro-residues as substrates. In recent years, the interest in xylanase, which plays an important role in the breakdown of xylan, has markedly increased due to its wide variety of biotechnological applications. Among several agro-industrial residues that have been intensively investigated, many, such as wheat bran, wheat straw, and sugarcane bagasse, are suitable and result in high yields of xylanase, leading to low production costs. In addition, many relatively unexplored residues, such as oil palm wastes, sorghum straw, and coffee by-products, are some of the most promising substrates for xylanase production, requiring further assessment.

Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus spp

Energy Technology Data Exchange (ETDEWEB)

Okazaki, W; Akahoshi, R; Akiba, T; Horikoshi, K

1984-05-01

Four strains (W1, W2, W3, and W4) of alkalophilic thermophilic bacteria which produced xylanase were isolated from soils. They were aerobic, spore-forming. Gram-positive, and rod-shaped bacteria and hence identified as the genus Bacillus. The optimal temperatures for growth of the four strains were between 45/sup 0/C and 50/sup 0/C and pH optima were between 9.0 and 10.0. No growth occurred below pH 7.0 or above 55/sup 0/C. The four strains produced xylanases in medium containing xylan or xylose under these conditions. The optimal pH and temperature for activities of the four xylanases ranged from 6.0 to 7.0 and from 65/sup 0/C to 70/sup 0/C, respectively. The four xylanases were stable in the wide pH range from 4.5 to 10.5 at 45/sup 0/C for 1 h. All xylanases split xylan to yield xylose and xylobiose.

PERAWATAN GIGI DENGAN KELAINAN ENDO-PERIO

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Andrew Chandra Luwuk

2015-07-01

Full Text Available Endo-perio lesion could occur due to the close relationship between the pulp and the periodontium. Therefore, pulpal lesion could cause a periodontium lesion. To decide on an appropriate diagnosis, a thorough and careful examination needs to be done in order to determine the right treatment. Most of the endo-perio cases should be approached with a root canal treatment because the source of the lesion is in the canal, and there is a possibility of healing of the periapical and periodontal ligament without surgical intervention. A report of a healing of an endo-perio case without surgical approach will be discussed.

Production of D-xylanases by thermophilic fungi using different methods of culture

Energy Technology Data Exchange (ETDEWEB)

Grajek, W

1986-01-01

Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, were H. lanuginosa and S. thermophile in liquid fermentation, and T. aurantiacus and H. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture of H. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.

Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase

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Liu Liangwei

2012-06-01

Full Text Available Abstract Background Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn with a hyper-thermophilic Thermotoga maritima glucanase (Glu to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity. Results When expressed in E. coli BL21(DE3, the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt at 50 °C and thermal in-activation half-life (t1/2 at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates. Conclusions Domain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.

High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen.

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Guozeng Wang

Full Text Available BACKGROUND: The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11 in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95% were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific. CONCLUSION/SIGNIFICANCE: The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen

Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis

DEFF Research Database (Denmark)

Wainø, M.; Ingvorsen, K.

2003-01-01

-xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic: P-xylanase activity exhibiting two optima at 55degrees and 70degreesC, while beta......The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed...... for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta...

Nucleolytic degradation of homologous and heterologous deoxyribonucleic acid molecules at the surface of competent pneumococci

International Nuclear Information System (INIS)

Seto, H.; Lopez, R.; Garrigan, O.; Tomasz, A.

1975-01-01

Competent pneumococci can catalyze the rapid and quantitative degradation of extracellular deoxyribonucleic acid (DNA) molecules through the activity of surface-located nucleases (endo- and, possibly, exonucleases as well). Both homologous and heterologous DNAs are degraded by a mechanism that seems to involve a cyclic process: (i) attachment of DNA to the cell surface followed by (ii) nucleolytic attack, and (iii) release to the medium. Processes (ii) and (iii) are both inhibited by ethylenediaminetetraacetate. Whereas surface nuclease activity is specific for competent cells, the bulk of this activity is not coupled to irreversible DNA uptake (deoxyribonuclease-resistant binding). Pneumococcal DNA treated with ultraviolet irradiation or nitrous acid (cross-linking) is selectively impaired in the ability to irreversibly bind to competent cells, whereas reversible binding is normal. (U.S.)

Cloning, sequencing and expression of a novel xylanase cDNA from ...

African Journals Online (AJOL)

A strain SH 2016, capable of producing xylanase, was isolated and identified as Aspergillus awamori, based on its physiological and biochemical characteristics as well as its ITS rDNA gene sequence analysis. A xylanase gene of 591 bp was cloned from this newly isolated A. awamori and the ORF sequence predicted a ...

Induction and catabolite repression of cellulase and xylanase synthesis in the selected white-rot basidiomycetes

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Aza Kobakhidze

2016-09-01

Full Text Available This paper reports regulation of endoglucanase (EC 3.2.1.4 and xylanase (EC 3.2.1.8 production in submerged cultivation of four white-rot basidiomycetes. Among carbon sources tested, the Avicel-based medium provided the highest levels of both hydrolases activities in all fungal cultures. However, the maximum endoglucanase and xylanase activities of the tested basidiomycetes varied from 3.9 U/ml and 7.4 U/ml in Fomes fomentarius to 34.2 U/ml and 29.5 U/ml in Pseudotrametes gibbosa, respectively (P. gibbosa specific cellulase and xylanase activities achieved 8.55 and 7.38 U/mg, respectively. Replacement of Avicel in the medium with carboxymethyl cellulose or xylan significantly lowered the enzyme yield of the tested fungi. Moreover, xylan did not ensure high xylanase activity of these fungi. Lignocellulosic substrates used as a carbon source provided poorer productivity (the specific CMCase activity was 1.12–3.62 U/mg and the specific xylanase activity was 1.95–3.32 U/mg. Expression of endoglucanase and xylanase synthesis in Panus lecometei and P. gibbosa was inducible; supplementation of the glycerol-containing medium with Avicel accompanied with a sharp increase of the fungal specific CMCase and xylanase activities from 0.02–0.04 U/mg to 1.30–8.55 U/mg. Supplementation of the Avicel-induced cultures with glucose or glycerol caused a catabolite repression of the cellulase and xylanase formation by P. gibbosa and P. lecometei. The enzyme synthesis resumed only after depletion of easily metabolizable carbon source, glucose or glycerol, from the medium. The data received suggest that in the tested fungi endoglucanase and xylanase synthesis is under control by a common regulatory mechanism.

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization.

Science.gov (United States)

Dutta, T; Sengupta, R; Sahoo, R; Sinha Ray, S; Bhattacharjee, A; Ghosh, S

2007-02-01

The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3.6. Stimulation of the activity by beta mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50 degrees C at pH 8.5. Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.

Benefits from additives and xylanase during enzymatic hydrolysis of bamboo shoot and mature bamboo.

Science.gov (United States)

Li, Kena; Wang, Xiao; Wang, Jingfeng; Zhang, Junhua

2015-09-01

Effects of additives (BSA, PEG 6000, and Tween 80) on enzymatic hydrolysis of bamboo shoot and mature bamboo fractions (bamboo green, bamboo timber, bamboo yellow, bamboo node, and bamboo branches) by cellulases and/or xylanase were evaluated. The addition of additives was comparable to the increase of cellulase loadings in the conversion of cellulose and xylan in bamboo fractions. Supplementation of xylanase (1 mg/g DM) with cellulases (10 FPU/g DM) in the hydrolysis of bamboo fractions was more efficient than addition of additives in the production of glucose and xylose. Moreover, addition of additives could further increase the glucose release from different bamboo fractions by cellulases and xylanase. Bamboo green exhibited the lowest hydrolyzability. Almost all of the polysaccharides in pretreated bamboo shoot fractions were hydrolyzed by cellulases with the addition of additives or xylanase. Additives and xylanase showed great potential for reducing cellulase requirement in the hydrolysis of bamboo. Copyright © 2015 Elsevier Ltd. All rights reserved.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

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Gupta Munishwar

2007-06-01

Full Text Available Abstract Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1 hydrolysis of pectin, 2 hydrolysis of xylan and 3 hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Production of xylanases and cellulases by aspergillus fumigatus ms16 using crude lignocellulosic substrates

International Nuclear Information System (INIS)

Naseeb, S.; Sohai, M.; Ahmad, A.; Khan, S.A.

2015-01-01

Xylanolytic and cellulolytic potential of a soil isolate, Aspergillus fumigatus (MS16) was studied by growing it on a variety of lignocellulosics, purified cellulose and xylan supplemented media. It was noted that carboxymethyl cellulose, salicin and xylan induce the -glucosidase and xylanase, respectively production of endoglucanase. The study revealed that Aspergillus fumigatus (MS16) co-secretes xylanase and cellulase in the presence of xylan; the ratio of the two enzymes was influenced by the initial pH of the medium. The maximum titers of xylanase and cellulase were noted at initial pH of 5.0. Relatively higher titers of both the enzymes were obtained when the fungus was cultivated at 35 degree C. Whereas, cellulase production was not detected when the fungus was cultivated at 40 degree C. The volumetric productivity (Qp) of xylanase was much higher than cellulases. The organism produced 2-3 folds higher titers of xylanase when grown on lignocellulosic materials in submerged cultivation than under solid-state cultivation, suggesting a different pattern of enzyme production in presence and in absence of free water. The partial characterization of enzymes showed that xylanase from this organism has -glucosidase. The higher melting temperature than endoglucanase and optimum temperature for activity was higher for xylanases than cellulases, whereas the optimum pH differed slightly i.e. in the range of 4.0-5.0. Enzyme preparation from this organism was loaded on some crude substrates and it showed that the enzyme preparation can be used to hydrolyze a variety of vegetable and agricultural waste materials. (author)

Improvement of xylanase production by a parasexual cross between Aspergillus niger strains

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Octavio Loera

2003-03-01

Full Text Available A diploid strain (D4 isolated via parasexual recombination between two Aspergillus niger xylanase overproducing mutants was characterised in terms of enzyme production and catabolite repression by glucose. This strain increased xylanase production (607 nkat/ml, which was nearly 100% higher than titers achieved by the wild type strain (305 nkat/ml and 28% higher than the best mutant used to induce parasexual cycle. Diploid D4 was also less sensitive to carbon catabolite repression by glucose, since xylanolytic activity was detected under conditions normally repressing production by the wild type strain. No decrease in maximal xylanase levels was observed in the presence of glucose for diploid D4.Um cepa diplóide (D4 isolada por combinação parasexual entre dois Aspergillus niger, mutantes superprodutores de xylanase foi caracterizado através da produção de (607 nkat/ml e repressão catabólica por glicose. Essa cepa aumenta a produção de xylanase em mais de 100% em comparação com uma cepa selvagem (305 nkat/ml e 28% superior do que o melhor mutante usado para induzir o ciclo parasexual. A cepa diplóide D4 foi também menos sensível a repressão catabólica pela glicose, sendo que a atividade xylanolitica foi detectada sob condições normalmente de produção repressiva pela cepa selvagem. Não foi observado um decréscimo na produção máxima de xylanase em presença de glicose para o diplóide D4.

Purification and Characterization of Haloalkaline, Organic Solvent Stable Xylanase from Newly Isolated Halophilic Bacterium-OKH

Science.gov (United States)

Sanghvi, Gaurav; Jivrajani, Mehul; Patel, Nirav; Jivrajani, Heta; Bhaskara, Govinal Badiger; Patel, Shivani

2014-01-01

A novel, alkali-tolerant halophilic bacterium-OKH with an ability to produce extracellular halophilic, alkali-tolerant, organic solvent stable, and moderately thermostable xylanase was isolated from salt salterns of Mithapur region, Gujarat, India. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. Maximum xylanase production was achieved at pH 9.0 and 37°C temperature in the medium containing 15% NaCl and 1% (w/v) corn cobs. Sugarcane bagasse and wheat straw also induce xylanase production when used as carbon source. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum xylanase activity was observed at 5% sodium chloride. Xylanase was purified with 25.81%-fold purification and 17.1% yield. Kinetic properties such as Km and Vmax were 4.2 mg/mL and 0.31 μmol/min/mL, respectively. The enzyme was stable at pH 6.0 and 50°C with 60% activity after 8 hours of incubation. Enzyme activity was enhanced by Ca2+, Mn2+, and Mg2+ but strongly inhibited by heavy metals such as Hg2+, Fe3+, Ni2+, and Zn2+. Xylanase was found to be stable in organic solvents like glutaraldehyde and isopropanol. The purified enzyme hydrolysed lignocellulosic substrates. Xylanase, purified from the halophilic bacterium-OKH, has potential biotechnological applications. PMID:27350996

Efficient utilization of xylanase and lipase producing thermophilic ...

African Journals Online (AJOL)

Efficient utilization of xylanase and lipase producing thermophilic marine actinomycetes ( Streptomyces albus and Streptomyces hygroscopicus ) in the production of ecofriendly alternative energy from waste.

Cellulase and Xylanase Production from Three Isolates of Indigenous Endophytic Fungi

Science.gov (United States)

Yopi; Tasia, W.; Melliawati, R.

2017-12-01

Cellulases and hemicellulases have good potential to be used in energy production, in pulp, paper, textile industries, as well as in animal feed industries. Moreover, its utilization in food industries also cannot be ignored, among others, cellulase and xylanase roles in bakery, wine, and fruit and vegetables juice production. One of the potential enzyme source is endophytic fungi. Object of this study is to explore the potency of endophytic fungi isolated from medicinal plants as source of cellulolytic and xylanolytic enzymes. HL.47F.216 is endophytic fungi isolated from traditional medicinal plants ironwood tree was determined as xylanase producer. HL.51F.235 from pin-flower tree is cellulase producer, while CBN.6F.29 which produces both xylanase and cellulase is originated from Madagascar periwinkle. HL.47F.216 showed 2.5 cm in clear zone diameter and its xylanase activity was 0.262 U/mL with optimum condition pH 7 at 50°C. HL.51F.235 showed 2.4 cm clear zone diameter and 0.239 U/mL of cellulase activity at pH 5 and 70°C. CBN.6F.29 showed 2.8 cm and 0.394 U/mL (pH 5, 40°C) for its cellulase activity, while 2.3 cm and 0.439 U/mL (pH 8, 70°C) for its xylanase activity. Xylanase from HL.47F.216 and CBN.6F.29 showed low molecular masses of 20 kDa and 37-50 kDa, respectively. Molecular masses for cellulases from HL.51F.235 and CBN.6F.29 were 25 and 50 kDa for HL.51F.235 and 100 kDa for CBN.6F.29. Based on macroscopic and microscopic identification, fungal isolate CBN.6F.29 is a member of Class Coelomycetes, while HL.47F.216 was Acremonium sp. and HL.51F.235 was Aspergillus nigri.

Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

Science.gov (United States)

Joshi, Chetna; Khare, S K

2011-01-01

Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization. Copyright © 2010 Elsevier Ltd. All rights reserved.

Purification and characterization of a thermostable hypothetical xylanase from Aspergillus oryzae HML366.

Science.gov (United States)

He, Haiyan; Qin, Yongling; Li, Nan; Chen, Guiguang; Liang, Zhiqun

2015-03-01

In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0-10.0 and at a temperature below 70 °C. Mg(2+), Mn(2+), Ca(2+), and K(+) enhanced the XynH1 xylanase activity to 146, 122, 114, and 108%, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 μmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Science.gov (United States)

Chao Zhang; Xinshu Zhuang; Zhao Jiang Wang; Fred Matt; Franz St. John; J.Y. Zhu

2013-01-01

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose...

Production and characterization of thermostable xylanase from ...

African Journals Online (AJOL)

ajl2

2013-02-20

Feb 20, 2013 ... produced from Trichoderma (Huitron et al., 2008). Only a ... around their colonies against a red background, were selected and .... Resistance to antibiotic ..... Xylanases of marine fungi potential use for biobleaching of paper.

Xylanase production by a local fungal isolate, Aspergillus niger USM AI 1 via solid state

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Ibrahim Che Omar

2005-03-01

Full Text Available Isolate USM A1 I which was identified to be Aspergillus niger was selected as a potential producer of xylanase via a solid state fermentation system (SSF using palm kernel cake (PKC as substrate. The modification of the physical conditions of the SSF system indicated that the xylanase activity was 23.97 U/g PKC at the moisture ratio of 1:0.75 of PKC: moistening agent with the inoculum size of 1¥104 spores/ml and cultivated at the ambient temperature (28±3ºC. The supplementation of additional carbon and nitrogen sources in the PKC medium could enhance enzyme productivity. The maximum production of xylanase and growth obtained with the supplementation of xylose at 0.75% (w/w were 25.40 U/g and 1.69 mg glucosamine/ g PKC. Moreover, the presence of NaNO3 at 0.075% (w/w as additional nitrogen source further enhanced xylanase production to 33.99 U/g PKC although the growth remained unchanged at about 1.67 mg glucosa- mine/g PKC. The optimized conditions showed an increased xylanase production by 157% compared to before the optimization of the SSF system. The xylanase productivity was 23.12 U/mg glucosamine after optimization and 11.72 U/mg glucosamine before optimization.

Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

Science.gov (United States)

Beitel, Susan Michelz; Fortkamp, Diana; Terrasan, César Rafael Fanchini; de Almeida, Alex Fernando

2013-01-01

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. PMID:23762855

Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

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Adriana Knob

2013-01-01

Full Text Available In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

Effects of structure and xylanase treatment of brewers' spent grain on performance and nutrient availability in broiler chickens.

Science.gov (United States)

Denstadli, V; Westereng, B; Biniyam, H G; Ballance, S; Knutsen, S H; Svihus, B

2010-06-01

1. A factorial (2 x 3) feeding trial was set up to investigate the effects of coarse or finely ground brewers' spent grain (BSG) and xylanase treatment, either with no xylanase, top-dressed with xylanase or pre-treated with xylanase. 2. The experimental diets shared the same basal formulation and were fed to male broiler chickens (Ross 308) housed in individual cages from 12 to 29 d of age. 3. Xylanase pre-treatment reduced the dietary concentration of arabinoxylan by 15-30%. Pellet durability increased when BSG was ground. 4. Feed utilisation was significantly higher (6%) when the birds were given coarse BSG rather than ground BSG, whereas there was no significant effect of enzyme treatment. Apparent metabolisable energy was unaffected by the dietary treatments. 5. The overall starch digestibility was high (99%), with no dietary differences, whereas ileal protein digestibility was low (57%). Xylanase top-dressing tended to improve ileal protein digestibility but, in general, xylanase treatment had no major effect on overall performance in male broilers given diets with BSG.

Reuse of wastewater from pulp industry for the optimization of fungal xylanase production

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Geisiany Maria de Queiroz-Fernandes

2017-05-01

Full Text Available The production of enzymes using agro-industrial waste is a low cost alternative for the reuse of byproducts, with the subsequent impact decrease on the environment. Current analysis produced xylanase using fungus Aspergillus niger, with two types of wastewater generated during the pulp chemical bleaching phase as inducers. Xylanase was produced by submerged liquid fermentation and factorial design optimized parameters that influence production (concentration of wastewater and production period. Initial culture conditions (pH, temperature and agitation were optimized independently. Alkaline wastewater was more effective than acidic wastewater for the induction of xylanase in optimized conditions: 50% of culture medium, 7-day production, 30°C, pH 6.0 and agitation at 160 rpm. Despite different results, acidic and alkaline wastewaters induced xylanase production by A. niger when employed in concentrations lower than or equal to 50% of culture medium and in the most optimal conditions described above. Alkaline wastewater is highlighted as the most efficient for such production.

Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.

Science.gov (United States)

Korkmaz, Melih N; Ozdemir, Sennur C; Uzel, Ataç

2017-10-01

Xylanases constitutes one the most important enzymes with diverse applications in different industries such as bioethanol production, animal feedstock production, production of xylo-oligosaccharides, baking industry, paper and pulp industry, xylitol production, fruit juice, and beer finishing, degumming, and agriculture. Currently, industrial xylanases are mainly produced by Aspergillus and Trichoderma members. Since the marine environments are less studied compared to terrestrial environments and harbors great microbial diversity we aimed to investigate the xylanase production of 88 marine-derived filamentous fungal strains. These strains are semi-quantitatively screened for their extracellular xylanase production and Trichoderma pleuroticola 08ÇK001 xylanase activity was further characterized. Optimum pH and temperature was determined as 5.0 and 50 °C, respectively. The enzyme preparation retained 53% of its activity at pH 5.0 after 1 h and have found resistant against several ions and compounds such as K + , Ba 2+ , Na + , β-mercaptoethanol, Triton X-100 and toluene. This study demonstrates that marine-derived fungal strains are prolific sources for xylanase production and presents the first report about the production and characterization of xylanase from a marine derived T. pleuroticola strain. The characteristics of T. pleuroticola 08ÇK001 xylanase activity indicate possible employment in some industrial processes such as animal feed, juice and wine industries or paper pulping applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phylogenetic diversity and environment-specific distributions of glycosyl hydrolase family 10 xylanases in geographically distant soils.

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Guozeng Wang

Full Text Available BACKGROUND: Xylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Partial xylanase genes of glycoside hydrolase (GH family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities. CONCLUSION/SIGNIFICANCE: These results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.

Long Term Success of Endo-DCR with Silicone Tube Catheter (STC versus Endo-DCR without STC: A Comparative Study

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Bidhan Ray

2016-08-01

Full Text Available Introduction   Epiphora  caused by  mechanical block at puncta, canaliculi, lacrimal sac and nasolacrimal duct need surgical correction. EndoDCR  is gaining  popularity over conventional surgery. This study aims at evaluating the role of Silicone Tube Catheter (STC to improve the long term outcome of endoDCR .               Materials and Method   This retrospective study was conducted on 58 consecutive endoDCR surgeries between 2012 and 2014. 22 subjects had endoDCR without STC, 36 subjects had endoDCR with STC, of whom in 20 cases STC was removed in  3 weeks and in 16 cases in  12 weeks. Clinical and endoscopic data collected post-operatively after 3 weeks,12 weeks and at 1 year. Analysis was done on intention to get suggestion for better result from endoDCR operations.                                           Results   Complications were identified as early and late. Complications like granulations, synaechia, echymosis, eyelid haematoma and surgical emphysema were searched for in every follow up. Development of obliterating scar was the main cause of failure. Epistaxis was negligible. No retrobulbar haematoma or rectus injury was noted. Minor synaechia between middle turbinate and nasal wall noted in 13.6 to 20%  of cases in all groups. Granulations were more in cases had STC for 12 weeks but managed with conservative measures. Obliterating scar was slightly less in this group in comparison to other groups.                                         Discussion    The present study emphasises the use of indwelling STC in endoDCR for a longer period .

Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

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Ekaterina A Ivanova

Full Text Available Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.

PERAWATAN GIGI DENGAN KELAINAN ENDO-PERIO

OpenAIRE

Andrew Chandra Luwuk; Winiarti Sidharta

2015-01-01

Endo-perio lesion could occur due to the close relationship between the pulp and the periodontium. Therefore, pulpal lesion could cause a periodontium lesion. To decide on an appropriate diagnosis, a thorough and careful examination needs to be done in order to determine the right treatment. Most of the endo-perio cases should be approached with a root canal treatment because the source of the lesion is in the canal, and there is a possibility of healing of the periapical and periodontal liga...

Production and characterization of xylanase from Bacillus thermoalkalophilus grown on agricultural wastes

Energy Technology Data Exchange (ETDEWEB)

Rajaram, S.; Varma, A. (Jawaharlal Nehru Univ., New Delhi (India). School of Life Sciences)

1990-10-01

Bacillus thermoalkalophilus isolated from termite-infested mound soils of the semi-arid zones of India had the ability to produce good amounts of xylanase(s) from cheap agricultural wastes. Of the two hemicellulosic substrates tested, bagasse was found to be the better inducer for xylanase production. Alkali treatment of bagasse and rice husk had varied effects on enzyme production. The enzyme preparation had activity optima at 60deg C and 70deg C and a half-life of 60 min at 65deg C. The enzyme was stable for 24 h over a pH range of 4.0-6.0, while maximum activity was observed at pH 6.0-7.0. Enzyme production and activity were inhibited by the end-product of xylan hydrolysis, xylose. (orig.).

Purification and characterization of xylanase from Aspergillus ...

African Journals Online (AJOL)

Xylanase was subjected to a three-step purification scheme involving ammonium sulphate precipitation, gel filtration chromatography and anion exchange chromatography. Purity was verified by running the extracted protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single band was ...

A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

Digital Repository Service at National Institute of Oceanography (India)

Khandeparker, R.; Verma, P.; Deobagkar, D.

A novel halotolerant xylanase from marine bacterium Bacillus subtilis cho40 isolated from Chorao island of Mandovi estuary Goa, India has been reported. Extracellular xylanase was produced by using agricultural residue such as wheat bran as carbon...

Statistical optimization of thermo-alkali stable xylanase production from Bacillus tequilensis strain ARMATI

Directory of Open Access Journals (Sweden)

Ameer Khusro

2016-07-01

Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis.

Optimization of biosynthesis conditions and catalitic behavior evaluation of cellulase-free xylanase produced by a new Streptomyces sp. strain

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GABRIELA BAHRIM

2011-07-01

Full Text Available Cellulase-free xylanase by Streptomyces sp.P12-137 was obtained bycultivation on the wheat bran as the sole carbon source. The effect of carbon and nitrogen sources and a ratio of them on the cellulase-free xylanase production was investigated. The new isolate Streptomyces sp. strain was able to grow in submerged system and to produce an increased level of xylanase. Wheat bran induced xylanase biosynthesis yield at a high level (9.27 UA/ml. For economical reasons cultivation was achieved on a cheap fermentative medium represented by agro-industrial wastes. The optima of the pH and temperature of the crude xylanase activity were 5.5 and 70°C,respectively.

Xylanase, CM-cellulase and avicelase production by the thermophilic fungus Sporotrichum thermophile

Energy Technology Data Exchange (ETDEWEB)

Margaritis, A; Merchant, R; Yaguchi, M

1983-01-01

When wheat straw was used as C source, S. thermophile produced large amounts of xylanase extracellularly in addition to CM-cellulase and Avicelase. These enzymes were isolated by alcohol precipitation, desalting, and column chromatography. The molecular weights were estimated to be 25,0065,000 and 84,000 for xylanase, CM-cellulase, and Avicelase, respectively. Serine and threonine were the most abundant amino acids and these enzymes are very acidic proteins.

Endo-lysosomal and autophagic dysfunction: a driving factor in Alzheimer's disease?

Science.gov (United States)

Whyte, Lauren S; Lau, Adeline A; Hemsley, Kim M; Hopwood, John J; Sargeant, Timothy J

2017-03-01

Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-β in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD. © 2016 International Society for Neurochemistry.

Influence of agitation speeds and aeration rates on the Xylanase activity of Aspergillus niger SS7

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Yasser Bakri

2011-08-01

Full Text Available In this study, the effect of agitation and aeration rates on xylanase activity of Aspergillus niger SS7 in 3-litre stirred tank bioreactor was investigated. The agitation rates tested were 100, 200 and 300 rpm at each airflow rates of 0.5, 1.0 and 1.5 vvm. The maximum xylanase activity in mono- agitator system was at the agitation speed of 200 rpm and aeration rate of 1.0 vvm. In bi-agitator system, at low agitation speed (100 rpm, the xylanase activity was enhanced by 13% compared to mono- agitator system for an aeration rate of 1.0 vvm. Xylanase productivity in continuous culture was higher by approximately 3.5 times than in batch culture.

Thermoactive cellulase-free xylanase production from alkaliphilic ...

African Journals Online (AJOL)

STORAGESEVER

2010-01-04

Jan 4, 2010 ... The enzymatic hydrolysis of xylan, a major hemicellulose ... agar (glucose replaced by 1.0% w/v Birch-Wood Xylan (BW-X, ..... Thermal stability was determined by preincubating the xylanase at pH 9.0; at 60.0°C for 1 h.

Solid-state Fermentation of Xylanase from Penicillium canescens 10-10c in a Multi-layer-packed Bed Reactor

Science.gov (United States)

Assamoi, Antoine A.; Destain, Jacqueline; Delvigne, Frank; Lognay, Georges; Thonart, Philippe

Xylanase is produced by Penicillium canescens 10-10c from soya oil cake in static conditions using solid-state fermentation. The impact of several parameters such as the nature and the size of inoculum, bed-loading, and aeration is evaluated during the fermentation process. Mycelial inoculum gives more production than conidial inoculum. Increasing the quantity of inoculum enhances slightly xylanase production. Forced aeration induces more sporulation of strain and reduces xylanase production. However, forced moistened air improves the production compared to production obtained with forced dry air. In addition, increasing bed-loading reduces the specific xylanase production likely due to the incapacity of the Penicillium strain to grow deeply in the fermented soya oil cake mass. Thus, the best cultivation conditions involve mycelial inoculum form, a bed loading of 1-cm height and passive aeration. The maximum xylanase activity is obtained after 7 days of fermentation and attains 10,200 U/g of soya oil cake. These levels are higher than those presented in the literature and, therefore, show all the potentialities of this stock and this technique for the production of xylanase.

Endo-luminal grafting for treatment of abdominal aortic aneurysms

International Nuclear Information System (INIS)

Fu Weiguo; Wang Yuqi; Chen Fuzhen; Ye Jianrong; Wang Jianhua; Yan Zhiping; Cheng Jiemin

2000-01-01

Objective: To evaluate the preliminary clinical results of endovascular procedures for abdominal aortic aneurysms (AAA) in a prospective study. Methods: Six patients (average age 70 years, range 56 to 78) with infrarenal AAA were enrolled in Shanghai Zhongshan hospital from February 1998 to February 1999. Computed tomography and angiography were done in every patient for measurement of the length, diameter, and angulation of the proximal and distal AAA necks, aneurysm sac, and common and external iliac arteries. The average diameter of the aneurysm was 6.3 cm (range 4.6 cm to 8.0 cm). The mean proximal neck diameter was 2.0 cm (range 1.8 cm to 2.2 cm) and proximal neck length was 3.0 cm (range 2.5 cm to 3.5 cm). All patients were treated with the endo-luminal grafting for exclusion of AAA. Results: Two tubular and 4 bifurcated endo-grafts were used. All endo-graft procedures were completed successfully. One patient died of renal failure 72 hours after the procedure because of the prolonged operative time and excessive contrast medium. Aortography after the procedure showed the AAA were excluded by endo-graft and no endo-leak in the proximal or distal connections was detected. The patients could take meal and were ambulatory on the first and second postoperative day, respectively. Clinical success (aneurysm exclusion with no death or endo-leak) at 30 days was 83.3%. In the 24 months follow-up in 5 cases, no migration, endo-leak, and increasing aneurysm size were detected with spiral CT or color Duplex ultrasound. Conclusion: Based on initial results and a short term mean follow-up period of 24 months, the endovascular treatment of AAA with stent-graft system is feasible and safe. Further study will be required to observe the long term result in the exclusion of AAA

Dynamical infomorphism: form of endo-perspective

International Nuclear Information System (INIS)

Gunji, Yukio-Pegio; Takahashi, Tatsuji; Aono, Masashi

2004-01-01

The essential feature of the endo-perspective is examined, and a formal model of the endo-perspective is proposed by introducing the mixture of intra- and inter-operations. Because such a mixture in its naive realization entails a paradox within a formal system, we weaken the inter-operation in order to allow the formal system to be endowed with that mixture without a contradiction. The weakened inter-operation is related to the infomorphism proposed by Barwise [Information Flow, The Logic of Distributed Systems, Cambridge Univ. Press, 1997]. The formal model of the endo-perspective is thereby expressed as the dynamical infomorphism driven by that mixture. The endo-perspective is described as a formal system that includes the outside of the occupied perspective. If such an inclusion is applied to the common definition of a set, it entails Russel's paradox. Retaining the outside can be expressed as the mixture of the intent and the extent of a set together with the mixture of intra-operations within the intent (or the extent) and inter-operations between the intent and the extent. The endo-perspective, therefore, consists of two subsystems corresponding to the intent and the extent, respectively, and is defined as a system involving a particular mathematical tool (i.e., infomorphism) that allows for retaining the outside without a contradiction. Within that framework, the mixture of the intra- and the inter-operation drives the dynamical transition of the system, however, it can be terminated by its collapse. This collapse can be predicted from the internal logic defined within the system. The model is constructed through the verification of 'a weakened paradox'. Because the definition of the system involves a weakened paradox only, it does not always lead to a contradiction, although the collapse of the system corresponds to a contradiction. The double standards can be embedded into the system, the domain with truth-values (the inside) and the domain in which the

Efficient Expression of an Acidic Endo-polygalacturonase from Aspergillus niger and Its Application in Juice Production.

Science.gov (United States)

Wang, Jiaojiao; Zhang, Yuhong; Qin, Xing; Gao, Lingyu; Han, Bin; Zhang, Deqing; Li, Jinyang; Huang, He; Zhang, Wei

2017-04-05

An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was 1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris GS115 for heterologous expression. The polygalacturonase showed a maximum activity level of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were 4.5 and 40 °C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the volume of pear juice by 41.8%, and improved its light transmittance 3-fold.

Emerging role of N- and C-terminal interactions in stabilizing (β/α8 fold with special emphasis on Family 10 xylanases

Directory of Open Access Journals (Sweden)

Amit Bhardwaj

2012-09-01

Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

EMERGING ROLE OF N- AND C-TERMINAL INTERACTIONS IN STABILIZING (β;/α8 FOLD WITH SPECIAL EMPHASIS ON FAMILY 10 XYLANASES

Directory of Open Access Journals (Sweden)

Amit Bhardwaj

2012-09-01

Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

Xylanase, protease and superdosing phytase interactions in broiler performance, carcass yield and digesta transit time

Directory of Open Access Journals (Sweden)

Tiago T. dos Santos

2017-06-01

Full Text Available The interaction of xylanase, protease and superdosing (1,500 FTU/kg phytase in a 2 × 2 × 2 factorial arrangement was studied in broilers fed sorghum-based diets. A total of 2,800 one-day-old unsexed Ross 308 chicks were housed in 56 pens with 50 birds per pen, with or without inclusion of xylanase, protease and phytase, totaling 8 treatments and 7 replicates per treatment. Body weight (BW and feed intake (FI were measured at 21 and 42 days of age, and mortality corrected feed conversion ratio (FCR was calculated for each period and cumulatively. Tibia ash and carcass yield were determined in 2 birds per replicate at 21 and 42 days of age, respectively. Digesta transit time was determined at 21, 28, 35 and 42 days of age using 5 birds per replicate. Results showed that superdosing phytase increased BW and FI at 42 days of age (P < 0.05 and xylanase improved FCR (P < 0.05. Xylanase and phytase also positively influenced carcass yield and breast weight, respectively. Overall, inclusion of superdosing phytase increased transit time when included in a diet containing xylanase, and no change with protease inclusion. In conclusion, the beneficial effects of xylanase, protease and superdosing phytase in broiler performance were not additive. This limitation is likely not related to the lack of efficacy of any one of the individual enzymes but to a limitation of the bird to respond additively to successive additions of enzymes.

Spores of the mycorrhizal fungus Glomus mosseae host yeasts that solubilize phosphate and accumulate polyphosphates.

Science.gov (United States)

Mirabal Alonso, Loreli; Kleiner, Diethelm; Ortega, Eduardo

2008-04-01

The present paper reports the presence of bacteria and yeasts tightly associated with spores of an isolate of Glomus mosseae. Healthy spores were surface disinfected by combining chloramine-T 5%, Tween-40, and cephalexin 2.5 g L(-1) (CTCf). Macerates of these spores were incubated on agar media, microorganisms were isolated, and two yeasts were characterized (EndoGm1, EndoGm11). Both yeasts were able to solubilize low-soluble P sources (Ca and Fe phosphates) and accumulate polyphosphates (polyPs). Sequence analysis of 18S ribosomal deoxyribonucleic acid showed that the yeasts belong to the genera Rhodotorula or Rhodosporidium (EndoGm1) and Cryptococcus (EndoGm11). Results from inoculation experiments showed an effect of the spore-associated yeasts on the root growth of rice, suggesting potential tripartite interactions with mycorrhizal fungi and plants.

Ecofriendly application of cellulase and xylanase producing marine ...

African Journals Online (AJOL)

windows

2012-06-05

Jun 5, 2012 ... producing marine Streptomyces clavuligerus as enhancer in ... pretreatment of cellulase, xylanase and the combination of enzymes. ... Energy from biomass holds a promising scope under ... investment, simplification of the fermentation media, ... biodegradation of lignocellulosic residues and enhanced ...

Polypeptides having xylanase activity and polynucleotides encoding same

Energy Technology Data Exchange (ETDEWEB)

Spodsberg, Nikolaj

2018-02-06

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

Energy Technology Data Exchange (ETDEWEB)

Bailey, M.J. (VTT, Biotechnical Lab., Espoo (Finland)); Buchert, J. (VTT, Biotechnical Lab., Espoo (Finland)); Viikari, L. (VTT, Biotechnical Lab., Espoo (Finland))

1993-11-01

Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivated on media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulase was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-1) fermentors. Downstream processing of the xylanase-rich, low-cellulase culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-1 pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. (orig.)

Cellulase-poor xylanases produced by Trichoderma reesei RUT C-30 on hemicellulose substrates

Energy Technology Data Exchange (ETDEWEB)

Gamerith, G.; Groicher, R. (Lenzing AG (Austria). Dept. of Research and Development); Zeilinger, S.; Herzog, P.; Kubicek, C.P. (Technische Univ., Vienna (Austria). Abt. fuer Mikrobielle Biochemie)

1992-12-01

Hemicellulose components from industrial viscose fibre production are characterized by a lower cellulose content than commerical xylan and the pressence of a carboxylic acid fraction originating from the alkaline degradation of carbohydrates during the process. This substrate, after neutralization, can be used by Trichoderma reesei RUT C-30 for the production of cellulase-poor xylanases, useful for the pulp and paper industry. The yields of xylanase ranged up to almost 400 units/ml, with a ratio of carboxymethylcellulase/xylanase of less than 0.015. This crude xylanase enzyme mixture was shown to be superior to that obtained on beech-wood xylan when used for bleaching and, particularly, upgrading of hard-wood chemical pulp by selective removal of the xylan components. Biochemical studies indicate that the low cellulase production by T. reesei grown on these waste hemicelluloses is the result of a combination of at least three factors: (a) The comparatively low content of cellulose in these hemicellulosic wastes, (b) the inhibitory action of the carboxylic acid fraction present in the hemicellulosic wastes on growth and sporulation of T. reesei, and (c) the use of a mycelial inoculum that is unable to initiate the atack on the cellulose components within the carbon source. (orig.).

Inexpensive, rapid procedure for bulk purification of cellulase-free. beta. -1,4-D-xylanase of high specific activity

Energy Technology Data Exchange (ETDEWEB)

Tan, L.U.L.; Yu, E.K.C.; Louis-Seize, G.W.; Saddler, J.N.

1987-01-01

A process has been developed for the bulk purification of cellulase-free ..beta..-14-D-xylanase from the fungus Tirchoderma harzianum E58. The process involved the primary step of ultrafiltering the culture filtrate via a 10,000-molecular-weight cut-off membrane to separate the cellulase (retentate) and xylanase (permeate) fractions. The cellulase component was concentrated by 40- to 60-fold, resulting in an enzyme complex that could effectively hydrolyze high concentrations of cellulose and xylan to glucose and xylose. The xylanase was concentrated and solvent exchanged by adsorption to a cationic exchanger, SP-ZetaPrep 250, followed by elution with a pH change in the buffer to give a purified and concentrated xylanase complex dissolved in a low-salt buffer. The resultant xylanase system was pure by the criteria of sodium dodecyl sulfate polyacrylamide electrophoresis, had a very high specific activity of 2400 IU/mg protein, was virtually free of filter paper activity, and had a ratio of contaminating filter paper activity of 2 x 10/sup -6/. Approximately 3.3 g protein, which contained in excess of 7 x 10/sup 6/ IU xylanase activity was obtained from 17 L original culture filtrate. The process scheme was designed to facilitate scale-up to an industrial level of production.

19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

Science.gov (United States)

Zhao, Kexin; van der Spoel, Aarnoud; Castiglioni, Claudia; Gale, Sarah; Fujiwara, Hideji; Ory, Daniel S; Ridgway, Neale D

2018-06-01

Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome. Copyright © 2018 Elsevier B.V. All rights reserved.

Partial purification and characterization of xylanase produced from aspergillus niger using wheat bran

International Nuclear Information System (INIS)

Ahmad, Z.; Butt, M.S.

2013-01-01

In present exploration, purification and characterization of xylanase was carried out to find its optimum conditions for maximum functionality. The xylanase (EC 3.2.1.8) synthesized by Aspergillus niger in submerged fermentation was partially purified and characterized for different parameters like temperature, pH and heat stability. The molecular mass determined through SDS-PAGE was found 30 kDa. The specific activity of the enzyme was raised from 41.85 to 613.13 with 48.63% yield just in a two step partial purification comprising ammonium sulphate precipitation and Sephadex gel filteration column chromatography. The partially purified enzyme was found to be optimally active at 60 degree C and 7.5 pH. Conclusively, for the application of xylanase in food, feed or paper manufacturing processes, it is necessary to consider its optimum pH and temperature. (author)

Heterologous production of peptides in plants: fusion proteins and beyond.

Science.gov (United States)

Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

2013-11-01

Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps.

Science.gov (United States)

Torres, Jeremy Martin O; Dela Cruz, Thomas Edison E

2013-04-01

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70-650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production

NARCIS (Netherlands)

Nikolaev, I.; Hansen, S.; Madrid, S.; de Vries, R.P.

2013-01-01

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

Directory of Open Access Journals (Sweden)

Ranganathan KAPILAN

2011-03-01

Full Text Available To optimize culture conditions for xylanase production by solid state fermentation (SSF using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K2HPO4, 2.5 g/L; KH2PO4, 1.0 g/L; NaCl, 0.1 g/L; (NH42SO4, 2.0 g/L, CaCl2·2H2O, 0.005 g/L; MgCl2·6H2O, 0.005 g/L; and FeCl3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity (142.0 ±0.47 U/g DM] was obtained on the 6th day at 30°C. The optimized paddy husk to liquid fermentation medium ratio was 2:9, and the optimized culture temperature was 40°C. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized (224.2 U/g DM in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production (290.7 U/g DM was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum (326.5±0.34 U/g DM. Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Modeling process for bioproduction of xylanase by Streptomyces spp. p12-137 on lignocelluloses agro-wastes

Directory of Open Access Journals (Sweden)

Gigi COMAN

2012-12-01

Full Text Available The production of xylanase without cellulase is required for the prebleaching of pulps, paper and food industry. The strain Streptomyces spp.P12-137 developed from the spores of the wild type organism was used in this work. Cultures in Erlenmeyer flasks, under shaking condition (150 rpm at temperature and pH values (28°C, 5.0 respectively revealed a xylanase activity of 27.77 IU·mL-1 after 120 h fermentation. This study demonstrates that Streptomyces spp. P12-137 is able to produce xylanase when wheat bran is used as a substrate. Fermentation was performed in a glass bioreactor withforced aeration. Data obtained have been compared to data from mathematical model obtained by numerical simulation using Matlab 7.9.0.529 (MathWorks, Inc. USA. The numerical simulation of the bioprocess could be a useful tool for adopting a control strategy to achieve increased xylanases yields under pilot or industrial conditions.

Bifunctional xylanases and their potential use in biotechnology

Digital Repository Service at National Institute of Oceanography (India)

Khandeparker, R.; Numan, M.Th.

. J Chromatography 919:389–394 33. Hong SY, Lee JS, Cho KM, Math RK, Kim YH, Hong SJ, Cho YU, Kim H, Yun HD (2006) Assembling a novel bifunctional cel- lulase–xylanase from Thermotoga maritima by end-to-end fusion. Biotechnol Lett 28:1857–1862 34...

Development of a bifunctional xylanase-cellulase chimera with enhanced activity on rice and barley straws using a modular xylanase and an endoglucanase procured from camel rumen metagenome.

Science.gov (United States)

Khalili Ghadikolaei, Kamran; Akbari Noghabi, Kambiz; Shahbani Zahiri, Hossein

2017-09-01

The camel rumen metagenome is an untapped source of glycoside hydrolases. In this study, novel genes encoding for a modular xylanase (XylC) and a cellulase (CelC) were isolated from a camel rumen metagenome and expressed in Escherichia coli BL21 (DE3). XylC with xylanase (Xyn), CBM, and carbohydrate esterase (CE) domains was characterized as a β-1,4-endoxylanase with remarkable catalytic activity on oat-spelt xylan (K cat  = 2919 ± 57 s -1 ). The implication of XylC's modular structure in its high catalytic activity was analyzed by truncation and fusion construction with CelC. The resulting fusions including Cel-CBM, Cel-CBM-CE, and Xyn-CBM-Cel showed remarkable enhancement in CMCase activity with K cat values of 742 ± 12, 1289 ± 34.5, and 2799 ± 51 s -1 compared to CelC with a K cat of 422 ± 3.5 s -1 . It was also shown that the bifunctional Xyn-CBM-Cel with synergistic xylanase/cellulase activities was more efficient than XylC and CelC in hydrolysis of rice and barley straws.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

Science.gov (United States)

Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

DEFF Research Database (Denmark)

Mosbech, Caroline; Holck, Jesper; Meyer, Anne S.

2018-01-01

Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Here, we show...... for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo......-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. The data verify the enzyme's unique ability to catalyze removal of all glucuronoxylan associated...

Effect of Cellulases and Xylanases on Refining Process and Kraft Pulp Properties.

Directory of Open Access Journals (Sweden)

Kamila Przybysz Buzała

Full Text Available Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp's susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time, increased water retention value (WRV and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.

Screening of Thermophilic Bacteria Produce Xylanase from Sapan Sungai Aro Hot Spring South Solok

Science.gov (United States)

Irdawati, I.; Syamsuardi, S.; Agustien, A.; Rilda, Y.

2018-04-01

xylanase is one of the enzymes with great prospects as hemicellulose hydrolyzing enzyme. Global annual market demand for this enzyme reach US 200 million. This enzyme catalyzes the xylan (hemicellulose) reactions breaking into xilooligosakarida and xylose. Xylanase can be applied to various industrial sectors such as bread, sugar xylose, biofuels, especially in bleaching paper (bleaching) pulp. Xylanase Isable to replace conventional chemical bleaching using chlorine that is not friendly for the environment. Currently xylanase production is extracted from the thermophilic bacteria for enzyme stability at high temperatures that are suitable for industrial applications. Thermophilic bacteria can be isolated from a hot spring, one of the which is a source of Sapan Sungai Aro Hot Spring, located in the district South Solok. The aim of this study was to select and identification of thermophilic bacteria can produce xylanase.This roomates is a descriptive study, which was Carried out in the Laboratory of Microbiology, Mathematic and Science Faculty of Padang State University, and Laboratory of Bacteriology, BasoVeterinary Research Center. The research procedure consisted of the preparation and sterilization of materials and tools, medium manufacturing, regeneration, selection and identification. Selection is performed by using a semiquantitative screening plate that contains xylan substrate. Identification is based on microscopic and biochemical characteristics until the genus level.Selection results Showed 12 out of 16 isolates had xilanolitik activity, with the highest activity is SSA2 with xilanolitik index of 0.74. The top five index producehigestxilanolitik isolates that are SSA2, SSA3 and SSA4 identified as Bacillus sp. 1., and SSAS6 and SSA7 is Bacillus sp. 2.

The collagen receptor uPARAP/Endo180

DEFF Research Database (Denmark)

Engelholm, Lars H; Ingvarsen, Signe; Jürgensen, Henrik J

2009-01-01

The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind...... and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem...... in collagen breakdown seems to be involved in invasive tumor growth Udgivelsesdato: 2009...

High xylanase production by Trichoderma viride using pineapple ...

African Journals Online (AJOL)

Xylanases are hydrolases which depolymerize the xylan components present in plants cell wall. Commercial applications for these enzymes include its use in the pulp bleaching, food and animal feed industries, among others. Recently, there is a great interest on the exploitation of agro-industrial wastes as low-cost raw ...

Screening of culture condition for xylanase production by ...

African Journals Online (AJOL)

The study demonstrated not only the importance of the nature of the substrate in obtaining a system resistant to catabolic repression, but also the importance of the culture conditions for biosynthesis of this enzyme. T. viride showed a high potential for xylanase production under the conditions presented in these assays.

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production.

Science.gov (United States)

Cheng, Chieh-Lun; Chang, Jo-Shu

2011-09-01

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H2) production by Clostridium pasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H2/mol glucose. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation.

Science.gov (United States)

Abdullah, Roheena; Nisar, Kinza; Aslam, Aafia; Iqtedar, Mehwish; Naz, Shagufta

2015-01-01

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.

Isolation, Purification, and Characterization of Xylanase Produced by a New Species of Bacillus in Solid State Fermentation

Directory of Open Access Journals (Sweden)

Rajashri D. Kamble

2012-01-01

Full Text Available A thermoalkalophilic new species of Bacillus, similar to Bacillus arseniciselenatis DSM 15340, produced extracellular xylanase under solid state fermentation when wheat bran is used as carbon source. The extracellular xylanase was isolated by ammonium sulfate (80% precipitation and purified using ion exchange chromatography. The molecular weight of xylanase was ~29.8 ;kDa. The optimum temperature and pH for the enzyme activity were 50°C and pH 8.0. The enzyme was active on birchwood xylan and little active on p-nitrophenyl xylopyranoside but not on Avicel, CMC, cellobiose, and starch, showing its absolute substrate specificity. For birchwood xylan, the enzyme gave a Km 5.26 ;mg/mL and Vmax 277.7 ;μmol/min/mg, respectively. In addition, the xylanase was also capable of producing high-quality xylo-oligosaccharides, which indicated its application potential not only in pulp biobleaching processes but also in the nutraceutical industry.

Potential of Laceyella sacchari strain B42 crude xylanase in ...

African Journals Online (AJOL)

rajmac

2013-02-06

Feb 6, 2013 ... sacchari strain B42. Maximal xylanase production was achieved at the incubation period of 48 h with ... chemicals for pulp processing and bleaching. The major ... industrial enzyme with great biotechnological application.

Evaluation of the effect of different wheats and xylanase supplementation on performance, nutrient and energy utilisation in broiler chicks

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Gemma González-Ortiz

2016-09-01

Full Text Available The aim of this study was to evaluate the performance, nutrient utilisation and energy metabolism of broiler chicks fed 8 different wheat samples, supplemented or not with xylanase. Seven-hundred sixty eight male broilers (1-day-old were distributed to 16 experimental treatments (6 replicates per treatment. The treatments were in a factorial arrangement with 8 different wheats and 2 levels of xylanase (0 or 16,000 BXU/kg. The predicted apparent metabolisable energy (AME of the wheat samples ranged from 13.0 to 13.9 MJ/kg and all diets were formulated to contain the same amount of wheat. Body weight gain (BWG and feed intake (FI were measured at 21 d, as was jejunal digesta viscosity, and feed conversion ratio (FCR calculated. On day 24, one representative bird per pen was selected to calculate whole body energetics. At 21 d, 3 chicks per replicate were randomly allocated to metabolism cages for energy and nutrient utilisation determinations, and were continued on the experimental diets until 24-d-old. No interactions were observed for any performance response variables, ileal nutrient utilisation or digesta viscosity. Xylanase improved BWG and reduced FCR and digesta viscosity (P < 0.05. Wheat influenced dry matter (DM utilisation and xylanase increased ileal digestible energy (P = 0.04. Xylanase also improved (P < 0.05 DM and nitrogen retention. Apparent metabolisable energy and AME corrected for nitrogen (AMEn were subject to an interaction whereby wheats 2 and 6, which returned the lowest AME and AMEn values, responded to xylanase supplementation and the remainder did not. Net energy for production and the efficiency of energy use for production were not influenced by xylanase, but were affected by wheat (P < 0.05. Despite the significant differences between wheats with regards to their nutrient utilisation and energy metabolism in birds, xylanase removed this variance and resulted in more homogeneous performance.

Optimization of xylanase production by Mucor indicus, Mucor hiemalis, and Rhizopus oryzae through solid state fermentation

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Sanaz Behnam

2016-03-01

Full Text Available Introduction: Xylan is the main hemicellulosic polymer in a number of lignocelluloses which can be hydrolyzed by xylanolytic enzymes. One of the main ways for enzymes production is solid state fermentation (SSF. The ability of three fungal strains (Mucor indicus, Mucor hiemalis, and Rhizopus oryzae for xylanase production on wheat bran by SSF was investigated. Materials and methods: The effects of cultivation temperature, medium moisture content, and cultivation time on the enzyme production were investigated. Experiments were designed with an orthogonal central composite design on three variables using response surface methodology (RSM. Analysis of variance was applied and the enzyme production was expressed with a mathematical equation as a function of the three factors. The optimum operating conditions for the enzyme production was obtained. Results: For xylanase production by M. indicus, M. hiemalis and R. oryzae the optimum temperatures were 40.0, 43.4 and 43.4ºC respectively. These values were 49.8, 54.2 and 71.8% for moisture percent and 51.3, 53.2 and 53.5 h for cultivation time. The highest enzyme activities per g of dry substrate (gds were 43.1, 43.8 and 25.9 U/gds for M. indicus, M. hiemalis and R. oryzae respectively. Discussion and conclusion: All the fungi were able to produce xylanase. Maximum xylanase production was predicted by M. indicus and M. hiemalis at similar optimum conditions, while R. oryzae produced relatively lower xylanase activity even at the best condition. 

Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis.

Science.gov (United States)

Sadaf, Ayesha; Khare, S K

2014-02-01

De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries. Copyright © 2013 Elsevier Ltd. All rights reserved.

Xylanase from Fusarium heterosporum: Properties and influence of ...

African Journals Online (AJOL)

Unioeste

2014-02-26

Feb 26, 2014 ... influence of thiol compounds on xylanase activity. Paulo Ricardo ... and the extraction of plant oil, coffee and starch (Ahmed ... of F. heterosporum on 5 g of various carbon sources ... brewing residue under solid-state fermentation (fungal strain and .... active within the acidic pH range of 4.5 to 5.5 and.

High xylanase production by Trichoderma viride using pineapple ...

African Journals Online (AJOL)

SAM

2014-05-28

May 28, 2014 ... T. viride xylanase was stable at 40°C, showing the half-life (T1/2) value of ... Many studies have demonstrated that the pulp ... disposed in the environment without an adequate .... one-way analysis of variance and compared through the Tukey test, .... production by T. harzianum with wheat straw increases.

A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

Science.gov (United States)

Nakazawa, Hikaru; Kawai, Tetsushi; Ida, Noriko; Shida, Yosuke; Shioya, Kouki; Kobayashi, Yoshinori; Okada, Hirofumi; Tani, Shuji; Sumitani, Jun-Ichi; Kawaguchi, Takashi; Morikawa, Yasushi; Ogasawara, Wataru

2016-01-01

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus β-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III. To attain optimal saccharifying potential in T. reesei, we constructed two new strains, C1AB1 and E1AB1, in which aabg1 was expressed heterologously by means of the cbh1 or egl1 promoters, respectively, so that the endogenous XYN III synthesis remained intact. Due to the presence of wild-type xyn3 in T. reesei E1AB1, enzymes prepared from this strain were 20-30% more effective in the saccharification of alkaline-pretreated rice straw than enzyme extracts from X3AB1, and also outperformed recent commercial cellulase preparations. Our results demonstrate the importance of XYN III in the conversion of alkaline-pretreated cellulosic biomass by T. reesei. Copyright © 2015 Elsevier Inc. All rights reserved.

Production of Xylanase by Recombinant Bacillus subtilis DB104 Cultivated in Agroindustrial Waste Medium

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Is Helianti

2016-07-01

Full Text Available A recombinant Bacillus subtilis DB104 strain harbouring recombinant plasmid pSKE194 containing an Open Reading Frame (ORF of endoxylanase and its indigenous promoter from the wild-type B. subtilis AQ1 strain was constructed. This recombinant B. subtilis DB104 strain had higher endoxylanase activity than the nonrecombinant B. subtilis DB104 strain in standard media, such as Luria Bertani (LB and LB with xylan. The agroindustrial wastes corncobs and tofu liquid waste were chosen as cost-effective carbon and nitrogen sources, respectively, to test the economics of xylanase production using the recombinant B. subtilis DB104 at a larger scale. Submerged fermentation using a 4.5 L working volume fermentor with tofu liquid waste and 4% corncobs produced maximum xylanase activity of 1296 ± 1.2 U/mg (601.7 ± 0.6 U/mL after 48-hour fermentation at 37°C with 150 rpm agitation; this is more than twofold higher than the activity produced in an Erlenmeyer flask. This is the first report of high xylanase activity produced from recombinant B. subtilis using inexpensive medium. During fermentation, the xylanase degrades corncobs into xylooligosaccharides, showing its potential as an enzyme feed additive or in xylooligosaccharide production.

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin

2017-08-10

The present invention relates to a method and system for dynamically analyzing, determining, predicting and displaying ranked suitable heterologous biosynthesis pathways for a specified host. The present invention addresses the problem of finding suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived and developed to systematically and dynamically search for, determine, analyze, and display promising heterologous pathways while considering competing endogenous reactions in a given host organism.

Polypeptides having xylanase activity and polynucleotides encoding same

Energy Technology Data Exchange (ETDEWEB)

Spodsberg, Nikolaj; Shaghasi, Tarana

2017-06-20

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Evaluation of xylanases from Aspergillus niger and Trichoderma sp ...

African Journals Online (AJOL)

Despite being present in relatively low amounts, pentosans and hemicelluloses play an important role in dough rheology and bread properties. The aim of this work is to understand how the xylanases from Aspergillus niger and Trichoderma sp. influence dough rheology, such as elasticity, extensibility, strength and stability.

Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

Science.gov (United States)

Malau, N. D.; Sianturi, M.

2017-03-01

Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21.

Science.gov (United States)

Parab, Pankaj; Khandeparker, Rakhee; Amberkar, Ujwala; Khodse, Vishwas

2017-10-01

Enzymatic hydrolysis of seaweed biomass was studied using xylanase produced from marine bacteria Bacillus sp. strain BT21 through solid-state fermentation of wheat bran. Three types of seaweeds, Ahnfeltia plicata , Padina tetrastromatica and Ulva lactuca , were selected as representatives of red, brown, and green seaweeds, respectively. Seaweed biomass was pretreated with hot water. The efficiency of pretreated biomass to release reducing sugar by the action of xylanase as well as the type of monosaccharide released during enzyme saccharification of seaweed biomass was studied. It was seen that pretreated biomass of seaweed A. plicata, U. lactuca , and P. tetrastroma , at 121 °C for 45 min, followed by incubation with 50 IU xylanase released reducing sugars of 233 ± 5.3, 100 ± 6.1 and 73.3 ± 4.1 µg/mg of seaweed biomass, respectively. Gas chromatography analysis illustrated the release of xylose, glucose, and mannose during the treatment process. Hot water pre-treatment process enhanced enzymatic conversion of biomass into sugars. This study revealed the important role of xylanase in saccharification of seaweed, a promising feedstock for third-generation bioethanol production.

A sequential approach in treatment of perio-endo lesion.

Science.gov (United States)

Narang, Sumit; Narang, Anu; Gupta, Ruby

2011-04-01

The success of a combined periodontal and endodontic lesion depends on the elimination of both of these disease processes. In the case of a combined endo-perio lesion, the endodontic therapy results in healing of the endodontic component of involvement while the prognosis of tooth would finally depend on the healing of the periodontal structures. This case report evaluates the efficacy of bioactive glass in the management of furcation defect associated with an endo-perio lesion in a right mandibular first molar. A 22-year-old male patient with an endo-perio lesion in the right mandibular first molar was initially treated with endodontic therapy. Following the endodontic treatment, the furcation defect was treated using bioactive glass in a putty form. At the end of 9 months, there was a gain in the clinical attachment level and reduction in probing depth. Radiographic evidence showed that there was a significant bony fill.

Shushaku Endo (1923-1996): his tuberculosis and his writings.

Science.gov (United States)

Sharma, O P

2006-03-01

Shushaku Endo, one of the finest 20th century Japanese novelists, is not as well known in the West as some of his contemporary Japanese writers including, Yukio Mishima, Yasunari Kawabata, Kobo Abe, and Kenzaburo Oe. Mishima deals with turmoil of adolescence laced with surrealistic historical romance; Abe conjures strange, evocative images in the Kafkaesque tradition; Kawabata writes about loneliness and desolation; and Oe creates imaginary themes of life and myth that entwine and portray human predicaments. Kawabata and Oe both received Nobel Prizes in Literature. Endo's writing is different. He confronts problems pertaining to faith, morality, and individual responsibility. In his early years Endo struggled as a loner, an outsider, and a misfit. This pattern continued to a large extent into his adult life, most of which was consumed with fighting his life threatening tuberculosis. The agonies of his illness intensified his talent for exploring the complexity of human relationships.

Altered sarco(endo)plasmic reticulum calcium adenosine triphosphatase 2a content: Targets for heart failure therapy.

Science.gov (United States)

Liu, Gang; Li, Si Qi; Hu, Ping Ping; Tong, Xiao Yong

2018-05-01

Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is responsible for transporting cytosolic calcium into the sarcoplasmic reticulum and endoplasmic reticulum to maintain calcium homeostasis. Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is the dominant isoform expressed in cardiac tissue, which is regulated by endogenous protein inhibitors, post-translational modifications, hormones as well as microRNAs. Dysfunction of sarco(endo)plasmic reticulum calcium adenosine triphosphatase is associated with heart failure, which makes sarco(endo)plasmic reticulum calcium adenosine triphosphatase a promising target for heart failure therapy. This review summarizes current approaches to ameliorate sarco(endo)plasmic reticulum calcium adenosine triphosphatase function and focuses on phospholamban, an endogenous inhibitor of sarco(endo)plasmic reticulum calcium adenosine triphosphatase, pharmacological tools and gene therapies.

Eficacia del proceso de esterilización de los Mini-Endo-bloc

Directory of Open Access Journals (Sweden)

Maritza Eraso Rodríguez

2017-01-01

Full Text Available El Mini-Endo-Bloc® es un elemento usado para mediar las limas durante la endodoncia, presenta muchas concavidades y orificios que dificultan el proceso de esterilización y pueden promover la infección cruzada o comprometer el pronóstico del tratamiento endodóntico. Objetivo: Observar la eficacia de la esterilización de los Mini-Endo-Bloc® utilizados en la clínica de la Federación Odontológica Colombiana (FOC. Métodos: Se recolectaron 60 Mini-Endo-Bloc® utilizados en pacientes que presentaban diagnósticos de necrosis pulpar y periodontitis apical, lo cuales se dividieron en dos grupos: Grupo control: 12 Mini-Endo-Bloc® desinfectados y no esterilizados. Grupo experimental: 48 Mini-Endo-Bloc® desinfectados y esterilizados, se realizó, según lo recomendado por la Farmacopea USPXXXV, prueba de esterilidad en Caldo Thioglycollate a 37°C, medio de caldo Casoy a 37°C y medio de caldo Casoy a 25°C para determinar presencia de bacterias, levaduras y hongos. Resultados: Al someter los Mini-Endo-Bloc® al protocolo de esterilización establecido por la FOC y teniendo en cuenta los parámetros de las pruebas de esterilidad, en el grupo experimental el 50% (n=24, fueron reprobadas (crecimiento microorganismos y 50% (n = 24 fueron aprobadas (no crecimiento de microorganismos. Discusión: la mitad de las muestras fueron reprobadas, estos resultados pueden ser por fallas en el proceso de desinfección mecánica manual directa, el cual se dificulta en los Mini-Endo-Bloc®, debido a su estructura y diseño, permitiendo que residuos biológicos permanezcan en la superficie del instrumento, evitando que el vapor saturado entre en contacto con el material o los microorganismos durante la esterilización.

76 FR 15321 - SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Inc.'s, Petition...

Science.gov (United States)

2011-03-21

...] SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Inc.'s, Petition for Review... an advisory committee Ethicon Endo- Surgery Inc.'s (EES's), petition for review of the Agency's... Foreman, FDA, CDRH, to Ken Charak, Ethicon Endo-Surgery, Inc., containing the order denying approval of...

Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

Science.gov (United States)

Bu, Yifan; Cui, Yinglu; Peng, Ying; Hu, Meirong; Tian, Yu'e; Tao, Yong; Wu, Bian

2018-04-01

Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

(Trametes sp.) in the production of cellulase and xylanase

African Journals Online (AJOL)

Nanda

2016-05-18

May 18, 2016 ... in solid-sate fermentation (SSF), in this work, the production of cellulase and xylanase by the fungus ... sugars can be converted to ethanol, lactic acid and ... substances; clarification of juices and wines; improving ..... SSF processes has a marked effect on growth kinetics, ..... Overview of applied solid-state.

Photosynthetic fuel for heterologous enzymes

DEFF Research Database (Denmark)

Mellor, Silas Busck; Vavitsas, Konstantinos; Nielsen, Agnieszka Janina Zygadlo

2017-01-01

of reducing power. Recent work on the metabolic engineering of photosynthetic organisms has shown that the electron carriers such as ferredoxin and flavodoxin can be used to couple heterologous enzymes to photosynthetic reducing power. Because these proteins have a plethora of interaction partners and rely...... on electrostatically steered complex formation, they form productive electron transfer complexes with non-native enzymes. A handful of examples demonstrate channeling of photosynthetic electrons to drive the activity of heterologous enzymes, and these focus mainly on hydrogenases and cytochrome P450s. However......, competition from native pathways and inefficient electron transfer rates present major obstacles, which limit the productivity of heterologous reactions coupled to photosynthesis. We discuss specific approaches to address these bottlenecks and ensure high productivity of such enzymes in a photosynthetic...

A sequential approach in treatment of perio-endo lesion

OpenAIRE

Sumit Narang; Anu Narang; Ruby Gupta

2011-01-01

The success of a combined periodontal and endodontic lesion depends on the elimination of both of these disease processes. In the case of a combined endo-perio lesion, the endodontic therapy results in healing of the endodontic component of involvement while the prognosis of tooth would finally depend on the healing of the periodontal structures. This case report evaluates the efficacy of bioactive glass in the management of furcation defect associated with an endo-perio lesion in a right man...

Comparison of several ethanol productions using xylanase, inorganic salts, surfactant

Science.gov (United States)

Wu, Yan; Lu, Jie; Yang, Rui-feng; Song, Wen-jing; Li, Hai-ming; Wang, Hai-song; Zhou, Jing-hui

2017-03-01

Liquid hot water (LHW) pretreatment is an effective and environmentally friendly method to produce bioethanol with lignocellulosic materials. Corn stover was pretreated with liquid hot water (LHW) and then subjected to semi-simultaneous saccharification and fermentation (S-SSF) to obtain high ethanol concentration and yield. The present study aimed to confirm the effect of several additives on the fermentation digestibility of unwashed WIS of corn stover pretreated with LHW. So we also investigated the process, such as enzyme addition, inorganic salts, surfactant and different loading Triton. Results show that high ethanol concentration is necessary to add xylanase in the stage of saccharification. The ethanol concentration increased mainly with magnesium ion on fermentation. Comparing with Tween 80, Span 80 and Polyethylene glycol, Triton is the best surfactant. In contrast to using xylanase and Triton respectively, optimization can make up the lack of stamina and improve effect of single inorganic salts.

A sequential approach in treatment of perio-endo lesion

Directory of Open Access Journals (Sweden)

Sumit Narang

2011-01-01

Full Text Available The success of a combined periodontal and endodontic lesion depends on the elimination of both of these disease processes. In the case of a combined endo-perio lesion, the endodontic therapy results in healing of the endodontic component of involvement while the prognosis of tooth would finally depend on the healing of the periodontal structures. This case report evaluates the efficacy of bioactive glass in the management of furcation defect associated with an endo-perio lesion in a right mandibular first molar. A 22-year-old male patient with an endo-perio lesion in the right mandibular first molar was initially treated with endodontic therapy. Following the endodontic treatment, the furcation defect was treated using bioactive glass in a putty form. At the end of 9 months, there was a gain in the clinical attachment level and reduction in probing depth. Radiographic evidence showed that there was a significant bony fill.

Direct Proof of Endo-Epicardial Asynchrony of the Atrial Wall During Atrial Fibrillation in Humans.

Science.gov (United States)

de Groot, Natasja; van der Does, Lisette; Yaksh, Ameeta; Lanters, Eva; Teuwen, Christophe; Knops, Paul; van de Woestijne, Pieter; Bekkers, Jos; Kik, Charles; Bogers, Ad; Allessie, Maurits

2016-05-01

The presence of focal fibrillation waves during atrial fibrillation (AF) can, besides ectopic activity, also be explained by asynchronous activation of the atrial endo- and epicardial layer and transmurally propagating fibrillation waves. To provide direct proof of endo-epicardial asynchrony, we performed simultaneous high-resolution mapping of the right atrial endo- and epicardial wall during AF in humans. Intraoperative mapping of the endo- and epicardial right atrial wall was performed during (induced) AF in 10 patients with AF (paroxysmal: n=3; persistent: n=4; and longstanding persistent: n=3) and 4 patients without a history of AF. A clamp made of 2 rectangular 8×16 electrode arrays (interelectrode distance 2 mm) was inserted into the incision in the right atrial appendage. Recordings of 10 seconds of AF were analyzed to determine the incidence of asynchronous endo-epicardial activation times (≥15 ms) of opposite electrodes. Asynchronous endo-epicardial activation ranged between 0.9 and 55.9% without preference for either side. Focal waves appeared equally frequent at endocardium and epicardium (11% versus 13%; ITALIC! P=0.18). Using strict criteria for breakthrough (presence of an opposite wave within 4 mm and ≤14 ms before the origin of the focal wave), the majority (65%) of all focal fibrillation waves could be attributed to endo-epicardial excitation. We provided the first evidence for asynchronous activation of the endo-epicardial wall during AF in humans. Endo-epicardial asynchrony may play a major role in the pathophysiology of AF and may offer an explanation why in some patients therapy fails. © 2016 American Heart Association, Inc.

Suitable conditions for xylanases activities from Bacillus sp. GA2(1 and Bacillus sp. GA1(6 and their properties for agricultural residues hydrolysis

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Sudathip Chantorn

2016-04-01

Full Text Available Bacillus sp. GA2(1 and Bacillus sp. GA1(6 were isolated from soybean field in Khon Kaen province, Thailand. Crude enzymes from both isolates showed the activities of cellulase, xylanase, and mannanase at 37°C for 24 h. The highest xylanase activities of Bacillus sp. GA2(1 and Bacillus sp. GA1(6 were 1.58±0.25 and 0.82±0.16 U/ml, respectively. The relative xylanase activities from both strains were more than 60% at pH 5.0 to 8.0. The optimum temperature of xylanases was 50°C in both strains. The residual xylanase activities from both strains were more than 70% at 60°C for 60 min. Five agricultural wastes (AWs, namely coffee residue, soybean meal, potato peel, sugarcane bagasse, and corn cobs, were used as substrates for hydrolysis properties. The highest reducing sugar content of 101±1.32 µg/ml was obtained from soybean meal hydrolysate produced by Bacillus sp. GA2(1 xylanase.

Optimization of Xylanase production from Penicillium sp.WX-Z1 by a two-step statistical strategy: Plackett-Burman and Box-Behnken experimental design.

Science.gov (United States)

Cui, Fengjie; Zhao, Liming

2012-01-01

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO(3), MgSO(4), and CaCl(2). The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO(3), 12.71; MgSO(4), 0.96; and CaCl(2), 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor.

Optimisation of amylase and xylanase addition in dependance of white flour amylase activity

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Lončar Davor M.

2016-01-01

Full Text Available In this study the effect of different quantities of added amylase to white wheat flours characterized with different activities of naturally existing amylases is tested. Response surface methodology is chosen to test the effects of main applied technological parameters on bread quality responses. Independent variables are chosen to be: quantity of added amylase and bulk fermentation time, while analysed responses are: specific volume, grain structure, bulk fermentation. Bread quality responses are statistically significant, while predicted and observed responses correspond very well, which allows good prediction of bread quality parameters based on applied technological parameters and flour characteristics. Score analysis shows that optimum quantity of amylase addition regarding bread quality depends on the activity of naturally existing amylases. Optimal quantity of added xylanase in bread samples made from both flour types is 0.004%. Xylanase improved properties of white wheat bread and higher effect is experienced with flour that has more active naturally existing amylases. Addition of amylase has statistically significantly increased a* values of crust. Addition of xylanase has statistically significantly decreased values of b* in comparison to the respective bread sample with only added amylase.

Ultrasounds pretreatment of olive pomace to improve xylanase and cellulase production by solid-state fermentation.

Science.gov (United States)

Leite, Paulina; Salgado, José Manuel; Venâncio, Armando; Domínguez, José Manuel; Belo, Isabel

2016-08-01

Olive mills generate a large amount of waste that can be revaluated. This work aim to improve the production lignocellulolytic enzymes by solid-state fermentation using ultrasounds pretreated olive mill wastes. The composition of olive mill wastes (crude and exhausted olive pomace) was compared and several physicochemical characteristics were significantly different. The use of both wastes in SSF was evaluated and a screening of fungi for xylanase and cellulase production was carried out. After screening, the use of exhausted olive pomace and Aspergillus niger led to the highest enzyme activities, so that they were used in the study of ultrasounds pre-treatment. The results showed that the sonication led to a 3-fold increase of xylanase activity and a decrease of cellulase activity. Moreover, the liquid fraction obtained from ultrasounds treatment was used to adjust the moisture of solid and a positive effect on xylanase (3.6-fold increase) and cellulase (1.2-fold increase) production was obtained. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of tannin with and without polyethylene glycol on in vitro ...

African Journals Online (AJOL)

PEG also increased the activity of protease enzymes, xylanase, endo- and exocellulases, indicating that tannins depress the digestibility of feeds partly by suppressing the activity of these enzymes. Consequently, condensed tannin depressed the digestibility of feed in the rumen by affecting the activity of enzymes involved ...

EFFECT OF XYLANASE ADDED TO A RYE-BASED DIET ON NUTRIENT UTILIZATION IN PIGS

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Jaroslav Heger

2012-02-01

Full Text Available The effect of enzyme xylanase derived from Trichoderma longibrachiatum supplemented to a rye-based diet on apparent ileal digestibility of amino acids and non-starch polysaccharides constituting sugars was studied. Enzymes supplementation at 200 mg.kg−1 increased (P˂0.05 the digestibility of total amino acids from 67.1 to 70.8. When the dietary concentration of enzyme increased from 0 to 100 mg.kg-1, the ileal digestibility of the NSP constituents gradually increased as well. No further increase was observed with the supplementation level of 200 mg.kg-1. The improvement in the digestibility of arabinose and xylose (685%, P˂0.05 was much higher in comparison with remaining sugars (110%, P˂0.05. The apparent ileal digestibility of galactose was positively influenced by xylanase but it remained negative in all dietary treatments, presumably due to the high concentration of galactose in endogenous secretions. It is concluded that xylanase effectively degrades non-starch polysaccharides in upper digestive tract and marginally improves amino acid availability in young pigs.

Xylanase production by a thermo-tolerant Bacillus species under solid-state and submerged fermentation

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Uma Gupta

2009-12-01

Full Text Available Effects of xylose on xylanase production by a thermophilic Bacillus sp showed diverse patterns on corn cob (CC and wheat bran (WB as sole carbon sources in solid- state fermentation (SSF and submerged fermentation (SmF. Supplementation of these media with either mineral salt solution (MSS or yeast extract peptone (YEP also exerted variable effects. While under SSF, xylose stimulated xylanase synthesis by 44.01%, on wheat bran supplemented with MSS, it decreased the enzyme activity by 12.89% with YEP supplementation. In SmF, however the enzyme synthesis was stimulated by xylose on supplementation with both MSS and YEP by 41.38% and 27.47%, respectively. On corn cob under SSF, xylose repression was significant both with MSS (26.92% and YEP (23.90% supplementation. Repression by xylose also took place on corn cob and YEP (19.69% under SmF, while significant stimulation (28.55% was observed by MSS supplementation. The possible role of media composition and fermentation conditions in the regulation of xylanase synthesis by xylose is discussed.

Enzymic hydrolysis of xylans. I. A high xylanase and beta-xylosidase producing strain of Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Conrad, D.

1981-01-01

Aspergillus niger, strain 110.42 (CBS) was selected as a producer of high xylanolytic activities. The time course of xylanase and beta-xylosidase production as well as the effect of pH and temperature on the activity of these enzymes were studied. High-performance liquid chromatography analysis of the enzymic degradation of arabinoxylan showed a nearly complete conversion to pentose sugars. Aspects of using crude xylanase preparations for enzymic saccharification of xylans are discussed.

Penatalaksanaan Lesi Endo-Perio dengan Perawatan Endodontik Non Bedah

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Irene Sulistio

2014-06-01

Full Text Available Secara anatomis pulpa dan periodontal saling berhubungan. Pada keadaan tertentu bisa terjadi inflamasi di pulpa dan periodontal. Hal ini disebut dengan lesi endodontik-periodontal. Perkembangan dan progresi lesi endo-perio ini dipengaruhi oleh faktor etiologi seperti bakteri, jamur, dan virus serta faktor pendukung seperti trauma, resorpsi akar, perforasi, dan malformasi gigi. Pada lesi endo-perio diperlukan rencana perawatan yang tepat agar prognosis perawatan dari gigi tersebut dapat baik. Artikel ini bertujuan memaparkan perawatan kasus lesi endo-perio yang berhasil setelah manajemen endodontik tanpa dilakukan bedah endodontik. Seorang pasien laki-laki berusia 21 tahun datang ke RSGM dengan keluhan gigi belakang kiri bawah sakit. Gigi tersebut pernah ditumpat 1 tahun yang lalu.Pada gambaran radiograf terdapat lesi radiolusen luas pada tulang alveolar sekitar akar distal. Perawatan endodontik dilakukan dengan pergantian bahan dressing kalsium hidroksida sebanyak 3 kali. Pada kontrol bulan kedua terlihat terjadi penulangan pada bagian lesi periodontal tersebut dan pasien tidak mengeluhkan rasa sakit. Kesimpulan hasil perawatan lesi endodontik periodontal dapat dirawat dengan perawatan endodontik non bedah. Management of Nonsurgical Endodontic Treatment on A Combined Endo-period Lesion. The pulp and periodontium have anatomic interrelationships. As the tooth matures, and the root is formed, three main avenues are created between pulp and periodontal ligament, i.e. dentinal tubules, lateral and accessory canals, apical foramen. These are the pathways that may provide a means by which pathological agent pass between the pulp and periodontium, thereby creating the endo - period lesion. Etiologic factors such as bacteria, fungi, and viruses as well as contributing factors such as trauma, root resorption, and dental malformations play a significant role in the development and progression of such lesions. In the endodontic - periodontal lesion is

Improvement for enhanced xylanase production by Cellulosimicrobium cellulans CKMX1 using central composite design of response surface methodology.

Science.gov (United States)

Walia, Abhishek; Mehta, Preeti; Guleria, Shiwani; Shirkot, Chand Karan

2015-12-01

The effects of yeast extract (X 1 ), NH 4 NO 3 (X 2 ), peptone (X 3 ), urea (X 4 ), CMC (X 5 ), Tween 20 (X 6 ), MgSO 4 (X 7 ), and CaCO 3 (X 8 ) on production of xylanase from Cellulosimicrobium cellulans CKMX1 were optimized by statistical analysis using response surface methodology (RSM). The RSM was used to optimize xylanase production by implementing the Central composite design. Statistical analysis of the results showed that the linear, interaction and quadric terms of these variables had significant effects. However, only the linear effect of X 4 , X 5 , interaction effect of X 1 X 7 , X 1 X 8 , X 2 X 3 , X 2 X 8 , X 3 X 6 , X 3 X 8 , X 4 X 6 , X 4 X 7 , X 5 X 7 , X 5 X 8 and quadratic effect of X 3 2 , X 5 2 and X 7 2 found to be insignificant terms in the quadratic model and had no response at significant level. The minimum and maximum xylanase production obtained was 331.50 U/g DBP and 1027.65 U/g DBP, respectively. The highest xylanase activity was obtained from Run No. 30, which consisted of yeast extract (X 1 ), 1.00 g (%); NH 4 NO 3 (X 2 ), 0.20 g (%); peptone (X 3 ), 1.00 g (%); urea (X 4 ), 10 mg (%); CMC (X 5 ), 1.00 g (%); Tween 20 (X 6 ), 0.02 mL (%); CaCO 3 (X 7 ), 0.50 g (%) and MgSO 4 (X 8 ), 9.0 g (%). The optimization resulted in 3.1-fold increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP after 72 h of incubation in stationary flask experiment. Application of cellulase-free xylanase in pulp biobleaching from C. cellulans CKMX1 under C-E P -D sequence has been shown to bring about a 12.5 % reduction of chlorine, decrease of 0.8 kappa points (40 %), and gain in brightness was 1.42 % ISO points in 0.5 % enzyme treated pulp as compared to control.

Optimization of Xylanase Production through Response Surface Methodology by Fusarium sp. BVKT R2 Isolated from forest soil and its applications in saccharification

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Ramanjaneyulu Golla

2016-09-01

Full Text Available AbstractXylanses are hydrolytic enzymes with wide applications in several industries like biofuels, paper and pulp, deinking, food and feed. The present study was aimed at hitting at high yield xylanase producing fungi from natural resources. Two highest xylanase producing fungal isolates - Q12 and L1were picked from collection of 450 fungal cultures for the utilization of xylan. These fungal isolates - Q12 and L1 were identified basing on ITS gene sequencing analysis as Fusarium sp. BVKT R2 (KT119615 and Fusarium strain BRR R6 (KT119619, respectively with construction of phylogenetic trees. Fusarium sp. BVKT R2 was further optimized for maximum xylanase production and the interaction effects between variables on production of xylanase were studied through response surface methodology. The optimal conditions for maximal production of xylanase were sorbitol 1.5%, yeast extract 1.5%, pH of 5.0, Temperature of 32.5ºC, and agitation of 175 rpm. Under optimal conditions, the yields of xylanase production by Fusarium sp. BVKT R2 was as high as 4560 U/ml in SmF. Incubation of different lignocellulosic biomasses with crude enzyme of Fusarium sp. BVKT R2 at 37°C for 72 h could achieve about 45% saccharification. The results suggest that Fusarium sp. BVKT R2 has potential applications in saccharification process of biomass.Key words: Fusarium sp., Optimization, Response Surface Methodology, Saccharification, Submerged fermentation, Xylanase

Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.

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Xiaoyu Wang

Full Text Available Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X, respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.

[Modern concepts of etiology, pathogenesis and treatment approaches to endo-perio lesions].

Science.gov (United States)

Grudianov, A I; Makeeva, M K; Piatgorskaia, N V

2013-01-01

A combination ofperiodontitis and pulp or periapical tissues inflammation in one tooth is known as endo-periodontal lesions. Such kind of lesion is serious problem of modern dentistry. It was found that pathogenic microflora of periodontal pocket and root canal of tooth with eno-perio lesion is almost the equal and consist of anaerobic microorganisms. Pathogenic effects have not only microorganisms but also their life products. Apical foramen, lateral and additional canals are physiological ways for pathogens migration. Inflammatory processes in these structures complicate each other. Lack of information among dentists about treatment possibilities of endo-perio lesions is a main reasons of extraction such kind of teeth. Simultaneous elimination of pathogens both from periodontal pocket and root canal is a key factor for effective treatment. Periodontal status is main factor for prognosis of tooth with endo-perio lesion, because of it treatment of endo-perio lesions should consist of two stages: infection elimination and regeneration of tooth-supported structures.

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

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Ashwani Sanghi

2010-06-01

Full Text Available The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 ºC. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

Science.gov (United States)

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included ...

Chapter Three

African Journals Online (AJOL)

TonukariJ

Nyerhovwo J. TonukariY, *, John S. Scott-Craig and Jonathan D. Walton. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824. Accepted 18 November 2002. The expression of four Cochliobolus carbonum endo-1,4-β-xylanase genes (XYL1, XYL2, XYL3, XYL4),.

Effect of Aspergillus niger xylanase on dough characteristics and bread quality attributes.

Science.gov (United States)

Ahmad, Zulfiqar; Butt, Masood Sadiq; Ahmed, Anwaar; Riaz, Muhammad; Sabir, Syed Mubashar; Farooq, Umar; Rehman, Fazal Ur

2014-10-01

The present study was conducted to investigate the impact of various treatments of xylanase produced by Aspergillus niger applied in bread making processes like during tempering of wheat kernels and dough mixing on the dough quality characteristics i.e. dryness, stiffness, elasticity, extensibility, coherency and bread quality parameters i.e. volume, specific volume, density, moisture retention and sensory attributes. Different doses (200, 400, 600, 800 and 1,000 IU) of purified enzyme were applied to 1 kg of wheat grains during tempering and 1 kg of flour (straight grade flour) during mixing of dough in parallel. The samples of wheat kernels were agitated at different intervals for uniformity in tempering. After milling and dough making of both types of flour (having enzyme treatment during tempering and flour mixing) showed improved dough characteristics but the improvement was more prominent in the samples receiving enzyme treatment during tempering. Moreover, xylanase decreased dryness and stiffness of the dough whereas, resulted in increased elasticity, extensibility and coherency and increase in volume & decrease in bread density. Xylanase treatments also resulted in higher moisture retention and improvement of sensory attributes of bread. From the results, it is concluded that dough characteristics and bread quality improved significantly in response to enzyme treatments during tempering as compared to application during mixing.

76 FR 71980 - SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Incorporated's...

Science.gov (United States)

2011-11-21

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2010-P-0176] SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Incorporated's Petition for... SEDASYS computer-assisted personalized sedation system (SEDASYS) submitted by Ethicon Endo-Surgery Inc...

Endo-periodontal lesion--endodontic approach.

Science.gov (United States)

Jivoinovici, R; Suciu, I; Dimitriu, B; Perlea, P; Bartok, R; Malita, M; Ionescu, C

2014-01-01

Endo-perio lesions might be interdependent because of the vascular and anatomic connections between the pulp and the periodontium. The aim of this study is to emphasise that primary endodontic lesion heals after a proper instrumentation, disinfection and sealing of the endodontic space. The primary endodontic lesion with a secondary periodontal involvement first requires an endodontic therapy and, in the second stage, a periodontal therapy. The prognosis is good, with an adequate root canal treatment; it depends on the severity of the periodontal disease, appropriate healing time and the response to the treatment. A correct diagnosis is sometimes difficult; an accurate identification of the etiologic factors is important for an adequate treatment. Primary perio-endo lesion may heal after a proper disinfection and sealing of the endodontic system, the one-year follow-up radiograph showing bonny repair. Invasive periodontal procedures should be avoided at that moment. The microorganisms and by-products from the infected root canal may cross accessory and furcal canals and determine sinus tract and loss of attachment. In both clinical cases presented in this article, successful healing was obtained after a proper disinfection and sealing of the endodontic system.

Catalytic performance of corn stover hydrolysis by a new isolate Penicillium sp. ECU0913 producing both cellulase and xylanase.

Science.gov (United States)

Shi, Qian-Qian; Sun, Jie; Yu, Hui-Lei; Li, Chun-Xiu; Bao, Jie; Xu, Jian-He

2011-07-01

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.

Influence of xylanase addition on the characteristics of loaf bread prepared with white flour or whole grain wheat flour

Directory of Open Access Journals (Sweden)

Leandra Zafalon Jaekel

2012-12-01

Full Text Available The aim of this study was to verify the influence of the addition of the enzyme xylanase (four concentrations: 0, 4, 8, and 12 g.100 kg-1 flour on the characteristics of loaf bread made with white wheat flour or whole grain wheat flour. Breads made from white flour and added with xylanase had higher specific volumes than those of the control sample (no enzyme; however, the specific volume did not differ significantly (p < 0.05 among the breads with different enzyme concentrations. All formulations made from whole grain wheat flour and added with xylanase also had specific volumes significantly higher than those of the control sample, and the highest value was found for the 8 g xylanase.100 kg-1 flour formulation. With respect to moisture content, the formulations with different enzyme concentrations showed small significant differences when compared to the control samples. In general, breads made with the addition of 8 g enzyme.100 kg-1 flour had the lowest firmness values, thus presenting the best technological characteristics.

Identification of over producer strain of endo-ß-1,4-glucanase in ...

African Journals Online (AJOL)

Cellulases are a group of hydrolytic enzymes capable of degrading cellulose to smaller sugar components like glucose units. These enzymes are produced by fungi and bacteria. The aim of this research was to identify a Aspergillus species with over production of endo-β-1,4-glucanase. Properties of endo-β-1 ...

Revisiting the stability of endo/exo Diels-Alder adducts between cyclopentadiene and 1,4-benzoquinone

International Nuclear Information System (INIS)

Tormena, Claudio F.; Lacerda Junior, Valdemar; Oliveira, Kleber T. de

2010-01-01

In this work it is presented a detailed theoretical analysis of the relative stability of endo/exo Diels-Alder adducts formed by the reaction between cyclopentadiene (1) and 1,4-benzoquinone (2). The intrinsic reaction coordinate (IRC) showed the existence of only one transition state for the reaction studied, for both endo 3 and exo 4 adducts. The energies of both adducts were obtained at high level of theory (CBS-Q) confirming that the endo adduct is more stable than exo, which is in the opposite way to the observed in reactions that usually follow Alder's rule. An electronic structure analysis was performed through NBO methodology, indicating that the attractive delocalization interaction predominates over the steric repulsive interaction in the endo adducts. In summary, for the studied cycloaddition reaction the endo adduct is the thermodynamic and kinetic product, which can be also confirmed by experimental data mentioned in this work. (author)

The Influence of Xylanase Supplementation on Dough Rheology Concerning its Consistograph Parameters

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Rodica Chereji

2010-05-01

Full Text Available In this study we determined the influence of xylanase supplementation on dough rheology concerning its consistograph parameters: maximum pressure (Pr max, (mb and water absorption (Wa, %. The consistograph analyses were conducted at constant hydration and consistency of 500UF. Determinations were made on 4 types of flour and optimal enzyme dosages were determined. Then we added the optimal enzyme dose for each type of flour as follows: F1, F2, F3, F4: P1-8100U.FXU/100kg flour, P2-16200U.FXU/100kg flour, P3-24300U.FXU/100kg flour. Fungal xylanase used in these concentrations led to the improvement of bread quality properties: finer texture of the crumb, extending freshness of bread, improving the colour and flavour, improving the slicing ability.

The influence of sorbitol on the production of cellulases and xylanases in an airlift bioreactor.

Science.gov (United States)

Ritter, Carla Eliana Todero; Fontana, Roselei Claudete; Camassola, Marli; da Silveira, Maurício Moura; Dillon, Aldo José Pinheiro

2013-11-01

The production of cellulases and xylanases by Penicillium echinulatum in an airlift bioreactor was evaluated. In batch production, we tested media with isolated or associated cellulose and sorbitol. In fed-batch production, we tested cellulose addition at two different times, 30 h and 48 h. Higher liquid circulation velocities in the downcomer were observed in sorbitol 10 g L(-1) medium. In batch production, higher FPA (filter paper activity) and endoglucanase activities were obtained with cellulose (7.5 g L(-1)) and sorbitol (2.5 g L(-1)), 1.0 U mL(-1) (120 h) and 6.4 U m L(-1) (100 h), respectively. For xylanases, the best production condition was cellulose 10 g L(-1), which achieved 5.5 U mL(-1) in 64 h. The fed-batch process was favorable for obtaining xylanases, but not for FPA and endoglucanases, suggesting that in the case of cellulases, the inducer must be added early in the process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of the EndoPAT as a Tool to Assess Endothelial Function

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M. Moerland

2012-01-01

Endothelial function was stable over a longer period of time in renally impaired patients (coefficient of variation 13%. Endothelial function in renally impaired and type 2 diabetic patients was not decreased compared to healthy volunteers (2.9±1.4 and 1.8±0.3, resp., versus 1.8±0.5, P>0.05. The EndoPAT did not detect an effect of robust interventions on endothelial function in healthy volunteers (glucose load: change from baseline 0.08±0.50, 95% confidence interval −0.44 to 0.60; smoking: change from baseline 0.49±0.92, 95% confidence interval −0.47 to 1.46. This suggests that at present the EndoPAT might not be suitable to assess (changes in endothelial function in early-phase clinical pharmacology studies. Endothelial function as measured by the EndoPAT could be physiologically different from endothelial function as measured by conventional techniques. This should be investigated carefully before the EndoPAT can be considered a useful tool in drug development or clinical practice.

Shushaku Endo (1923–1996): his tuberculosis and his writings

Science.gov (United States)

Sharma, O P

2006-01-01

Shushaku Endo, one of the finest 20th century Japanese novelists, is not as well known in the West as some of his contemporary Japanese writers including, Yukio Mishima, Yasunari Kawabata, Kobo Abe, and Kenzaburo Oe. Mishima deals with turmoil of adolescence laced with surrealistic historical romance; Abe conjures strange, evocative images in the Kafkaesque tradition; Kawabata writes about loneliness and desolation; and Oe creates imaginary themes of life and myth that entwine and portray human predicaments. Kawabata and Oe both received Nobel Prizes in Literature. Endo's writing is different. He confronts problems pertaining to faith, morality, and individual responsibility. In his early years Endo struggled as a loner, an outsider, and a misfit. This pattern continued to a large extent into his adult life, most of which was consumed with fighting his life threatening tuberculosis. The agonies of his illness intensified his talent for exploring the complexity of human relationships. PMID:16517794

76 FR 75887 - SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Incorporated's...

Science.gov (United States)

2011-12-05

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2010-P-0176] SEDASYS Computer-Assisted Personalized Sedation System; Ethicon Endo-Surgery, Incorporated's Petition for... system (SEDASYS) submitted by Ethicon Endo-Surgery Inc. (EES), the sponsor for SEDASYS. This meeting has...

Concurrent production of cellulase and xylanase from Trichoderma reesei NCIM 1186: enhancement of production by desirability-based multi-objective method.

Science.gov (United States)

Jampala, Preethi; Tadikamalla, Satish; Preethi, M; Ramanujam, Swathy; Uppuluri, Kiran Babu

2017-05-01

Application of multiple response optimizations using desirability function in the production of microbial metabolites improves economy and efficiency. Concurrent production of cellulase and xylanase in Trichoderma reesei NCIM 1186 using an agricultural weed, Prosopis juliflora pods, was studied. The main aim of the study was to optimize significant medium nutrient parameters for maximization of cellulase and xylanase by multi-objective optimization strategy using biomass. Process parameters such as the nutrient concentrations (pods, sucrose, and yeast extract) and pH were investigated to improve cellulase and xylanase activities by one factor at a time approach, single response optimization and multi-objective optimization. At the corresponding optimized process parameters in single response optimization, the maximum cellulase activity observed was 3055.65 U/L where xylanase highest activity was 422.16 U/L. Similarly, the maximum xylanase activity, 444.94 U/L, was observed with the highest cellulase activity of 2804.40 U/L. The multi-objective optimization finds a tradeoff between the two objectives and optimal activity values in between the single-objective optima were achieved, 3033.74 and 439.13 U/L for cellulase and xylanase, respectively.

Enhancement of Cellulase and Xylanase Production Using pH-Shift and Dissolved Oxygen Control Strategy with Streptomyces griseorubens JSD-1.

Science.gov (United States)

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-01-01

In this study, the production of cellulase and xylanase by Streptomyces griseorubens JSD-1 was improved by integrating the pH-shift and dissolved oxygen (DO)-constant control strategies. The pH-shift control strategy was carried out by analyzing the specific cell growth rate (μ) and specific enzyme formation rate (Q p) of S. griseorubens JSD-1. The pH was controlled at 8.0 during the first 48 h to maintain high cell growth, which then shifted to 7.5 after 48 h to improve the production of cellulase and xylanase. Using this method, the maximum activities of cellulase, xylanase, and filter paper enzyme (FPase) increased by 47.9, 29.5, and 113.6 %, respectively, compared to that obtained without pH control. On the basis of pH-shift control, the influence of DO concentrations on biomass and enzyme production was further investigated. The maximum production of cellulase, xylanase, and FPase reached 114.38 ± 0.96 U mL(-1), 330.57 ± 2.54 U mL(-1), and 40.11 ± 0.38 U mL(-1), which were about 1.6-fold, 0.6-fold, and 3.2-fold higher than that of neutral pH without DO control conditions. These results supplied a functional approach for improving cellulase and xylanase production.

Production of ethanol from wet oxidised wheat straw by Thermoanaerobacter mathranii

DEFF Research Database (Denmark)

Ahring, B.K.; Licht, D.; Schmidt, A.S.

1999-01-01

evaluated with respect to total sugars, xylose, and 2-furfural produced. The concentration of sugars tended to be highest in hydrolysates produced at high oxygen pressures, whereas the concentration of 2-furfural was lowest in hydrolysates produced at low oxygen pressures and high carbonate concentrations...... with the commercial enzyme Celluclast(R) or with acid hydrolysis improved the ethanol yield from the hydrolysates. Treatment with Pentopan(TH) Mono BG or Pulpzyme(R) HC, both endo-1,4-beta-xylanases, had no effect neither had co-cultivation with the xylanase-producing Dictyoglomus B4. (C) 1998 Published by Elsevier...

Revisiting the stability of endo/exo Diels-Alder adducts between cyclopentadiene and 1,4-benzoquinone

Energy Technology Data Exchange (ETDEWEB)

Tormena, Claudio F. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil). Inst. de Quimica. Dept. de Quimica Organica; Lacerda Junior, Valdemar [Universidade Federal do Espirito Santo (UFES), Vitoria, ES (Brazil). Centro de Ciencias Exatas. Dept. de Quimica; Oliveira, Kleber T. de [Universidade Federal do ABC (UFABC), Santo Andre, SP (Brazil). Centro de Ciencias Naturais e Humanas

2010-07-01

In this work it is presented a detailed theoretical analysis of the relative stability of endo/exo Diels-Alder adducts formed by the reaction between cyclopentadiene (1) and 1,4-benzoquinone (2). The intrinsic reaction coordinate (IRC) showed the existence of only one transition state for the reaction studied, for both endo 3 and exo 4 adducts. The energies of both adducts were obtained at high level of theory (CBS-Q) confirming that the endo adduct is more stable than exo, which is in the opposite way to the observed in reactions that usually follow Alder's rule. An electronic structure analysis was performed through NBO methodology, indicating that the attractive delocalization interaction predominates over the steric repulsive interaction in the endo adducts. In summary, for the studied cycloaddition reaction the endo adduct is the thermodynamic and kinetic product, which can be also confirmed by experimental data mentioned in this work. (author)

Kinetics and substrate selectivity of a Triticum aestivum xylanase inhibitor (TAXI) resistant D11F/R122D variant of Bacillus subtilis XynA xylanase

DEFF Research Database (Denmark)

Rasmussen, Louise Enggaard; Sørensen, Jens F.; Meyer, Anne S.

2010-01-01

This study examined the kinetics and substrate selectivity of a GH11 Bacillus subtilis XynA xylanase (BsX) sensitive to inhibition by TAXI and an engineered variant, which is much less inhibited by TAXI (BsX(mut)). The main purpose of the work was to elucidate any influence of the structural point...

Xylanase production by Trichoderma longibrachiatum

Energy Technology Data Exchange (ETDEWEB)

Royer, J C; Nakas, J P [State Univ. of New York, Syracuse, NY (USA). Coll. of Environmental Science and Forestry

1989-07-01

Xylan comprises up to 25% of hardwood biomass and is found in a variety of agricultural residues. It therefore represents a significant renewable resource which should be utilized to improve the economics of bioconversion of plant biomass to useful products. Before fermentation to fuels or solvents, xylan must be hydrolysed to xylose or short-chain oligomers of xylose. The effects of carbon source, substrate concentration, culture pH, and spore inoculum concentration on production of extracellular xylanase and cellulase were examined. Very low enzyme activities were obtained with growth on glucose, xylose, and cellobiose, while significantly higher levels were produced from lactose and arabinose. Higher levels of both enzymes were generated from alpha cellulose, wood pulp, and fibrous paper waste than from purified xylan. (author).

Optimization of Cellulase and Xylanase Production by Micrococcus Species under Submerged Fermentation

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Ziyanda Mmango-Kaseke

2016-11-01

Full Text Available This paper reports on the optimization of culture conditions for cellulase and xylanase production by bacterial isolate from lignocellulosic biomass. The bacterial isolate was screened for cellulase and xylanase production on carboxyl methyl cellulose (CMC and birch wood xylan as substrates, respectively. One bacterial isolate showing the highest halo zone diameter (isolate PLY1 was selected for detailed studies. The analysis of the 16S ribosomal ribonucleic acid (rRNA gene nucleotide sequence of PLY1 revealed it to have 98% similarity to Micrococcus luteus strain Fse9 and the sequence was deposited in the GenBank as Micrococcus luteus strain SAMRC-UFH3 with accession number KU171371. Cellulase production was achieved in the presence of CMC (1% w/v under an incubation temperature of 25 °C (198 U/mL, pH 5 (173 U/mL, agitation speed 50 rpm (173 U/mL and incubation period of 96 h (102 U/mL. Xylanase was produced maximally when birch wood xylan (1% w/v was used as the substrate at 25 °C (1007 U/mL, pH 10 (2487 U/mL, 200 rpm (1814 U/mL, and under an incubation period of 84 h (1296 U/mL. Our findings showed that Micrococcus sp. SAMRC-UFH3 appears to be a potentially important candidate for lignocellulosic waste degradation and other relevant industrial applications.

CLONING, PURIFICATION AND CHARACTERIZATION OF HALOTOLERANT XYLANASE FROM Geobacillus Thermodenitrificans C5

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Muhammad Irfan

2016-06-01

Full Text Available High levels of extracellular xylanase activity (994.50 IU/ml produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C and low pH (4.0 to 8.0. Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

Cyclic Fatigue Resistance of 3 Proprietary Rotary File Brands and their Analogous EdgeEndo Counterparts.

Science.gov (United States)

2018-04-26

Resistance of 3 Proprietary Rotary File Brands and their Analogous EdgeEndo Counterparts. 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Sd...Endodontics 14. ABSTRACT Cyclic Fatigue Resistance of 3 Proprietary Rotary File Brands and their Analogous EdgeEndo Counterparts. David J. Weyh DDS...Resistance of 3 Proprietary Rotary File Brands and their Analogous EdgeEndo Counterparts. David J. Weyh DDS Jarom J. Ray DDS Introduction: The aim of this

Enhanced sugar production from pretreated barley straw by additive xylanase and surfactants in enzymatic hydrolysis for acetone-butanol-ethanol fermentation.

Science.gov (United States)

Yang, Ming; Zhang, Junhua; Kuittinen, Suvi; Vepsäläinen, Jouko; Soininen, Pasi; Keinänen, Markku; Pappinen, Ari

2015-01-01

This study aims to improve enzymatic sugar production from dilute sulfuric acid-pretreated barley straw for acetone-butanol-ethanol (ABE) fermentation. The effects of additive xylanase and surfactants (polyethylene glycol [PEG] and Tween) in an enzymatic reaction system on straw hydrolysis yields were investigated. By combined application of 2g/100g dry-matter (DM) xylanase and PEG 4000, the glucose yield was increased from 53.2% to 86.9% and the xylose yield was increased from 36.2% to 70.2%, which were considerably higher than results obtained with xylanase or surfactant alone. The ABE fermentation of enzymatic hydrolysate produced 10.8 g/L ABE, in which 7.9 g/L was butanol. The enhanced sugar production increased the ABE yield from 93.8 to 135.0 g/kg pretreated straw. The combined application of xylanase and surfactants has a large potential to improve sugar production from barley straw pretreated with a mild acid and that the hydrolysate showed good fermentability in ABE production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preservation of Bacillus pumilus PU4-2 xylanases by immobilization technique into pollard and cation addition

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T Haryati

2010-03-01

Full Text Available Utilization of by-product from agriculture as alternative source of feedstuff has been widely practiced. However their usage is limited due to high fiber content and low nutrient digestibility. The use of specific hydrolizing enzymes, xylanases are gaining importance because of their wide application in various industrial sectors especially in bioconversion of hemicellulosic material. This experiment was done to evaluate the effect of cation addition and immobilization of enzyme into pollard on stability of B. pumilus xylanase. The enzyme extract was purified by precipitation with 75% ammonium sulphate. Four kinds of cation (Ca2+, Fe3+, Mg2+, Zn2+ were added to the purified enzyme, at concentration of 1m M and stored at 4 and 27˚C. For immobilization process, the optimum enzyme concentration that will be added to pollard has been evaluated by analysis of xylanase activity and their recovery. The specific activity of enzyme after precipitation increased 1.8 times, from 420.3 to 765.2 U/mg protein. All cations act as activator which relative activity become 130.6; 139.0; 103.8 and 163.5% respectively. Concentration of 0.5mM Ca2+ and Fe3+ were most able to keep xylanases activity stable at 4˚C. The optimum composition of enzymes and pollard was 1.5 ml for 5 gram of pollard with recovery of xylanases activity of 82.2%. In immobilized enzyme, the activity of enzyme without cation addition is higher than that with addition of Ca2+ and Fe3+. Activity of enzyme stored at 4˚C is more stable than that at 27˚C. Immobilized enzyme is more stable for storage, which lasted for 7 weeks at 27˚C and 12 weeks at 4˚C compared to liquid enzyme which lasted for only 7 days at 27˚C and 13 days at 4˚C.

The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity, not with its catalytic activity

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González Celedonio

2010-02-01

Full Text Available Abstract Background The Botrytis cinerea xylanase Xyn11A has been previously shown to be required for full virulence of this organism despite its poor contribution to the secreted xylanase activity and the low xylan content of B. cinerea hosts. Intriguingly, xylanases from other fungi have been shown to have the property, independent of the xylan degrading activity, to induce necrosis when applied to plant tissues, so we decided to test the hypothesis that secreted Xyn11A contributes to virulence by promoting the necrosis of the plant tissue surrounding the infection, therefore facilitating the growth of this necrotroph. Results We show here that Xyn11A has necrotizing activity on plants and that this capacity is conserved in site-directed mutants of the protein lacking the catalytic activity. Besides, Xyn11A contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity, as the catalytically-impaired Xyn11A variants were able to complement the lower virulence of the xyn11A mutant. The necrotizing activity was mapped to a 30-amino acids peptide in the protein surface, and this region was also shown to mediate binding to tobacco spheroplasts by itself. Conclusions The main contribution of the xylanase Xyn11A to the infection process of B. cinerea is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates binding to plant cells.

Xylanase production from Thermomyces lanuginosus VAPS-24 using low cost agro-industrial residues via hybrid optimization tools and its potential use for saccharification.

Science.gov (United States)

Kumar, Vishal; Chhabra, Deepak; Shukla, Pratyoosh

2017-11-01

The xylanase production from Thermomyces lanuginosus VAPS-24 has been optimized using OFAT (One factor at a time) approach using agro-industrial substrates. Further, central composite design (CCD) has been employed to optimize various process parameters such as temperature (45-55°C), carbon source concentration (1.5-2.5%), fermentation time (72-120h) and production medium pH (6-8). Maximum xylanase yield after RSM optimization was approximately double (119.91±2.53UmL -1 ) than un-optimized conditions (61.09±0.91UmL -1 ). Several hybrid statistical tools such as Genetic Algorithm-Response Surface Methodology (GA-RSM), Artificial Neural Network (ANN), Genetic Algorithm-Artificial Neural Network (GA-ANN) were employed to obtain more optimized process parameters to maximize the xylanase production and observed an increase of 10.50% xylanase production (132.51±3.27UmL -1 ) as compared to RSM response (119.91±2.53UmL -1 ). The various pretreated and untreated agricultural residues were subjected to saccharification by using crude xylanase in which the pretreated rice straw yielded maximum fermentable sugars 126.89mgg -1 . Copyright © 2017 Elsevier Ltd. All rights reserved.

Production of high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Energy Technology Data Exchange (ETDEWEB)

Gomes, J [Institute of Biotechnology, Technical Univ. of Graz (Austria); Purkarthofer, H [Institute of Biotechnology, Technical Univ. of Graz (Austria); Hayn, M [Institute of Biochemistry, Univ. of Graz (Austria); Kapplmueller, J [Voest-Alpine Industrieanlagen GmbH, Zellstofftechnik und Biomasseverwertung, Linz (Austria); Sinner, M [Voest-Alpine Industrieanlagen GmbH, Zellstofftechnik und Biomasseverwertung, Linz (Austria); Steiner, W [Institute of Biotechnology, Technical Univ. of Graz (Austria)

1993-08-01

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m[sup 3] fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4-30 C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70-75 C. The enzyme was almost thermostable (91-92%) at pH 6.5 and 9.0 after 41 h preincubation at 55 C and lost only 20-33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0 Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55 C and pH 6.5. (orig.)

Surgical intervention of complex endo-perio lesions.

Science.gov (United States)

Adcock, John E; Bright, David

2007-08-01

Complex endo-perio lesions are infrequent, but pose treatment dilemmas. The lesions are complex with bone loss involving adjacent teeth that are not part of the initial endodontic lesion. The aggressive bone loss is not clearly understood and apparently has some differences from the usual apical periodontitis.

A case series associated with different kinds of endo-perio lesions.

Science.gov (United States)

Aksel, Hacer; Serper, Ahmet

2014-02-01

Pulpal and periodontal problems are responsible for more than half of the tooth mortality. There are some articles published in the literature about this issue. Many of them are quite old. There has been also lack of knowledge about the effect of endodontic treatment on the periodontal tissue healing and suitable treatment interval between endodontic and periodontal treatments. In this case report, different kinds of endo-perio lesion were treated with sequential endodontic and periodontal treatment. The follow-up radiographs showed complete healing of the hard and soft tissue lesions. The tooth with endo-perio lesions should be evaluated thoroughly in terms of any cracks and fracture, especially furcation areas for a long term prognosis. In this case report, it was showed that 3 months treatment intervals between endodontic treatment and periodontal surgery has no harmful effect on periodontal tissue healing. Key words:Endo-perio lesion, furcation, mandibular molar, bone graft, crack line, treatment interval.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

International Nuclear Information System (INIS)

Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

2007-01-01

The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4 3 2 1 2, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2 1 , with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution

Endo-perio dilemma: a brief review.

Science.gov (United States)

Singh, Preetinder

2011-01-01

The actual relationship between periodontal and pulpal disease was first described by Simring and Goldberg in 1964. Since then, the term "perio-endo" lesion has been used to describe lesions due to inflammatory products found in varying degrees in both the periodontium and the pulpal tissues. The pulp and periodontium have embryonic, anatomic and functional inter-relationships. The simultaneous existence of pulpal problems and inflammatory periodontal disease can complicate diagnosis and treatment planning. A perio-endo lesion can have a varied pathogenesis which ranges from quite simple to relatively complex one. Knowledge of these disease processes is essential in coming to the correct diagnosis. This is achievable by careful history taking, examination and the use of special tests. The prognosis and treatment of each endodontic-periodontal disease type varies. Primary periodontal disease with secondary endodontic involvement and true combined endodontic-periodontal diseases require both endodontic and periodontal therapies. The prognosis of these cases depends on the severity of periodontal disease and the response to periodontal treatment. This enables the operator to construct a suitable treatment plan where unnecessary, prolonged or even detrimental treatment is avoided.

Endo-periodontal lesion – endodontic approach

Science.gov (United States)

Jivoinovici, R; Suciu, I; Dimitriu, B; Perlea, P; Bartok, R; Malita, M; Ionescu, C

2014-01-01

Endo-perio lesions might be interdependent because of the vascular and anatomic connections between the pulp and the periodontium. The aim of this study is to emphasise that primary endodontic lesion heals after a proper instrumentation, disinfection and sealing of the endodontic space. The primary endodontic lesion with a secondary periodontal involvement first requires an endodontic therapy and, in the second stage, a periodontal therapy. The prognosis is good, with an adequate root canal treatment; it depends on the severity of the periodontal disease, appropriate healing time and the response to the treatment. A correct diagnosis is sometimes difficult; an accurate identification of the etiologic factors is important for an adequate treatment. Primary perio-endo lesion may heal after a proper disinfection and sealing of the endodontic system, the one-year follow-up radiograph showing bonny repair. Invasive periodontal procedures should be avoided at that moment. The microorganisms and by-products from the infected root canal may cross accessory and furcal canals and determine sinus tract and loss of attachment. In both clinical cases presented in this article, successful healing was obtained after a proper disinfection and sealing of the endodontic system. PMID:25713618

Endo-perio dilemma: A brief review

Directory of Open Access Journals (Sweden)

Preetinder Singh

2011-01-01

Full Text Available The actual relationship between periodontal and pulpal disease was first described by Simring and Goldberg in 1964. Since then, the term "perio-endo" lesion has been used to describe lesions due to inflammatory products found in varying degrees in both the periodontium and the pulpal tissues. The pulp and periodontium have embryonic, anatomic and functional inter-relationships. The simultaneous existence of pulpal problems and inflammatory periodontal disease can complicate diagnosis and treatment planning. A perio-endo lesion can have a varied pathogenesis which ranges from quite simple to relatively complex one. Knowledge of these disease processes is essential in coming to the correct diagnosis. This is achievable by careful history taking, examination and the use of special tests. The prognosis and treatment of each endodontic-periodontal disease type varies. Primary periodontal disease with secondary endodontic involvement and true combined endodontic-periodontal diseases require both endodontic and periodontal therapies. The prognosis of these cases depends on the severity of periodontal disease and the response to periodontal treatment. This enables the operator to construct a suitable treatment plan where unnecessary, prolonged or even detrimental treatment is avoided.

Heterologous expression of a rice metallothionein isoform (OsMTI-1b in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance

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Zahra Ansarypour

Full Text Available Abstract Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

Science.gov (United States)

Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

2010-11-01

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

Influence of a direct-fed microbial and xylanase enzyme on the dietary energy uptake efficiency and performance of broiler chickens.

Science.gov (United States)

Murugesan, Ganapathi Raj; Persia, Michael E

2015-09-01

Efficacy of a multi-strain direct-fed microbial product (PoultryStar(®) ME; PS) and a xylanase enzyme product on the dietary energy utilization efficiency and resulting performance in broiler chickens was evaluated. Apart from performance parameters, cecal and serum metabolites and activities of hepatic enzymes involved in energy metabolism were also determined. Ross 308 chicks were fed one of four experimental diets [control (CON), CON + PS, CON + xylanase and CON + PS + xylanase] using a 2 × 2 factorial arrangement from 1-21 days of age. Cecal proportions of propionate and butyrate, as well as total short-chain fatty acid concentration were increased (P energy uptake and hepatic energy retention. The combination additively increased the FCR, suggesting involvement of synergistic modes of actions. © 2014 Society of Chemical Industry.

A Computational Experiment of the Endo versus Exo Preference in a Diels-Alder Reaction

Science.gov (United States)

Rowley, Christopher N.; Woo, Tom K.

2009-01-01

We have developed and tested a computational laboratory that investigates an endo versus exo Diels-Alder cycloaddition. This laboratory employed density functional theory (DFT) calculations to study the cycloaddition of N-phenylmaleimide to furan. The endo and exo stereoisomers of the product were distinguished by building the two isomers in a…

A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

Science.gov (United States)

Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin

2016-05-15

A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect?

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Saddler Jack N

2011-10-01

Full Text Available Abstract Background We and other workers have shown that accessory enzymes, such as β-glucosidase, xylanase, and cellulase cofactors, such as GH61, can considerably enhance the hydrolysis effectiveness of cellulase cocktails when added to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors that hamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, a major problem lies in our incomplete understanding of the cooperative action of the different enzymes acting on pretreated lignocellulosic substrates. Results The reported work assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low protein loadings. When xylanases were added to the minimum amount of cellulase enzymes required to achieve 70% cellulose hydrolysis of steam pretreated corn stover (SPCS, or used to partially replace the equivalent cellulase dose, both approaches resulted in enhanced enzymatic hydrolysis. However, the xylanase supplementation approach increased the total protein loading required to achieve significant improvements in hydrolysis (an additive effect, whereas the partial replacement of cellulases with xylanase resulted in similar improvements in hydrolysis without increasing enzyme loading (a synergistic effect. The enhancement resulting from xylanase-aided synergism was higher when enzymes were added simultaneously at the beginning of hydrolysis. This co-hydrolysis of the xylan also influenced the gross fiber characteristics (for example, fiber swelling resulting in increased accessibility of the cellulose to the cellulase enzymes. These apparent increases in accessibility enhanced the steam pretreated corn stover digestibility, resulting in three times faster cellulose and xylan hydrolysis, a seven-fold decrease in cellulase loading and

Wheat dough rheology at low water contents and the influence of xylanases

NARCIS (Netherlands)

Hardt, N.A.; Boom, R.M.; Goot, van der A.J.

2014-01-01

The effect of low water contents and xylanases on wheat dough rheology is reported. Farinograph, dynamic oscillation, and creep-recovery measurements were performed using water concentrations from 34 to 44.8% (total basis). A water reduction from 43.5–44.8% to 34% increased resistance upon mixing as

Aktivitas endo-β-mannanase pada perkecambahan biji Parkia roxburghii dengan pemberian variasi konsentrasi giberelin

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AJENG EDITA SUBANDI

2015-05-01

Full Text Available Subandi AE, Sari SLA, Anggarwulan E, Solichatun. 2015. Enzyme activities of endo-β–mannanase on seed germination of Parkia roxburghii with grant of concentration variation giberellin. Bioteknologi 12: 8-15. Kedawung (Parkia roxburghii G. Don is member of leguminous plants used as medicinal plants. Parkia roxburghii seeds have rigid seed coats and this can inhibit germination process. Seed germination barriers can be broken by adding external hormones such as gibberellin and auxin. Gibberellin hormone has been known to has capabilities to accelerate seed germination. The purpose of this study was to (i determine the effect of various concentrations of the gibberellin hormone on seed germination of P. roxburghii and (ii investigate the activity of endo-β-mannanase enzyme in breaking the endosperm on seed germination P. roxburghii. The experiment was conducted by randomized design (CRD with 4 different gibberellin concentrations namely 0, 100, 300 and 500 ppm respectively. Seeds were incubated in waterbath at 55oC for 1 hour as a pretreatment. Seeds then soaked in a solution of gibberellin hormone for 24 hours. After rinsed with distilled water, seeds subsequently germinated in petri dishes for 30 days. Germination was performed at room temperature (28oC. Germination parameters observed were seed imbibition, germination rate, germination percentage, and activity of endo-β-mannanase enzyme. Measurement of endo-β-mannanase activity was using spectrophotometer. The results showed that the highest germination percentage of P.roxburghii seed at 300 ppm was 47%. During germination process, the activity endo-β-mannanase enzyme has been changing, in which the closer to the germination time, enzyme activity increased. The highest peak of activity endo-β-mannanase enzyme at 300 ppm was 20.34 units/2.4 ml on the third day of germination.

Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

Science.gov (United States)

Franz J. St John; James F. Preston; Edwin Pozharski

2012-01-01

The Gram-positive bacterium Paenibacillus sp. JDR-2 (PbJDR2) has been shown to have novel properties in the utilization of the abundant but chemically complex hemicellulosic sugar glucuronoxylan. Xylanase A1 of PbJDR2 (PbXynA1) has been implicated in an efficient process in which extracellular...

Effect of degradation of xylan constituent in Mitsumata (Edgeworthia papyrifera Sieb. et Zucc. ) bast on its pulping by pectinolytic enzymes form Erwinia carotovora

Energy Technology Data Exchange (ETDEWEB)

Tanabe, Hiroyuki; Matsuo, Ryukichi; Kobayashi, Yoshinari

1988-01-01

Pulping of mitsumata (Edgeworthia papyrifera Sieb. et Zucc.) bast by the crude enzyme from a bacterium Erwinia carotovora FERM P-7576, was more effective by a stepwise treatment at pH 6.5 and subsequently at pH 9.5 and eluted greater amount of xylose constituent than a constant pH treatment at pH 9.5 where only the maceration enzymes, endo-pectate lyase and endo-pectin lyase, among the crude enzyme are operative. The crude enzymes obtained from the cultivation of this bacterial strain on mitsumata bast fibers were more effective for the stepwise pH pulping method than those from the cultivation on soluble pectin. Xylanase activity in the mitsumata bast-induced enzyme at pH 6.5 was twice as high as that in the soluble pectin-induced one. The activities of other hemicellulases and cellulase were, high as that in the soluble pectin-induced one. The activities of other hemicellulases and cellulase were, however, independent on the inducing materials. Purified exo-type xylanase prepared from the crude enzyme acted comparably to the entire crude enzyme in the first step of the combination pulping, but the xylanase per se showed no maceration activity. These results suggests that the degradation of xylan constituent within the bast fibers effects the acceleration of the subsequent enzymatic pulping by the pectinolytic maceration enzymes. The maceration mechanism involving xylan degradation was also discussed.

Complete NMR assignment of a bisecting hybrid-type oligosaccharide transferred by Mucor hiemalis endo-β-N-acetylglucosaminidase.

Science.gov (United States)

Yamanoi, Takashi; Oda, Yoshiki; Katsuraya, Kaname; Inazu, Toshiyuki; Yamamoto, Kenji

2016-06-02

This study describes the complete nuclear magnetic resonance (NMR) spectral assignment of a bisecting hybrid-type oligosaccharide 1, transferred by Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M). Through (1)H- and (13)C-NMR, DQF-COSY, HSQC, HMBC, TOCSY, and NOESY experiments, we determine the structure of the glycoside linkage formed by the Endo-M transglycosylation, i.e., the connection between GlcNAc and GlcNAc in oligosaccharide 1. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation.

Science.gov (United States)

Dwivedi, Pallavi; Vivekanand, V; Pareek, Nidhi; Sharma, Amit; Singh, Rajesh P

2011-10-01

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching. Copyright © 2011 Elsevier B.V. All rights reserved.

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

Science.gov (United States)

Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

2018-05-01

Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

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Bey Mathieu

2011-12-01

Full Text Available Abstract Background Cellobiose dehydrogenase (CDH is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i the production of a large amount of gluconic acid, (ii increased hemicellulose degradation, and (iii increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM. Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

DEFF Research Database (Denmark)

Jørgensen, Henning; Eriksson, T.; Borjesson, J.

2003-01-01

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l(-1) cellulose and 10 g l(-1) xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis...... studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity...... the cellulose-binding domain or an essential part of it. The basic xylanase (pI > 9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12...

Cellulase and xylanase productions by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation

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Heck Júlio X.

2002-01-01

Full Text Available In Brazil, a large amount of a fibrous residue is generated as result of soybean (Glycine max protein production. This material, which is rich in hemicellulose and cellulose, can be used in solid state cultivations for the production of valuable metabolites and enzymes. In this work, we studied the bioconversion of this residue by bacteria strains isolated from water and soil collected in the Amazon region. Five strains among 87 isolated bacteria selected for their ability to produce either celullases or xylanases were cultivated on the aforementioned residue. From strain BL62, identified as Bacillus subtilis, it was obtained a preparation showing the highest specific cellulase activity, 1.08 UI/mg protein within 24 hours of growth. Concerning xylanase, the isolate BL53, also identified as Bacillus subtilis, showed the highest specific activity for this enzyme, 5.19 UI/mg protein within 72 hours of cultivation. It has also been observed the production of proteases that were associated with the loss of cellulase and xylanase activities. These results indicated that the selected microorganisms, and the cultivation process, have great biotechnological potential.

Xylanase and cellulase activities during anaerobic decomposition of three aquatic macrophytes.

Science.gov (United States)

Nunes, Maíra F; da Cunha-Santino, Marcela B; Bianchini, Irineu

2011-01-01

Enzymatic activity during decomposition is extremely important to hydrolyze molecules that are assimilated by microorganisms. During aquatic macrophytes decomposition, enzymes act mainly in the breakdown of lignocellulolytic matrix fibers (i.e. cellulose, hemicellulose and lignin) that encompass the refractory fraction from organic matter. Considering the importance of enzymatic activities role in decomposition processes, this study aimed to describe the temporal changes of xylanase and cellulose activities during anaerobic decomposition of Ricciocarpus natans (freely-floating), Oxycaryum cubense (emergent) and Cabomba furcata (submersed). The aquatic macrophytes were collected in Óleo Lagoon, Luiz Antonio, São Paulo, Brazil and bioassays were accomplished.  Decomposition chambers from each species (n = 10) were set up with dried macrophyte fragments and filtered Óleo Lagoon water. The chambers were incubated at 22.5°C, in the dark and under anaerobic conditions. Enzymatic activities and remaining organic matter were measured periodically during 90 days. The temporal variation of enzymes showed that C. furcata presented the highest decay and the highest maximum enzyme production. Xylanase production was higher than cellulase production for the decomposition of the three aquatic macrophytes species.

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune.

Science.gov (United States)

Metreveli, Eka; Kachlishvili, Eva; Singer, Steven W; Elisashvili, Vladimir

2017-10-01

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus. Copyright © 2017 Elsevier Ltd. All rights reserved.

A sequential approach in treatment of endo-perio lesion a case report.

Science.gov (United States)

Kambale, Sharanappa; Aspalli, Nagaveni; Munavalli, Anil; Ajgaonkar, Nishant; Babannavar, Roopa

2014-08-01

Endo-perio lesions primarily occur by way of the intimate anatomic and vascular connections between the pulp and the periodontium. Endodontic-periodontal combined lesion is a clinical dilemma because making a differential diagnosis and deciding a prognosis are difficult. An untreated primary endodontic lesion may become secondarily involved with periodontal breakdown, which clinically present unusual signs and symptoms. This may delay the diagnosis and hence the correct treatment. This case report describes diagnosis and treatment protocol for an endo-perio lesion of primary endodontic with secondary periodontal involvement.

Endo-Perio Lesion and Uncontrolled Diabetes

OpenAIRE

Dhoum, Sara; Laslami, Kaoutar; Rouggani, Fatimazahraa; El Ouazzani, Amal; Jabri, Mouna

2018-01-01

This work is to discuss the management of an endo-perio lesion, which represents a challenge to clinicians when it comes to diagnosis and prognosis of the involved teeth and especially with an altered general condition. A 50-year-old female patient with uncontrolled diabetes type 2 is suffering from a purulent discharge coming from the upper right canine. Endodontic and periodontal treatments were realized with 36 months radiological and clinical follow-up with the collaboration of her intern...

The endo-lysosomal system of brain endothelial cells is influenced by astrocytes in vitro

DEFF Research Database (Denmark)

Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J

2018-01-01

Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes......, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture....... Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain....

Application of xylanases from Amazon Forest fungal species in bleaching of eucalyptus kraft pulps

Directory of Open Access Journals (Sweden)

Roseli Garcia Medeiros

2007-03-01

Full Text Available Crude xylanase preparations from Penicillium corylophilum, Aspergillus niger and Trichoderma longibrachiatum were used to treat Eucalyptus kraft pulp, prior to chlorine dioxide and alkaline bleaching sequences. The enzyme pretreatment improved brightness and delignification of non-delignified and oxygen-bleached samples of eucalyptus kraft pulp. Xylanase preparations from T. longibrachiatum and P. corylophilum were more effective to reduce pulp kappa number. A small reduction in viscosity was obtained when the oxygen-bleached pulp was treated with xylanase preparation from A. niger. For all enzyme samples, the best release of chromophoric material from the pulp was at 237 nm. The enzyme preparation from P. corylophilum was responsible for the highest release of reducing sugar at a dosage interval of 10-20 IU/g dry weight pulp. Scanning electron microscopy studies of oxygen-bleached pulp after xylanase treatment revealed morphological changes, including holes, cracks, filament forming and peeling.Amostras de xilanases de extratos brutos de Penicillium corylophilum, Aspergillus niger e Trichoderma longibrachiatum foram utilizadas no branqueamento de polpa kraft de eucalipto antes das seqüências alcalina e dióxido de cloro. O pré-tratamento enzimático melhorou a alvura e o processo de deslignificação de amostras de polpa kraft de eucalipto não-tratada e tratada com oxigênio. Amostras de xilanases de T. longibrachiatum e P. corylophilum foram mais efetivas na redução do número kappa da polpa. A polpa tratada com oxigênio sofreu uma pequena redução na sua viscosidade quando incubada com amostra de xilanase de A. niger. Para todas as amostras de xilanases, a maior liberação de cromóforos da polpa foi a 237 nm. A amostra de xilanase de P. corylophilum liberou maior quantidade de açúcar redutor da polpa, utilizando dosagem de 10-20 UI/g de peso seco da polpa. Estudos de microscopia eletrônica de varredura revelaram várias altera

RECOVERY OF ASPERGILLUS ENDO-GLUCANASE PRODUCED ON SOLID SUBSTRATE: A DOE BASED APPROACH

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Sibabrata Mukherjee

2014-10-01

Full Text Available The endo-glucanase (E.C. 3.2.1.4 was produced by Aspergillus terreus adopting solid state fermentation (SSF using agro residues as main substrate. To recover the enzyme from the fermented mass, different extraction liquids were tried and 10% aqueous solution of glycerol was found to be superior. When the selected extractant was applied at different ratio to the fermented solid mass, maximum enzyme was recovered at 1:5 (w/v ratio. The other process parameters (time, temperature and mixing speed effects on the enzyme recovery were subsequently studied by response surface methodology (RSM. Box-Bhenken Design of experiment (BBDOE was exploited for the analysis of interactive effects of the independent variables. The optimization was done following the numerical approach focusing reduction in utility cost without compromising the endo-glucanse activity. Based on the predicted solution the validation experiments were carried out and finally 32 IU/g of endo-glucanase was recovered at room temperature, at a mixing speed of 100 rpm in 2.65 h which was very close to the predicted response. The optimization evidenced more than two times betterment in enzyme recovery than the un-optimized state. The model developed was found to be robust for process analysis. Repetitive extraction had revealed that maximum endo-glucanase recovery was required of two cycles of extraction at optimized conditions.

Comparison between syringe irrigation and RinsEndo in reduction of Enterococcus faecalis in experimentally infected root canal

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Sharareh Mousavi Zahed

2015-05-01

Full Text Available Background and Aims: To ensure root canal treatment success, endodontic microbiota should be efficiently reduced. Several irrigation devices have been recently introduced with the main objective of improving root canal disinfection.The purpose of this study was to evaluate the rinsing effect of RinsEndo system in reduction of enterococcus faecalis in comparison with conventional hand syringe in infected root canals.   Materials and Methods: 60 extracted single canal anterior teeth were infected with enterococcus faecalis and divided into 3 groups: RinsEndo system, conventional hand syringe and control group. The enterococcus faecalis colonies were counted in each group before and after rinsing. Data were analyzed using Variance and Kruskal Wallis test.   Results: The mean of enterococcus faecalis growth after rinsing was 3.50×103 in group with conventional syring rinsing, 2.04×103 in group with RinsEndo washing and 6.11×103 in control group. Reduction of enterococcus faecalis after rinsing was statistically significant in each group (P<0.001. The amount of reduction in number of colonies with RinsEndo and conventional syringe rinsing was higher in comparison with control group and this difference was significant (P<0.001. RinsEndo rinsing effect was statistically significantly higher in comparison to conventional syringe as well (P<0.001.   Conclusion: Rinsing with RinsEndo system was significantly more efficient in reduction of enterococcus faecalis from root canal in comparison with hand syringe washing.

Efficacy of XP-endo Finisher File in Removing Calcium Hydroxide from Simulated Internal Resorption Cavity.

Science.gov (United States)

Keskin, Cangül; Sariyilmaz, Evren; Sariyilmaz, Öznur

2017-01-01

The aim of this study was to evaluate the effect of supplementary use of XP-endo Finisher file, passive ultrasonic activation (PUI), EndoActivator (EA), and CanalBrush (CB) on the removal of calcium hydroxide (CH) paste from simulated internal resorption cavities. The root canals of 110 extracted single-rooted teeth with straight canals were prepared up to size 50. The specimens were split longitudinally, and standardized internal resorption cavities were prepared with burs. The cavities and root canals were filled with CH paste. The specimens were divided into 5 groups as follows: XP-endo Finisher, EA, PUI, CB, and syringe irrigation (SI). The root canals were irrigated with 5.25% NaOCl and 17% EDTA for 2 minutes, respectively. Apart from the SI group, both solutions were activated by using tested techniques for 1 minute. The quantity of CH remnants on resorption cavities was scored. Data were analyzed by using Kruskal-Wallis H and Mann-Whitney U tests. XP-endo Finisher and PUI removed significantly more CH than SI, EA, and CB (P  .05). Differences among SI, EA, and CB were also non-significant (P > .05). None of the tested techniques render the simulated internal resorption cavities free of CH debris. XP-endo Finisher and PUI were superior to SI, CB, and EA. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

Science.gov (United States)

Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

Science.gov (United States)

Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

2007-10-15

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

Xylanase and Protease Increase Solubilization of Non-Starch Polysaccharides and Nutrient Release of Corn- and Wheat Distillers Dried Grains with Solubles

DEFF Research Database (Denmark)

Pedersen, Mads Brøgger; Dalsgaard, Søren; Arent, Susan

2015-01-01

The use of distiller dried grains with solubles (DDGS) as alternative to conventional animal feed for non-ruminants is challenged by the high content of non-starch polysaccharides and varying protein quality. In this study the enzymatic degradation of corn- and wheat DDGS was evaluated, in vitro...... of this xylanase. The current in vitro results indicate a high potential of xylanase in combination with protease to efficiently degrade DDGS and promote nutrient release in diets for non-ruminant animals....

An explanation for the combined effect of xylanase-glucose oxidase in dough systems

NARCIS (Netherlands)

Primo-Martín, C.; Wang, M.; Lichtendonk, W.J.; Plijter, J.J.; Hamer, R.J.

2005-01-01

In the bakery industry, glucose oxidase is usually used in combination with xylanase. Although many theories exist on the mechanism of action of each enzyme, the positive effect of combining the two is as yet unexplained. In this paper we studied a possible basis for this synergy by focusing on the

Endo-Perio Lesion and Uncontrolled Diabetes

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Sara Dhoum

2018-01-01

Full Text Available This work is to discuss the management of an endo-perio lesion, which represents a challenge to clinicians when it comes to diagnosis and prognosis of the involved teeth and especially with an altered general condition. A 50-year-old female patient with uncontrolled diabetes type 2 is suffering from a purulent discharge coming from the upper right canine. Endodontic and periodontal treatments were realized with 36 months radiological and clinical follow-up with the collaboration of her internist doctor.

Endo-Perio Lesion and Uncontrolled Diabetes.

Science.gov (United States)

Dhoum, Sara; Laslami, Kaoutar; Rouggani, Fatimazahraa; El Ouazzani, Amal; Jabri, Mouna

2018-01-01

This work is to discuss the management of an endo-perio lesion, which represents a challenge to clinicians when it comes to diagnosis and prognosis of the involved teeth and especially with an altered general condition. A 50-year-old female patient with uncontrolled diabetes type 2 is suffering from a purulent discharge coming from the upper right canine. Endodontic and periodontal treatments were realized with 36 months radiological and clinical follow-up with the collaboration of her internist doctor.

Combining endoscopic ultrasound with Time-Of-Flight PET: The EndoTOFPET-US Project

CERN Document Server

Frisch, Benjamin

2013-01-01

The EndoTOFPET-US collaboration develops a multimodal imaging technique for endoscopic exams of the pancreas or the prostate. It combines the benefits of high resolution metabolic imaging with Time-Of-Flight Positron Emission Tomography (TOF PET) and anatomical imaging with ultrasound (US). EndoTOFPET-US consists of a PET head extension for a commercial US endoscope and a PET plate outside the body in coincidence with the head. The high level of miniaturization and integration creates challenges in fields such as scintillating crystals, ultra-fast photo-detection, highly integrated electronics, system integration and image reconstruction. Amongst the developments, fast scintillators as well as fast and compact digital SiPMs with single SPAD readout are used to obtain the best coincidence time resolution (CTR). Highly integrated ASICs and DAQ electronics contribute to the timing performances of EndoTOFPET. In view of the targeted resolution of around 1 mm in the reconstructed image, we present a prototype dete...

Cloning, sequencing and expression of a xylanase gene from the maize pathogen Helminthosporium turcicum

DEFF Research Database (Denmark)

Degefu, Y.; Paulin, L.; Lübeck, Peter Stephensen

2001-01-01

A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and ...

Endo-buccal dental radiology - Radiation protection: medical sheet ED 4249

International Nuclear Information System (INIS)

Celier, D.; Megnigbeto, C.; Aubert, B.; Talbot, A.; Vidal, J.P.; Biau, A.; Lahaye, T.; Gauron, C.; Ariscon, J.M.; Barret, C.; Devaux, M.J.; Dohan, D.; Gambini, D.; Guerin, C.; Rocher, P.

2009-04-01

This document presents the various aspects and measures related to radiation protection when performing endo-buccal examinations. It presents the concerned personnel, describes the operational process of snapshot taking, indicates the associated hazards and the risk related to ionizing radiation, and describes how the risk is to be assessed and how exposure levels are to be determined (elements of risk assessment, delimitation of controlled and monitored areas, personnel classification, and choice of the dose monitoring method). It describes the various components of a risk management strategy (risk reduction, technical measures regarding the installation and the personnel, training and information, prevention and medical monitoring). It briefly presents how risk management is to be assessed, and mentions other related risks. In appendix, a table indicates radiation measurements about two endo-buccal radiography devices in specific use conditions

Bi-functional fusion enzyme EG-M-Xyn displaying endoglucanase and xylanase activities and its utility in improving lignocellulose degradation.

Science.gov (United States)

Chen, Chin-Chung; Gao, Guo-Jhan; Kao, Ai-Ling; Tsai, Zheng-Chia

2018-05-01

In this study, the gene fusion of endoglucanase (EG, one of cellulases) from Teleogryllus emma and xylanase (Xyn, one of hemicellulases) from Thermomyces lanuginosus was constructed to generate a fusion enzyme (EG-M-Xyn). Through the expression and purification by ultrafiltration and size-exclusion chromatography, the purified EG-M-Xyn had a molecular weight of 75.5 kDa and exhibited the specific activity of CMCase and xylanase as 306.8 U/mg and 1227.3 U/mg, respectively. The K m values (CMC and beechwood xylan) were 6.8 and 60.6 mg mL -1 while catalytic efficiency (k cat /K m ) values of CMCase and xylanase were 3280 and 38,797 min -1  mg -1  mL, respectively. EG-M-Xyn exerted great properties for its great potential in improving the enzymatic hydrolysis of lignocellulosics to produce fermentable sugars. First, EG-M-Xyn showed mild reaction pH and temperature of 5.5 and 50 °C, respectively. Secondly, EG-M-Xyn exhibited great heat tolerance of T 1/2 values of 173 (CMCase) and 693 min (xylanase). Lastly and most importantly, application of EG-M-Xyn in combination with Ctec2 (commercial enzyme) in the saccharification led to a 10-20% net increase in fermentable sugars liberated from pretreated rice straw in comparison to the Ctec2 alone group. In conclusion, EG-M-Xyn had great potential in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industry. Copyright © 2017 Elsevier B.V. All rights reserved.

[Posterior lamellar keratoplasty with DSEK technique and use of the Tan EndoGlide - short-term results].

Science.gov (United States)

Kałuiny, Bartłomiej J; Piotrowiak, Ilona; Sołdańska, Beata; Grzybek, Katarzyna; Czajkowska, Monika; Galas, Małgorzata; Malukiewicz, Grazyna

2013-01-01

To present the differences in surgical technique of DSEK (Descemet's Stripping Endothelial Keratoplasty) with the use of Tan EndoGlide (Coronet, UK) and Busin Glide (Moria, FR). Short-term results will also be presented, DSEK was performed in 24 eyes, in 8 cases the surgery was combined with cataract phacoemulsification and lOL implantation. Surgery course and 6 months postoperative results of first 12 eyes performed with the use of Tan EndoGlide were compared with 12 consecutive eyes preformed with Busin Glide. Tan EndoGlide provided much more stable anterior chamber, donor tissue unfolding process was better controlled but the incision was wider incision. Surgically induced mean refractory cylinder 6. months after the surgery was 1.56 - 1.15 Dsph in Tan EndoGlide group and 1.18 +/- 1.10 Dsph in Busin Glide group (P 0.05). Mean CDVA was 0.65+/- 0.27 and 0.63 +/- 0.25, respectively (P>0,05). Statistically significant differences in intra- and post-operative complications between both groups were not found. The Tan EndoGlide used during posterior lamellar keratoplasty with DSEK technique is a good alternative to currently used methods. It provides better stabilization of the anterior chamber, however its use is linked with higher postoperative astigmatism in comparison with Busin Glide. The visual outcomes and endothelial cell loss 6 months after the surgery were similar in both groups.

Radioimmunoassay with heterologous antibody (hetero-antibody RIA)

International Nuclear Information System (INIS)

Iwasawa, Atsushi; Hayashi, Hiroaki; Itoh, Zen; Wakabayashi, Katsumi

1991-01-01

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a 'hetero-antibody' RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCGβ or anti-ovine LHβ was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration. (author)

Partial purification and characterization of endo-β-1,4- mannanases ...

African Journals Online (AJOL)

STORAGESEVER

2008-07-04

Jul 4, 2008 ... Available online at http://www.academicjournals.org/AJB. ISSN 1684–5315 © 2008 Academic ... In the current study we report on the purification and characterization of endo-1 ... MATERIALS AND METHODS. Fungal isolates.

Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation

International Nuclear Information System (INIS)

Wojas, Sylwia; Hennig, Jacek; Plaza, Sonia; Geisler, Markus; Siemianowski, Oskar; Sklodowska, Aleksandra; Ruszczynska, Anna; Bulska, Ewa; Antosiewicz, Danuta M.

2009-01-01

Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications. - AtMRP7 expression in tobacco enhances Cd-tolerance and increases Cd storage in vacuoles

The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review)

DEFF Research Database (Denmark)

Carlsen Melander, Eva Maria; Jürgensen, Henrik J; Madsen, Daniel H

2015-01-01

The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen...... as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important...

An approach to industrial application: influence of black liquor and pH on xylanase efficiency in bleaching of eucalyptus kraft pulp

OpenAIRE

Fillat Latorre, Úrsula; Roncero Vivero, María Blanca; Bassa, Alexandre; Sacón, Vera Maria

2010-01-01

To obtain a more realistic appraisal of the potential efficiency of xylanases in the industrial bleaching, the influence of pH and the presence of black liquor (measured as COD) on the bleaching efficiency of two commercial xylanases was studied at high temperature. These pH’s, CODs, and temperatures are close to those used in the storage tower of the B fiber line in Jacareı´ unit of Fibria (Brazil). The pulp samples obtained after each bleaching stage were analyzed for kappa number, brigh...

Synthesis and Transformations of di-endo-3-Aminobicyclo-[2.2.2]oct-5-ene-2-carboxylic Acid Derivatives

Directory of Open Access Journals (Sweden)

Márta Palkó

2011-09-01

Full Text Available all-endo-3-amino-5-hydroxybicyclo[2.2.2]octane-2-carboxylic acid (13 and all-endo-5-amino-6-(hydroxymethylbicyclo[2.2.2]octan-2-ol (10 were prepared via dihydro-1,3-oxazine or g-lactone intermediates by the stereoselective functionalization of an N-protected derivative of endo-3-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acid (2. Ring closure of b-amino ester 4 resulted in tricyclic pyrimidinones 15 and 16. The structures, stereochemistry and relative configurations of the synthesized compounds were determined by IR and NMR.

Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

Science.gov (United States)

Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

2015-12-15

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

Science.gov (United States)

Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

2010-01-01

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

Amenability of Acacia and Eucalyptus Hardwood Pulps to Elemental Chlorine-Free Bleaching: Application and Efficacy of Microbial Xylanase

Directory of Open Access Journals (Sweden)

Avdhesh Kumar Gangwar

2015-10-01

Full Text Available This study outlines the results of a biobleaching study of acacia (A. mangium and eucalyptus (E. globulus hardwood kraft pulps with commercial xylanase (Optimase CX 72 L. The comparative study was carried out using an elemental chlorine-free (ECF bleaching sequence (D0EPD1D2 after the enzyme (X stage. The enzyme treatment resulted in improved optical properties with a reduction in bleach chemical consumption. At an equivalent bleach chemical consumption, a brightness gain of 2.1 and 1.7 units and a whiteness gain of 2.7 and 2.3 units were observed with xylanase treatment in acacia and eucalyptus pulps, respectively. In ECF bleaching using the D0EPD1D2 sequence, a final brightness was achieved to the extent of 90% ISO and 89% ISO for acacia and eucalyptus, respectively, at an equivalent charge of bleach chemicals. The post-color (PC number was also reduced by up to 45% for both hardwood pulps compared with the control. The bleachability of acacia was observed to be significantly higher than that of eucalyptus. In addition, a 17.0% and 23.0% reduction in chlorine dioxide and sodium hydroxide, respectively, were obtained for both hardwood pulps after xylanase pre-bleaching, thus indicating an environmentally friendly approach to the process.

Effects of Xylanase Supplementation on Growth Performance, Nutrient Digestibility and Non-starch Polysaccharide Degradation in Different Sections of the Gastrointestinal Tract of Broilers Fed Wheat-based Diets

Directory of Open Access Journals (Sweden)

L. Zhang

2014-06-01

Full Text Available This experiment was performed to investigate the effects of exogenous xylanase supplementation on performance, nutrient digestibility and the degradation of non-starch polysaccharides (NSP in different sections of the gastrointestinal tract (GIT of broilers fed wheat-based diets. A total of 120 7-day-old Arbor Acres broiler chicks were randomly allotted to two wheat-based experimental diets supplemented with 0 or 1.0 g/kg xylanase. Each treatment was composed of 6 replicates with 10 birds each. Diets were given to the birds from 7 to 21 days of age. The results showed that xylanase supplementation did not affect feed intake, but increased body weight gain of broiler at 21 day of age by 5.8% (pjejunum>duodenum>>gizzard> caecum. The supplementation of xylanse increased ileal isomaltriose concentration (p<0.05, but did not affect the concentrations of isomaltose, panose and 1-kestose in the digesta of all GIT sections. These results suggest that supplementation of xylanase to wheat-based diets cuts the arabinoxylan backbone into small fragments (mainly arabinose and xylose in the ileum, jejunum and duodenum, and enhances digestibilites of nutrients by decreasing digesta viscosity. The release of arabinose and xylose in the small intestine may also be the important contributors to the growth-promoting effect of xylanase in broilers fed wheat-based diets.

Two new xylanases with different substrate specificities from the human gut bacterium Bacteroides intestinalis DSM 17393.

Science.gov (United States)

Hong, Pei-Ying; Iakiviak, Michael; Dodd, Dylan; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac

2014-04-01

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

KAUST Repository

Hong, Pei-Ying

2014-01-24

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

KAUST Repository

Hong, Pei-Ying; Iakiviak, M.; Dodd, D.; Zhang, M.; Mackie, R. I.; Cann, I.

2014-01-01

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X5) and xylohexaose (X6) to a mixture of xylobiose (X2) and xylotriose (X3), while Rex8A hydrolyzed X3 through X6 to a mixture of xylose (X1) and X2. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X2 to X1. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.

STUDIES ON XYLANASE AND LACCASE ENZYMATIC PREBLEACHING TO REDUCE CHLORINE-BASED CHEMICALS DURING CEH AND ECF BLEACHING

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Vasanta V. Thakur,

2012-02-01

Full Text Available The biobleaching efficiency of xylanase and laccase enzymes was studied on kraft pulps from wood and nonwood based raw materials employed in the Indian paper industry. Treatment of these pulps with xylanase enzyme could result in improved properties, showing 2.0% ISO gain in pulp brightness and/or reducing the demand of chlorine-based bleach chemicals by up to 15% with simultaneous reduction of 20 to 25% in AOX generation in bleach effluents. Further, mill-scale trial results revealed that enzymatic prebleaching can be successfully employed with xylanases to reach the same bleach boosting efficacy. Laccase bleaching was also studied on hardwood pulp at a pH around 8.0, where most of the pulp mills in India are operating, in contrast to earlier studies on laccase enzyme bleaching, which were conducted at acidic pHs, i.e. 4.0 to 5.0. In case of laccase bleaching, interesting results were found wherein a bleach-boosting effect was observed even at pH 8.0. Further studies carried out with HOBT as mediator in comparison to the commonly used and expensive ABTS laccase mediator system (LMS resulted in improvement of the bleaching efficiency with reduction in demand of chlorine dioxide by more than 35%. Potential for further reduction was indicated by the brightness gain, when compared with a control using the DE(pD bleach sequence.

Method for determining heterologous biosynthesis pathways

KAUST Repository

Gao, Xin; Kuwahara, Hiroyuki; Alazmi, Meshari Saud; Cui, Xuefeng

2017-01-01

suitable pathways for the endogenous metabolism of a host organism because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. The present invention is called MRE (Metabolic Route Explorer), and it was conceived

Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs.

Science.gov (United States)

Winger, A M; Heazlewood, J L; Chan, L J G; Petzold, C J; Permaul, K; Singh, S

2014-11-01

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

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Sigoillot Jean-Claude

2009-11-01

Full Text Available Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS. Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78, commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5, was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity, locust bean gum galactomannan, carob galactomannan (low viscosity, and 1,4-β-D-mannan (from carob are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

Irreversible endo-Selective Diels–Alder Reactions of Substituted Alkoxyfurans: A General Synthesis of endo-Cantharimides

Science.gov (United States)

Foster, Robert W; Benhamou, Laure; Porter, Michael J; Bučar, Dejan-Krešimir; Hailes, Helen C; Tame, Christopher J; Sheppard, Tom D

2015-01-01

The [4+2] cycloaddition of 3-alkoxyfurans with N-substituted maleimides provides the first general route for preparing endo-cantharimides. Unlike the corresponding reaction with 3H furans, the reaction can tolerate a broad range of 2-substitued furans including alkyl, aromatic, and heteroaromatic groups. The cycloaddition products were converted into a range of cantharimide products with promising lead-like properties for medicinal chemistry programs. Furthermore, the electron-rich furans are shown to react with a variety of alternative dienophiles to generate 7-oxabicyclo[2.2.1]heptane derivatives under mild conditions. DFT calculations have been performed to rationalize the activation effect of the 3-alkoxy group on a furan Diels–Alder reaction. PMID:25756502

Over expression of beta-1, 4-xylanase by auto-induction in E. coli

International Nuclear Information System (INIS)

Khan, M.I.K.; Sajjad, M.; Akhtar, W.

2013-01-01

Catalytic domain of β-1, 4-xylanase gene, (xynZ.CD) of Clostridium thermocellum was cloned in pET28a expression vector and over-expressed in Escherichia colt BL21 CodonPlus (RIL). The production of XynZ.CD in E. colt was optimized using different concentrations of lactose and induction of the enzyme at different stages of growth. The maximum growth of the cells and the enzyme activity were observed when the cells were induced with 10mM lactose after 8 hours of incubation. The enzyme was found to constitute >40% of the total cell proteins in the supernatant of the lysed cells transformed with recombinant pET28a/xynZ.CD. It was purified by heating the cell lysate at 65 degree C for 30 m followed by fractionation through FPLC. Molecular weight of XynZ.CD was found to be approximately 38,524 D by MALDI-TOF analysis. The enzyme variant was quite stable within broad pH range of 5.5 - 8.0 and it retained >85% of xylanase activity after 2 h incubation at 70 degre C. (author)

Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669.

Science.gov (United States)

Liu, Wanli; Shi, Pengjun; Chen, Qiang; Yang, Peilong; Wang, Guozeng; Wang, Yaru; Luo, Huiying; Yao, Bin

2010-09-01

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets.

Science.gov (United States)

Yang, Y Y; Fan, Y F; Cao, Y H; Guo, P P; Dong, B; Ma, Y X

2017-01-01

Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (pdigestibility of energy by 0.6 percentage units (pdigestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (pdigestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (pdigestibility of phosphorus (pdigestibility (pdigestibility but decreased ADF digestibility. Adding xylanase increased crude protein digestibility and pig performance. Phytase increased the apparent total tract digestibility of phosphorus and calcium. The combination of

Biochemical mechanisms determine the functional compatibility of heterologous genes

DEFF Research Database (Denmark)

Porse, Andreas; Schou, Thea S.; Munck, Christian

2018-01-01

-gene libraries have suggested that sequence composition is a strong barrier for the successful integration of heterologous genes. Here we sample 200 diverse genes, representing >80% of sequenced antibiotic resistance genes, to interrogate the factors governing genetic compatibility in new hosts. In contrast...... factors governing the functionality and fitness of antibiotic resistance genes. These findings emphasize the importance of biochemical mechanism for heterologous gene compatibility, and suggest physiological constraints as a pivotal feature orienting the evolution of antibiotic resistance....

An in silico platform for the design of heterologous pathways in nonnative metabolite production

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Chatsurachai Sunisa

2012-05-01

Full Text Available Abstract Background Microorganisms are used as cell factories to produce valuable compounds in pharmaceuticals, biofuels, and other industrial processes. Incorporating heterologous metabolic pathways into well-characterized hosts is a major strategy for obtaining these target metabolites and improving productivity. However, selecting appropriate heterologous metabolic pathways for a host microorganism remains difficult owing to the complexity of metabolic networks. Hence, metabolic network design could benefit greatly from the availability of an in silico platform for heterologous pathway searching. Results We developed an algorithm for finding feasible heterologous pathways by which nonnative target metabolites are produced by host microorganisms, using Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as templates. Using this algorithm, we screened heterologous pathways for the production of all possible nonnative target metabolites contained within databases. We then assessed the feasibility of the target productions using flux balance analysis, by which we could identify target metabolites associated with maximum cellular growth rate. Conclusions This in silico platform, designed for targeted searching of heterologous metabolic reactions, provides essential information for cell factory improvement.

Biocompatibility Evaluation of EndoSequence Root Repair Paste in the Connective Tissue of Rats.

Science.gov (United States)

Taha, Nessrin A; Safadi, Rima A; Alwedaie, Manal S

2016-10-01

The aim of this study was to evaluate the subcutaneous connective tissue response to EndoSequence root repair paste (Brasseler, Savannah, GA) compared with mineral trioxide aggregate (MTA). Thirty-six Wistar rats each received 3 sterile tubes, containing 1 of the tested materials and control. The animals were killed 1, 3 and 6 weeks after implantation. The specimens were evaluated histologically for type of inflammation, intensity and extent of inflammatory cells, foreign body reaction, fibrous capsule thickness, perivascular fragments, calcific deposits and vascular congestion. EndoSequence provoked severe inflammation after 1 week, which was significantly different from MTA and control (P ˂ .05), with fragmented particles and foreign body reaction. MTA showed tissue-tolerance features almost comparable to control. EndoSequence was significantly more irritating than MTA and control at 1 and 3 weeks in terms of severity and extent of inflammation. After 6 weeks it displayed more biocompatible characteristics. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

DEFF Research Database (Denmark)

Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

2004-01-01

Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

Science.gov (United States)

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Enzymatic production of wheat and ryegrass derived xylooligosaccharides and evaluation of their in vitro effect on pig gut microbiota

DEFF Research Database (Denmark)

Dotsenko, Gleb; Meyer, Anne S.; Canibe, Nuria

2017-01-01

This study examines enzymatic production of linear xylooligosaccharides (XOS) and branched arabinoxylooligosaccharides (AXOS) from monocotyledonous biomass, wheat straw and ryegrass, and compares the in vitro effects of these XOS and AXOS on pig gut microbiota. XOS and AXOS were obtained from...... the biomass by treatment with different endo-1,4-β-xylanases. XOS of DP2-6 from wheat straw, obtained after treatment with Aspergillus niger endo GH11, suppressed growth of Clostridium perfringens and resulted in a high level of lactic acid production when fermented in vitro by pig fecal microbiota...... (GH11). These results indicate that wheat straw as well as green grass biomass such as ryegrass have potential as new sources of putative prebiotics for pig feed....

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

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Lotta E Andersson

Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

Mannans and endo-β-mannanase transcripts are located in different seed compartments during Brassicaceae germination.

Science.gov (United States)

Carrillo-Barral, Néstor; Matilla, Angel J; Rodríguez-Gacio, María Del Carmen; Iglesias-Fernández, Raquel

2018-03-01

Mannans but not endo-β-mannanases are mainly found in the mucilage layer of two Brassicaceae seeds. Nonetheless, mannanase mobilization from inner to outer seed layers cannot be ruled out. The contribution of endo-β-mannanase (MAN) genes to the germination of the wild-type Sisymbrium officinale and cultivated Brassica rapa (Brassicaceae) species has been explored. In both species, mannans have been localized to the imbibed external seed coat layer (mucilage) by fluorescence immunolocalization and MAN enzymatic activity increases in seeds as imbibition progresses, reaching a peak before 100% germination is achieved. The MAN gene families have been annotated and the expression of their members analyzed in vegetative and reproductive organs. In S. officinale and B. rapa, MAN2, MAN5, MAN6, and MAN7 transcripts accumulate upon seed imbibition. SoMAN7 is the most expressed MAN gene in S. officinale germinating seeds, as occurs with its ortholog in Arabidopsis thaliana, but in B. rapa, the most abundant transcripts are BrMAN2 and BrMAN5. These genes (MAN2, MAN5, MAN6, and MAN7) are localized, by mRNA in situ hybridization, to the micropylar at the endosperm layer and to the radicle in S. officinale, but in B. rapa, these mRNAs are faintly found to the micropylar living seed coat layer and are mainly present at the radicle tip and the vascular bundles. If the domestication process undergone by B. rapa is responsible for these different MAN expression patterns, upon germination remains to be elucidated. Since mannans and MAN genes are not spatially distributed in the same seed tissues, a movement of MAN enzymes that are synthesized with typical signal peptides from the embryo tissues to the mucilage layer (via apoplastic space) is necessary for the mannans to be hydrolyzed.

Laser induced biostimulation: A possible healing prospect in endo-perio lesion

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Mithra N Hegde

2012-01-01

Full Text Available The health of the tooth is governed by both endodontic tissue and periodontal apparatus. "Endo-perio lesion" is the term used to describe the lesions in which inflammatory products involves both pulpal and periodontal tissues in varying degrees. The disease of endodontium may lead to the involvement of the periodontium and vice versa. Endo-perio lesions are the clinical conditions that are often difficult to diagnose and persistent if not treated appropriately. Lasers have been used successfully in endodontic as well as periodontal procedures. With endodontic treatment alone, only part of the lesion will heal to the level of the secondary periodontal lesion. Overall prognosis depends upon the severity of periodontal damage and the efficacy of the periodontal treatment. Laser can be considered as an efficacious tool and an adjunct to conventional periodontal therapy both for its decontaminating and biostimulating effects.

Intracellular collagen degradation mediated by uPARAP/Endo180 is a major pathway of extracellular matrix turnover during malignancy

DEFF Research Database (Denmark)

Curino, Alejandro C; Engelholm, Lars H; Yamada, Susan S

2005-01-01

We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/...

Production of a heterologous proteinase A by Saccharomyces kluyveri

DEFF Research Database (Denmark)

Møller, Kasper; Tidemand, L.D.; Winther, J.R.

2001-01-01

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a refer......In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter...

Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

Science.gov (United States)

Bai, Wenqin; Xue, Yanfen; Zhou, Cheng; Ma, Yanhe

2015-01-01

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

Energy Technology Data Exchange (ETDEWEB)

McIntosh, Lawrence P., E-mail: mcintosh@chem.ubc.ca; Naito, Daigo; Baturin, Simon J.; Okon, Mark; Joshi, Manish D. [University of British Columbia, Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre (Canada); Nielsen, Jens E. [University College Dublin, School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute (Ireland)

2011-09-15

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK{sub A} values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain {sup 13}C{sup {gamma}} nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK{sub A} values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK{sub Ai} values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK{sub A} values and hence catalytic roles of these two residues result from their electrostatic coupling.

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

International Nuclear Information System (INIS)

McIntosh, Lawrence P.; Naito, Daigo; Baturin, Simon J.; Okon, Mark; Joshi, Manish D.; Nielsen, Jens E.

2011-01-01

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK A values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain 13 C γ nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK A values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK Ai values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK A values and hence catalytic roles of these two residues result from their electrostatic coupling.

Single-step purification and characterization of an extreme halophilic, ethanol tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with application potential in the food industry.

Science.gov (United States)

Yegin, Sirma

2017-04-15

An extracellular xylanase from Aureobasidium pullulans NRRL Y-2311-1 produced on wheat bran was purified by a single-step chromatographic procedure. The enzyme had a molecular weight of 21.6kDa. The optimum pH and temperature for xylanase activity were 4.0 and 30-50°C, respectively. The enzyme was stable in the pH range of 3.0-8.0. The inactivation energy of the enzyme was calculated as 218kJmol -1 . The xylanase was ethanol tolerant and kept complete activity in the presence of 10% ethanol. Likewise, it retained almost complete activity at a concentration range of 0-20% NaCl. In general, the enzyme was resistant to several metal ions and reagents. Mg 2+ , Zn 2+ , Cu 2+ , K 1+ , EDTA and β-mercaptoethanol resulted in enhanced xylanase activity. The K m and V max values on beechwood xylan were determined to be 19.43mgml -1 and 848.4Uml -1 , respectively. The enzyme exhibits excellent characteristics and could, therefore, be a promising candidate for application in food and bio-industries. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Endo- and ectoparasites of South American camelids and their control].

Science.gov (United States)

Schmäschke, R

2015-01-01

In a literature review, common endo- and ectoparasites of South American camelids are described, presenting morphological details and clinical signs important for diagnosis. Based on the life cycle of the parasites, possibilities for prophylaxis and therapy are indicated. The review should aid the veterinarian to diagnose and control common parasitic infections in South American camelids.

Endo-β-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

Science.gov (United States)

Gonçalves, Ana Maria D; Silva, Catarina S; Madeira, Tânia I; Coelho, Ricardo; de Sanctis, Daniele; San Romão, Maria Vitória; Bento, Isabel

2012-11-01

The crystal structure of wild-type endo-β-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (β/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Heterologous expression of a ketohexokinase in potato plants leads to inhibited rates of photosynthesis, severe growth retardation and abnormal leaf development

DEFF Research Database (Denmark)

Geigenberger, P.; Regierer, B.; Lytovchenko, A.

2004-01-01

of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of (14)CO(2) assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis......In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity...

Heterologous Protein Expression by Lactococcus lactis

NARCIS (Netherlands)

Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

2012-01-01

This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

Use of Residual Biomass from the Textile Industry as Carbon Source for Production of a Low-Molecular-Weight Xylanase from Aspergillus oryzae

Directory of Open Access Journals (Sweden)

Gilvan Caetano Duarte

2012-10-01

Full Text Available Pretreated dirty cotton residue (PDCR from the textile industry was used as an alternative carbon source for the submerged cultivation of Aspergillus oryzae and the production of xylanases. The filtered culture supernatant was fractionated by ultrafiltration followed by three chromatographic steps, which resulted in the isolation of a homogeneous low-molecular-weight xylanase (Xyl-O1 with a mass of 21.5 kDa as determined by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE co-polymerized with 0.1% oat spelt xylan. Enzyme catalysis was the most efficient at 50 °C and pH 6.0. The Km values (mg·mL−1 for the soluble fraction of oat spelt and birchwood xylans were 10.05 and 3.34, respectively. Xyl-O1 was more stable in the presence of 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB, 1,4-dithiothreitol (DTT, l-cysteine or β-mercaptoethanol, which increased the rate of catalysis by 40%, 14%, 40% or 37%, respectively. The enzyme stability was improved at pH 7.0 in the presence of 20 mM l-cysteine, with the retention of nearly 100% of the activity after 6 h at 50 °C. Xyl-O1 catalyzed the cleavage of internal β-1,4 linkages of the soluble substrates containing d-xylose residues, with a maximum efficiency of 33% for the hydrolysis of birchwood xylan after 12 h of incubation. Identification of the hydrolysis products by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD indicated the predominance of the hydrolysis products X2-X6 during the first 12 h of incubation and the accumulation of higher xylooligomers after the elution of the last xylooligomer standard, xylohexaose.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

Science.gov (United States)

Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

2016-07-01

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

Normal ovarian cycle in endo vaginal ultrasound

International Nuclear Information System (INIS)

Martinez, F.; Dualde, D.; Labrador, T.; Morales, F.J.; Vidal, P.; Gordo, G.

1995-01-01

The changing morphology of the endometrium and ovaries during the menstrual cycle can be viewed with great richness of image using high frequency (5-6-7.5 Mhz) probes in endo vaginal ultrasound. The radiological findings associated with the menstrual cycle are reviewed in terms of four phases (follicular, preovulatory, ovulatory and luteal) and a study is made of the changes that can lead to different morphologies, some of which, especially those of the corpus luteus, are peculiar. The need to be familiar with these changes in order to avoid confusing them with pathological signs is pointed out. (Author)

Characterization of the arabinoxylan-degrading machinery of the thermophilic bacterium Herbinix hemicellulosilytica-Six new xylanases, three arabinofuranosidases and one xylosidase.

Science.gov (United States)

Mechelke, M; Koeck, D E; Broeker, J; Roessler, B; Krabichler, F; Schwarz, W H; Zverlov, V V; Liebl, W

2017-09-10

Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one β-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55-65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA 2+3 XX. β-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS. Copyright © 2017. Published by Elsevier B.V.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

Science.gov (United States)

Rahmani, Nanik; Kahar, Prihardi; Lisdiyanti, Puspita; Hermiati, Euis; Lee, Jaemin; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2018-02-23

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers

DEFF Research Database (Denmark)

Nielsen, Christoffer Fagernæs; van Putten, Sander Maarten; Lund, Ida Katrine

2017-01-01

A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelia......A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non...... into the endosomal-lysosomal system, thus opening a potential route of entry into receptor-positive cells. This combination of specific expression and endocytic function appears well suited for targeting of uPARAP/Endo180-positive cancers by antibody-drug conjugate (ADC) mediated drug delivery. Therefore, we...... model with human uPARAP/Endo180-positive leukemic cells, obtaining a complete cure of all tested mice following intravenous ADC treatment with no sign of adverse effects. Our study identifies uPARAP/Endo180 as a promising target for novel therapy against several highly malignant cancer types....

Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

Science.gov (United States)

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.

Ethylene evolution and endo-beta-mannanase activity during lettuce seed germination at high temperature Evolução de etileno e atividade da enzima endo-beta-mananase durante a germinação de sementes de alface sob altas temperaturas

Directory of Open Access Journals (Sweden)

Warley Marcos Nascimento

2004-04-01

Full Text Available High temperatures during lettuce seed imbibition can delay or completely inhibit germination and the endosperm layer appears to restrict the radicle protrusion. The role of endo-beta-mannanase during lettuce seed germination at 35°C and the influence of ethylene in endo-beta-mannanase regulation were investigated. Seeds of 'Dark Green Boston' (DGB and 'Everglades' (EVE were germinated in water, or 10 mmol L-1 of 1-aminocyclopropane-1-carboxylic acid (ACC, or 10 mmol L-1 of aminoethoxyvinylglycine (AVG, or 20 mmol L-1 of silver thiosulphate (STS. Seeds were also primed in polyethylene glycol (PEG, or PEG + ACC, PEG + AVG, or PEG + STS. Untreated seeds germinated 100% at 20°C. At 35°C, EVE seeds germinated 100%, whereas DGB seeds germinated only 33%. Seed priming or adding ACC during incubation increased germination at 35°C. Higher ethylene evolution was detected in EVE than in DGB during germination at 35°C. AVG did not inhibit seed germination of DGB at 35°C, but STS did. Higher endo-beta-mannanase activity was observed in EVE compared with DGB seeds. Providing ACC either during priming or during germination increased endo-beta-mannanase activity, whereas AVG and STS led to decreased or no activity. Ethylene may overcome the inhibitory effect of high temperature in thermosensitive lettuce seeds due to increased endo-beta-mannanase, possibly leading to weakening of the endosperm.Altas temperaturas durante a embebição das sementes de alface podem atrasar ou inibir a germinação e o endosperma parece ser o responsável na restrição da protrusão da radícula. O envolvimento da enzima endo-beta-mananase durante a germinação de sementes de alface a 35°C e a influência do etileno na regulagem desta enzima foram estudados. Sementes das cultivares Dark Green Boston (DGB e Everglades (EVE foram germinadas em água ou em soluções de 10 mmol L-1 de 1-aminociclopropano-1-ácido carboxilico (ACC, 10 mmol L-1 de amino-etoxi-vinil-glicina (AVG

A colorimetric method to quantify endo-polygalacturonase activity.

Science.gov (United States)

Torres, Sebastián; Sayago, Jorge E; Ordoñez, Roxana M; Isla, María Inés

2011-02-08

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities. Copyright © 2010 Elsevier Inc. All rights reserved.

Face, content, and construct validity of the EndoViS training system for objective assessment of psychomotor skills of laparoscopic surgeons.

Science.gov (United States)

Escamirosa, Fernando Pérez; Flores, Ricardo Manuel Ordorica; García, Ignacio Oropesa; Vidal, Cristian Rubén Zalles; Martínez, Arturo Minor

2015-11-01

The aim of this study is to present face, content, and constructs validity of the endoscopic orthogonal video system (EndoViS) training system and determines its efficiency as a training and objective assessment tool of the surgeons' psychomotor skills. Thirty-five surgeons and medical students participated in this study: 11 medical students, 19 residents, and 5 experts. All participants performed four basic skill tasks using conventional laparoscopic instruments and EndoViS training system. Subsequently, participants filled out a questionnaire regarding the design, realism, overall functionality, and its capabilities to train hand-eye coordination and depth perception, rated on a 5-point Likert scale. Motion data of the instruments were obtained by means of two webcams built into a laparoscopic physical trainer. To identify the surgical instruments in the images, colored markers were placed in each instrument. Thirteen motion-related metrics were used to assess laparoscopic performance of the participants. Statistical analysis of performance was made between novice, intermediate, and expert groups. Internal consistency of all metrics was analyzed with Cronbach's α test. Overall scores about features of the EndoViS system were positives. Participants agreed with the usefulness of tasks and the training capacities of EndoViS system (score >4). Results presented significant differences in the execution of three skill tasks performed by participants. Seven metrics showed construct validity for assessment of performance with high consistency levels. EndoViS training system has been successfully validated. Results showed that EndoViS was able to differentiate between participants of varying laparoscopic experience. This simulator is a useful and effective tool to objectively assess laparoscopic psychomotor skills of the surgeons.

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

DEFF Research Database (Denmark)

Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

2004-01-01

appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

Endo-perio lesions: Diagnosis and clinical considerations

Directory of Open Access Journals (Sweden)

Shenoy Nina

2010-01-01

Full Text Available The interrelationship between periodontal and endodontic disease has aroused confusion, queries and controversy. Differentiating between periodontal and endodontic problems can be difficult. A symptomatic tooth may have pain of periodontal and/or pulpal origin. The nature of that pain is often the first clue in determining the etiology of such a problem. Radiographic and clinical evaluation can help clarify the nature of the problem. In some cases, the influence of pulpal pathology may create periodontal involvement. In others, periodontal pathology may create pulpal pathology. This review article discusses the various clinical aspects to be considered for accurately diagnosing and treating endo-perio lesions.

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation

International Nuclear Information System (INIS)

Dwivedi, Pallavi; Vivekanand, V.; Ganguly, Ruma; Singh, Rajesh P.

2009-01-01

The use of congress grass (Parthenium sp.) and water hyacinth (Eichhornia crassipes) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU E -3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 ± 6.0 IU ml -1 ) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 ± 6.5 IU ml -1 ) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 deg. C with its stability at 80 deg. C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme

An Intramolecular Heck reaction that Prefers a 5-endo- to a 6-exo-trig Cyclization Pathway

DEFF Research Database (Denmark)

Vital, Paulo; Norrby, Per-Ola; Tanner, David Ackland

2006-01-01

A regioselective aromatic Claisen rearrangement was used to prepare 17a, the precursor of triflate 17e. The intramolecular Heck reaction of 17e is promoted only by bidentate phosphine ligands, giving exclusively and in excellent yield 20, the product of a 5-endo-trig cyclization, despite the poss......A regioselective aromatic Claisen rearrangement was used to prepare 17a, the precursor of triflate 17e. The intramolecular Heck reaction of 17e is promoted only by bidentate phosphine ligands, giving exclusively and in excellent yield 20, the product of a 5-endo-trig cyclization, despite...

Results of the ANCHOR prospective, multicenter registry of EndoAnchors for type Ia endoleaks and endograft migration in patients with challenging anatomy.

Science.gov (United States)

Jordan, William D; Mehta, Manish; Varnagy, David; Moore, William M; Arko, Frank R; Joye, James; Ouriel, Kenneth; de Vries, Jean-Paul

2014-10-01

Proximal attachment site complications continue to occur after endovascular repair of abdominal aortic aneurysms (EVAR), specifically type Ia endoleak and endograft migration. EndoAnchors (Aptus Endosystems, Sunnyvale, Calif) were designed to enhance endograft proximal fixation and sealing, and the current study was undertaken to evaluate the potential benefit of this treatment. During the 23-month period ending in December 2013, 319 subjects were enrolled at 43 sites in the United States and Europe. EndoAnchors were implanted in 242 patients (75.9%) at the time of an initial EVAR procedure (primary arm) and in 77 patients with an existing endograft and proximal aortic neck complications (revision arm). Technical success was defined as deployment of the desired number of EndoAnchors, adequate penetration of the vessel wall, and absence of EndoAnchor fracture. Procedural success was defined as technical success without a type Ia endoleak at completion angiography. Values are expressed as mean ± standard deviation and interquartile range. The 238 male (74.6%) and 81 female (25.4%) subjects had a mean age of 74.1 ± 8.2 years. Aneurysms averaged 58 ± 13 (51-63) mm in diameter at the time of EndoAnchor implantation (core laboratory measurements). The proximal aortic neck averaged 16 ± 13 (7-23) mm in length (42.7% <10 mm and 42.7% conical) and 27 ± 4 mm (25-30 mm) in diameter; infrarenal neck angulation was 24 ± 15 (13-34) degrees. The number of EndoAnchors deployed was 5.8 ± 2.1 (4-7). Technical success was achieved in 303 patients (95.0%) and procedural success in 279 patients (87.5%), 217 of 240 (89.7%) and 62 of 77 (80.5%) in the primary and revision arms, respectively. There were 29 residual type Ia endoleaks (9.1%) at the end of the procedure. During mean follow-up of 9.3 ± 4.7 months, 301 patients (94.4%) were free from secondary procedures. Among the 18 secondary procedures, eight were performed for residual type Ia endoleaks and the others

[Endothelial keratoplasty: Descemet stripping (DSEK) using TAN EndoGlide™ device: case series].

Science.gov (United States)

Pazos, Henrique Santiago Baltar; Pazos, Paula Fernanda Morais Ramalho Baltar; Nogueira Filho, Pedro Antônio; Grisolia, Ana Beatriz Diniz; Silva, André Berger Emiliano; Gomes, José Álvaro Pereira

2011-01-01

To report the results of Descemet stripping endothelial keratoplasty (DSEK) using the TAN EndoGlideTM device to facilitate the insertion of the endothelial membrane. Prospective clinical study that included nine patients presenting corneal edema secondary to endothelial dysfunction. Best corrected visual acuity, refraction, central corneal thickness, endothelial cell density and complications were analyzed after a six-month follow-up. There was a significant improvement in the corneal edema and visual acuity in 7 patients (77.78%). The best corrected visual acuity ranged between 20/40 and 20/200. The average density of endothelial cells in six months varied between 1,305 cells/mm² and 2,346 cells/mm² with an average loss of 33.14% cells. Detachment of part of the graft was observed in one eye (11.11%) and primary failure of the endothelial transplantation occurred in 2 eyes (22.22%). The device TAN EndoGlideTM facilitates the introduction of the graft in Descemet stripping endothelial keratoplasty.

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

Science.gov (United States)

Vogt, Cédric M; Schraner, Elisabeth M; Aguilar, Claudio; Eichwald, Catherine

2016-08-11

Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Isolation, purification and characterization of xylanase produced by Arthrobacter sp. MTCC 5214 when grown in solid-state fermentation

Digital Repository Service at National Institute of Oceanography (India)

Khandeparker, R.; Bhosle, N.B.

%) fractionation, and purified to homogeneity using size exclusion and ion exchange chromatography. The molecular mass of xylanase was approx. 20 kDa. Enzyme retained 100% activity at pH 7 and 8 for 24 h. It was interesting to note that at higher pH such as 9, 10...

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans

Directory of Open Access Journals (Sweden)

Wei-Lin Hu

2017-11-01

Full Text Available Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF and endonuclease G (EndoG in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid. Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro.

Science.gov (United States)

Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J; Venø, Susanne T; Thomsen, Louiza B; Moos, Torben; Lohi, Hannes T; Madsen, Peder; Lykke-Hartmann, Karin; Nielsen, Morten S

2018-03-20

Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

METHOD FOR THE PRODUCTION OF HETEROLOGOUS POLYPEPTIDES IN TRANSFORMED YEAST CELLS

DEFF Research Database (Denmark)

2000-01-01

The invention describes industrial fermentation of a $i(Saccharomyces) yeast species for production of a heterologous product encoded by a plasmid or DNA contained in said $i(Saccharomyces) yeast species with method utilizes the substrate more efficiently and without fermentative metabolism...... resulting in formation of ethanol and other unwanted primary products of fermentative activity whereby high yields of the heterologous product are obtained. The $i(Saccharomyces) yeast species is preferably a Crabtree negative $i(Saccharomyces species) in particular $i(Saccharomyces kluyveri)....

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

Science.gov (United States)

Ramoni, Jonas; Marchetti-Deschmann, Martina; Seidl-Seiboth, Verena; Seiboth, Bernhard

2017-05-01

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Influence of organic nitrogen amendment, containing amino acids on the cellulase and xylanase, produced by Trichoderma spp. isolates

Directory of Open Access Journals (Sweden)

D. Draganova

2017-09-01

Full Text Available Abstract. Cellulases and hemicellulases are amount the main hydrolytic enzymes, involved in the bioconversion of lignocellulose material by microorganisms. Filamentous fungi of the genus Trichoderma are one of the most studied and good producer of cellulases and hemicellulases. The nutrients balance, especially carbon to nitrogen ratio, is one of the main factors of the biodegradation. The ability of 37 local isolates of Trichoderma sp. to produce cellulases and xylanase were tested in solid state cultivation on wheat straw as a substrate whit two variants: 1. the straw was only moistured with destilated water (CN 80:1; 2. the C:N ratio of the straw was adjusted to 30:1 using organic nitrogen amendment. There is a significant difference in the enzymatic activity of the isolates in their cultivation on straw with CN 80 and CN 30. The highest carboxymethylcellulase (CMCase activity at CN 80 showed T1T (110.19U/ml, and in the variant at CN 30 - TD (369.07U/ml. The highest β-glucosidase activity on both variants CN 80 and CN 30 was established for TG (2743.1U/ml - 12679.9U/ml. The highest xylanase activity at CN 80 and CN 30 was measured on T4I (21311.5U/ml – 47937.5U/ml. After ONA addition, all enzymes activities have increased several times, indicating the enhancing effect of the additive. The average activity of CMCase increased 6.1 times, the average β - glucosidase activity increased 5.1 times, while the xylanase activity increased 4.9 times for all tested isolates. The increase in activity of the investigated enzymes showed different patterns.

Spatial organization of heterologous metabolic system in vivo based on TALE.

Science.gov (United States)

Zhu, Lv-yun; Qiu, Xin-Yuan; Zhu, Ling-Yun; Wu, Xiao-Min; Zhang, Yuan; Zhu, Qian-Hui; Fan, Dong-Yu; Zhu, Chu-Shu; Zhang, Dong-Yi

2016-05-17

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

DEFF Research Database (Denmark)

Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

2012-01-01

Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029.

Science.gov (United States)

Bian, Xiaoying; Tang, Biao; Yu, Yucong; Tu, Qiang; Gross, Frank; Wang, Hailong; Li, Aiying; Fu, Jun; Shen, Yuemao; Li, Yue-Zhong; Stewart, A Francis; Zhao, Guoping; Ding, Xiaoming; Müller, Rolf; Zhang, Youming

2017-07-21

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L -1 . These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.

Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature.

Science.gov (United States)

Farinas, Cristiane S; Loyo, Marcel Moitas; Baraldo, Anderson; Tardioli, Paulo W; Neto, Victor Bertucci; Couri, Sonia

2010-12-31

Ethanol from lignocellulosic biomass has been recognized as one of the most promising alternatives for the production of renewable and sustainable energy. However, one of the major bottlenecks holding back its commercialization is the high costs of the enzymes needed for biomass conversion. In this work, we studied the enzymes produced from a selected strain of Aspergillus niger under solid state fermentation. The cellulase and xylanase enzymatic cocktail was characterized in terms of pH and temperature by using response surface methodology. Thermostability and kinetic parameters were also determined. The statistical analysis of pH and temperature effects on enzymatic activity showed a synergistic interaction of these two variables, thus enabling to find a pH and temperature range in which the enzymes have a higher activity. The results obtained allowed the construction of mathematical models used to predict endoglucanase, β-glucosidase and xylanase activities under different pH and temperature conditions. Optimum temperature values for all three enzymes were found to be in the range between 35°C and 60°C, and the optimum pH range was found between 4 and 5.5. The methodology employed here was very effective in estimating enzyme behavior under different process conditions. Copyright © 2010 Elsevier B.V. All rights reserved.

The role of heterologous chloroplast sequence elements in transgene integration and expression.

Science.gov (United States)

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-04-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

Adaption of Saccharomyces cerevisiae expressing a heterologous protein

DEFF Research Database (Denmark)

Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars

2008-01-01

Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger

Directory of Open Access Journals (Sweden)

C. S. Farinas

2011-03-01

Full Text Available In order to develop cost effective processes for converting biomass into biofuels, it is essential to improve enzyme production yields, stability and specific activity. In this context, the aim of this work was to evaluate the concentration of two enzymes involved in the hydrolysis of biomass, endoglucanase and xylanase, through precipitation. Statistical experimental design was used to evaluate the influence of precipitant agent concentration (ammonium sulfate and ethanol, aging time, and temperature on enzyme activity recovery. Precipitant agent concentration and aging time showed a statistically significant effect at the 95% confidence level, on both enzyme activity recoveries. The recovery of endoglucanase with ammonium sulfate and ethanol reached values up to 65 and 61%, respectively. For xylanase, the recovery rates were lower, 27 and 25% with ammonium sulfate and ethanol, respectively. The results obtained allowed the selection of the variables relevant to improving enzyme activity recovery within operational conditions suitable for industrial applications.

Isolation, screening and characterization of a novel extracellular xylanase from Aspergillus niger (KP874102.1) and its application in orange peel hydrolysis.

Science.gov (United States)

Uday, Uma Shankar Prasad; Majumdar, Ria; Tiwari, Onkar Nath; Mishra, Umesh; Mondal, Abhijit; Bandyopadhyay, Tarun Kanti; Bhunia, Biswanath

2017-12-01

In the present work, a potent xylanase producing fungal strain Aspergillus niger (KP874102.1) was isolated through cultural and morphological observations from soil sample of Baramura forest, Tripura west, India. 28S rDNA technique was applied for genomic identification of this fungal strain. The isolated strain was found to be phylogenetically closely related to Aspergillus niger. Kinetic constants such as K m and V max for extracellular xylanase were determined using various substrate such as beech wood xylan, oat spelt xylan and CM cellulose through Lineweaver-Burk plot. K m , V max and K cat for beech wood xylan are found to be 2.89mg/ml, 2442U and 426178Umlmg -1 respectively. Crude enzyme did not show also CM cellulose activity. The relative efficiency of oat spelt xylan was found to be 0.819 with respect to beech wood xylan. After acid hydrolysis, enzyme was able to produce reducing sugar with 17.7, 35.5, 50.8 and 65% (w/w) from orange peel after 15, 30, 45 and 60min incubation with cellulase free xylanase and maximum reducing sugar formation rate was found to be 55.96μg/ml/min. Therefore, the Aspergillus niger (KP874102.1) is considered as a potential candidate for enzymatic hydrolysis of orange peel. Copyright © 2017 Elsevier B.V. All rights reserved.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

Science.gov (United States)

2011-01-01

Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

Crystallization and Preliminary Crystallographic Characterization of Endo-polygalacturonase II from Aspergillus niger

NARCIS (Netherlands)

Schröter, K.-H.; Arkema, A.; Kester, H.C.M.; Visser, J.; Dijkstra, B.W.

1994-01-01

The endo-polygalacturonase II from Aspergillus niger has been crystallized from an ammonium sulfate solution by the hanging drop method. The crystals belong to the monoclinic space group P2(1), with cell dimensions a = 60.6 Angstrom, b = 152.6 Angstrom, c = 74.0 Angstrom and beta = 91.2 degrees with

Xylanase Increased the Ileal Digestibility of Non-Starch Polysaccharides and Concentration of Low Molecular Weight Non-Digestible Carbohydrates in Pigs Fed High Levels of wheat DDGS

DEFF Research Database (Denmark)

Pedersen, Mads Brøgger; Yu, Shukun; Arent, Susan

2015-01-01

The objective was to study the effect of a commercially available xylanase (CAX), an experimental xylanase (EX), and EX in combination with protease (EXP) on the degradation of nondigestible carbohydrates (NDC) and apparent ileal digestibility (AID) of nutrients in wheat distillers dried grains...... with solubles (wDDGS). The control and 3 enzyme diets contained 96% wDDGS supplemented with vitamins, minerals, l-lysine, and chromic oxide as a digestibility marker in addition to enzyme premix. Eight ileal cannulated pigs were fed 4 experimental diets containing 96% wDDGS—a control diet or 1 of 3 diets...

Production of cellulase and xylanase in a bubble gum column using immobilized Aspergillus niger KKS

Energy Technology Data Exchange (ETDEWEB)

Kang, Seong-Woo; Kim, Seung-Woo [Univ. of Suwon (Korea, Republic of); Lee, Jin-Suk [Korea Institute of Energy Research, Daejeon (Korea, Republic of)

1995-05-01

Aspergillus niger KKS, isolated from a farmland near Suwon, was immobilized on Celite and polyurethane foams. Enzyme activities produced by the immobilized cell system in a bubble column were higher than that of shake-flask culture. The enzyme productivities were twice as high. {Beta}-Glucosidase, {Beta}-xylosidase, and xylanase activities obtained in a bubble column were significant when the ground rice straw was used as a substrate. 9 refs., 2 figs., 3 tabs.

Inspired by nonenveloped viruses escaping from endo-lysosomes: a pH-sensitive polyurethane micelle for effective intracellular trafficking

Science.gov (United States)

Song, Nijia; Zhou, Lijuan; Li, Jiehua; Pan, Zhicheng; He, Xueling; Tan, Hong; Wan, Xinyuan; Li, Jianshu; Ran, Rong; Fu, Qiang

2016-03-01

A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core exposure, resulting from the detachment of the hydrophilic polyethylene glycol (PEG) shell, are similar to the behavior of a nonenveloped virus when trapped in acidic endo-lysosomes. Moreover, the degradation mechanism was verified by gel permeation chromatography (GPC). The endo-lysosomal membrane rupture induced by these transformed micelles is clearly observed by transmission electron microscopy. Consequently, excellent antitumor activity is confirmed both in vitro and in vivo. The results verify that the pHPM could be a promising new drug delivery tool for the treatment of cancer and other diseases.A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

Science.gov (United States)

Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System

Directory of Open Access Journals (Sweden)

Kexin Zhao

2017-05-01

Full Text Available Lipoprotein cholesterol is delivered to the limiting membrane of late endosomes/lysosomes (LELs by Niemann-Pick C1 (NPC1. However, the mechanism of cholesterol transport from LELs to the endoplasmic reticulum (ER is poorly characterized. We report that oxysterol-binding protein-related protein 1L (ORP1L is necessary for this stage of cholesterol export. CRISPR-mediated knockout of ORP1L in HeLa and HEK293 cells reduced esterification of cholesterol to the level in NPC1 knockout cells, and it increased the expression of sterol-regulated genes and de novo cholesterol synthesis, indicative of a block in cholesterol transport to the ER. In the absence of this transport pathway, cholesterol-enriched LELs accumulated in the Golgi/perinuclear region. Cholesterol delivery to the ER required the sterol-, phosphatidylinositol 4-phosphate-, and vesicle-associated membrane protein-associated protein (VAP-binding activities of ORP1L, as well as NPC1 expression. These results suggest that ORP1L-dependent membrane contacts between LELs and the ER coordinate cholesterol transfer with the retrograde movement of endo-lysosomal vesicles.

Habib EndoHPB: a novel endobiliary radiofrequency ablation device. An experimental study.

Science.gov (United States)

Zacharoulis, Dimitris; Lazoura, Olga; Sioka, Eleni; Potamianos, Spyros; Tzovaras, George; Nicholls, Joanna; Koukoulis, George; Habib, Nagy

2013-02-01

The Habib EndoHPB is a bipolar radiofrequency (RF) catheter developed to be introduced across malignant strictures of the bile ducts, so that RF energy can locally ablate the tumor prior to stent placement. This experiment aims to assess the ability of the catheter to coagulate the wall of the common bile duct (CBD) in a porcine model, to establish power requirement and time parameters and correlate them to the depth of thermal injury, and to assess the ease of operation of the device. The CBD was catheterized using the device in 20 pigs. RF energy was applied to the CBD wall with various generator settings. The pigs were sacrificed 24 hr after the application and the CBD was excised for histological analysis. The device was easy to handle. Statistically significant correlations between the power, the time of RF application, and the thermal injury depth were found. The Habib EndoHPB catheter can effectively deliver RF energy intraluminally in the porcine CBD. Clinical studies are warranted in order to define proper settings for safe and efficient use in malignant biliary obstruction.

The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

Science.gov (United States)

Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

2017-11-24

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAU{sub E}-3.510 in submerged fermentation

Energy Technology Data Exchange (ETDEWEB)

Dwivedi, Pallavi; Vivekanand, V.; Ganguly, Ruma; Singh, Rajesh P. [Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667 (India)

2009-04-15

The use of congress grass (Parthenium sp.) and water hyacinth (Eichhornia crassipes) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU{sub E}-3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 {+-} 6.0 IU ml{sup -1}) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 {+-} 6.5 IU ml{sup -1}) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 C with its stability at 80 C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme. (author)

A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

Directory of Open Access Journals (Sweden)

Gordon Ng

Full Text Available BACKGROUND: Nitrogen (N(2 fixation also yields hydrogen (H(2 at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2 as sole N-source bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase, has nevertheless been presumed responsible for recycling such endogenous hydrogen. METHODS AND FINDINGS: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase. An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. CONCLUSIONS: Representative of aerobic N(2-fixing and H(2-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2, specifically that produced by N(2 fixation. To benefit human civilization, H(2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As

A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

Science.gov (United States)

Ng, Gordon; Tom, Curtis G S; Park, Angela S; Zenad, Lounis; Ludwig, Robert A

2009-01-01

Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such

Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

Directory of Open Access Journals (Sweden)

Leavell Sarah

2010-05-01

Full Text Available Abstract Background Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control and then cats were vaginally challenged with cell-associated or cell-free FIV. Results Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls. Conclusions The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus can be modulated by mucosal exposure to uninfected heterologous cells.

A Simple and Efficient Approach to the Synthesis of Endo and Exo Bicyclo[6.1.0]nona-3,5-diene-9-carboxaldehyde

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Gholamreza Mashayekhi

2007-10-01

Full Text Available Monobromination of 1,5-cyclooctadiene, followed by cyclopropanation with ethyl diazoacetate, led to the formation of endo and exo ethyl 4,5-dibromobicyclo[6.1.0]nonane-9-carboxylates 3a and 3b. Bis-dehydrobromination of 3a and 3b using 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU afforded the endo and exo ethyl bicyclo[6.1.0]nona-3,5-diene-9-carboxylates 4a and 4b. Reduction of these compounds to the corresponding alcohols 5a and 5b and subsequent oxidation with pyridinium chlorochromate (PCC resulted in the formation of the target compounds endo and exo bicyclo[6.1.0]nona-3,5-diene-9-carboxaldehydes 6a and 6b.

A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

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Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

2011-09-01

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

Introduction of caveolae structural proteins into the protozoan Toxoplasma results in the formation of heterologous caveolae but not caveolar endocytosis.

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Bao Lige

Full Text Available Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae 'precursors' in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma.

Past Life and Future Effects—How Heterologous Infections Alter Immunity to Influenza Viruses

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Aisha Souquette

2018-05-01

Full Text Available Influenza virus frequently mutates due to its error-prone polymerase. This feature contributes to influenza virus’s ability to evade pre-existing immunity, leading to annual epidemics and periodic pandemics. T cell memory plays a key protective role in the face of an antigenically distinct influenza virus strain because T cell targets are often derived from conserved internal proteins, whereas humoral immunity targets are often sites of increased mutation rates that are tolerated by the virus. Most studies of influenza T cell memory are conducted in naive, specific pathogen free mice and do not account for repetitive influenza infection throughout a lifetime, sequential acute heterologous infections between influenza infections, or heterologous chronic co-infections. By contrast to these mouse models, humans often experience numerous influenza infections, encounter heterologous acute infections between influenza infections, and are infected with at least one chronic virus. In this review, we discuss recent advances in understanding the effects of heterologous infections on the establishment and maintenance of CD8+ T cell immunological memory. Understanding the various factors that affect immune memory can provide insights into the development of more effective vaccines and increase reproducibility of translational studies between animal models and clinical results.

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

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Hoang, Huy-Dung; Maruyama, Jun-ichi

2014-01-01

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

Cellulase and Xylanase Production by Penicillium echinulatum in Submerged Media Containing Cellulose Amended with Sorbitol

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Carla Eliana Todero Ritter

2013-01-01

Full Text Available The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v soy bran; 0.1% (w/v wheat bran; and a solution of salts. The highest filter paper activity (FPA ( IU·mL−1 was obtained on the seventh day in the medium containing 0.5% (w/v sorbitol and 0.5% (w/v cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( IU·mL−1 in the medium containing 0.75% (w/v sorbitol and 0.75% (w/v cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v sorbitol and 0.25% (w/v cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems.

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

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Egel-Mitani; Andersen; Diers; Hach; Thim; Hastrup; Vad

2000-06-01

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

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Qing Qing

2011-06-01

Full Text Available Abstract Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy.

A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry.

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Cheng, Zhong; Chen, Dong; Lu, Bo; Wei, Yutuo; Xian, Liang; Li, Yi; Luo, Zhenzhen; Huang, Ribo

2016-06-28

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

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Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Heterologous Infection of Pregnant Mice Induces Low Birth Weight and Modifies Offspring Susceptibility to Malaria.

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Ankur Sharma

Full Text Available Pregnancy malaria (PM is associated with poor pregnancy outcomes, and can arise due to relapse, recrudescence or a re-infection with heterologous parasites. We have used the Plasmodium chabaudi model of pregnancy malaria in C57BL/6 mice to examine recrudescence and heterologous infection using CB and AS parasite strains. After an initial course of patent parasitemia and first recrudescence, CB but not AS parasites were observed to recrudesce again in most animals that became pregnant. Pregnancy exacerbated heterologous CB infection of AS-experienced mice, leading to mortality and impaired post-natal growth of pups. Parasites were detected in placental blood without evidence of sequestration, unlike P. falciparum but similar to other malaria species that infect pregnant women. Inflammatory cytokine levels were elevated in pregnant females during malaria, and associated with intensity of infection and with poor outcomes. Pups born to dams during heterologous infection were more resistant to malaria infections at 6-7 weeks of age, compared to pups born to malaria-experienced but uninfected dams or to malaria-naïve dams. In summary, our mouse model reproduces several features of human PM, including recrudescences, heterologous infections, poor pregnancy outcomes associated with inflammatory cytokines, and modulation of offspring susceptibility to malaria. This model should be further studied to explore mechanisms underlying PM pathogenesis.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

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Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Hordeum vulgare cysteine protease heterologous expressed in yeast

DEFF Research Database (Denmark)

Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

, (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression...

N-Cyano-7α-methoxycarbonyl-6,14-endo-ethenotetrahydronorthebaine

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Mustafa Odabaşoğlu

2009-09-01

Full Text Available In the title compound (systematic name: methyl 17-cyano-3,6-dimethoxy-4,5α-epoxy-6,14-endo-ethenomorphinan-7-carboxylate, C23H24N2O5, the dihydrofuran ring adopts a twist conformation, while the piperidine ring is in a chair conformation. The benzene-fused cyclohexene ring adopts an envelope conformation. An intramolecular C—H...O hydrogen bond is observed. Intermolecular C—H...N and C—H...O hydrogen bonds form C(5 chains along the a and b axes, respectively, and together they form a three-dimensional network.

Debromination of endo-(+)-3-bromocamphor with primary amines

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Markovic, Svetlana; Markovic, Violeta; Joksovic, Milan D.; Joksovic, Ljubinka, E-mail: ljubinka@kg.ac.rs [Department of Chemistry, Faculty of Science, University of Kragujevac (Serbia); Todorovic, Nina [Institute for Chemistry, Technology and Metallurgy, Belgrade (Serbia); Divjakovic, Vladimir [Department of Physics, University of Novi Sad (Serbia); Trifunovic, Snezana [Faculty of Chemistry, University of Belgrade, Belgrade, (Serbia)

2013-07-15

Reductive debromination of endo-(+)-3-bromocamphor with different primary amines followed by imine formation was investigated. This reaction requires simple experimental procedure without any organic solvent, metal or conventional reducing agent. A strong influence of amine polarity on the efficacy of debromination process was observed, and ethanolamine and ethylene diamine having sufficiently high boiling points can debrominate 3-bromocamphor giving corresponding camphanimines in good isolated yields. The mechanisms of debromination of 3-bromocamphor with ethanolamine and n-hexylamine were investigated at the B3LYP/6-311+G(d,p) level of theory. The radical mechanism was revealed, and it was shown that the reaction with more polar ethanolamine is energetically more favorable. (author)

Ovarian cryopreservation after laparoscopic ovariectomy using the Endo-GIA stapling device and LAPRO-clip absorbable ligating clip in a woman: a case report

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Messner Alexandra

2011-02-01

Full Text Available Abstract Introduction Several options are available for preserving fertility before cytotoxic treatment, including ovarian tissue cryopreservation. Most reported surgical techniques include electrocoagulation. Our hypothesis is that avoidance of electrocoagulation may decrease ovarian cortex injury during cryopreservation procedures. Case presentation We report a laparoscopic technique of whole-ovary removal without coagulation using Endo-GIA forceps and clips. Laparoscopic ovariectomy was performed for cryopreservation in a 37-year-old Caucasian woman with breast cancer and for whom chemotherapy was planned. The procedure was completed quickly and without complication. This Endo-GIA procedure was of short duration with a short period of ischemia before freezing. Conclusion Laparoscopic ovariectomy using the Endo-GIA stapling device procedure without coagulation may diminish ovary injury before ovarian cryopreservation.

Ovarian cryopreservation after laparoscopic ovariectomy using the Endo-GIA stapling device and LAPRO-clip absorbable ligating clip in a woman: a case report

Science.gov (United States)

2011-01-01

Introduction Several options are available for preserving fertility before cytotoxic treatment, including ovarian tissue cryopreservation. Most reported surgical techniques include electrocoagulation. Our hypothesis is that avoidance of electrocoagulation may decrease ovarian cortex injury during cryopreservation procedures. Case presentation We report a laparoscopic technique of whole-ovary removal without coagulation using Endo-GIA forceps and clips. Laparoscopic ovariectomy was performed for cryopreservation in a 37-year-old Caucasian woman with breast cancer and for whom chemotherapy was planned. The procedure was completed quickly and without complication. This Endo-GIA procedure was of short duration with a short period of ischemia before freezing. Conclusion Laparoscopic ovariectomy using the Endo-GIA stapling device procedure without coagulation may diminish ovary injury before ovarian cryopreservation. PMID:21291518

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

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Selma Majerowicz

1994-09-01

Full Text Available Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11. Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Upper respiratory tract infection, heterologous immunisation and meningococcal disease

NARCIS (Netherlands)

Scholten, R. J.; Bijlmer, H. A.; Tobi, H.; Dankert, J.; Bouter, L. M.

1999-01-01

To test the hypothesis that an episode of upper respiratory tract infection or heterologous immunisation is a predisposing factor for the occurrence of meningococcal disease, data from 377 cases of meningococcal disease and their household contacts (n = 1124) were analysed by conditional logistic

Endo-laparoscopic rendezvous approach for pericardia with gastric posterior wall of gastrointestinal stromal tumor: analysis of 52 consecutive cases.

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Ding, Po; Zhao, Yongjie

2014-12-01

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract and most frequently developed in the stomach, and surgical therapy is limited on removal of the tumor lesion. The aim of this study was to investigate the clinical values of endo-laparoscopic rendezvous approach for pericardial GISTs within gastric posterior wall. Surgical outcome and clinical data of 52 patients with pericardial GISTs within gastric posterior wall treated at Tianjin Peoples' Hospital from January 2004 to October 2013 were analyzed. Endo-laparoscopic rendezvous approach was used as an operative procedure for tumor resection ranged from 10 to 50 mm. Endoscopic ultrasound, computed tomography and microscopic findings all certified the gastric spindle type GIST locating in the submucosa to muscle proper. Endo-laparoscopic rendezvous approach was attempted in 52 patients (male/female: 31/21) with median age of 51 years (25-71 years). The median operating time was 80 min (range: 40-120 min) and median intra-operative blood loss was 26 ml (range: 10-50 ml). The median hospital stay was 5 days (range: 4-6 days), while the median tumor size was 25 mm (range: 7-50 mm). All operative margins were clear. There were no recurrences or metastases of all patients in a median follow-up of 24 months (range: 6-36 months). Endo-laparoscopic rendezvous approach is considered to represent the next revolution in surgery. The new technique is reliable and effective in clinical application, due to the advantages of accurate and quick localization for pericardial GIST within gastric posterior wall.

The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity.

Science.gov (United States)

Tena Fernández, Fátima; González, Inmaculada; Doblas, Paula; Rodríguez, César; Sahana, Nandita; Kaur, Harpreet; Tenllado, Francisco; Praveen, Shelly; Canto, Tomas

2013-06-01

In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase (HCPro). In silencing suppression patch assays, we found that Potato virus Y (PVY) HCPro expressed from a P1-HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was higher when expressed from P1-HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from P1-HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However, P1 could still have an indirect effect favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Uptake and Persistence of Homologous and Heterologous Zooxanthellae in the Temperate Sea Anemone Cereus pedunculatus (Pennant).

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Davy, S K; Lucas, I A N; Turner, J R

1997-04-01

The uptake and persistence of symbiotic dinoflagellates (zooxanthellae) were measured in the temperate sea anemone Cereus pedunculatus (Pennant). Aposymbiotic specimens of C. pedunculatus were inoculated with zooxanthellae freshly isolated from a range of temperate and subtropical Anthozoa. Each inoculate consisted of zooxanthellae from a single host species and was either homologous (zooxanthellae from a host of the same species as the one being inoculated) or heterologous (from a host of a different species than the one being inoculated). The densities of zooxanthellae in host tissues were determined at regular intervals. C. pedunculatus took up homologous and heterologous zooxanthellae to similar degrees, except for zooxanthellae from the temperate Anthopleura ballii, which were taken up to a lesser extent. The densities of all zooxanthellae declined between 4 hours and 4 days after uptake, indicating that zooxanthellae were expelled, digested, or both during this period. The densities of all zooxanthellae increased between 2 and 8 weeks after inoculation, indicating zooxanthella growth. Over the entire 8-week period after uptake, densities of homologous zooxanthellae were always greater than those of heterologous zooxanthellae. Between 8 and 36 weeks after infection, densities of homologous zooxanthellae declined markedly and densities of some heterologous zooxanthellae increased further, resulting in homologous and heterologous zooxanthella densities being the same at 36 weeks. These densities were the same as those in naturally infected C. pedunculatus of similar size. The results suggest that zooxanthellae from a range of host species and environments can establish symbioses with C. pedunculatus and that, over long periods under laboratory conditions, heterologous zooxanthellae may populate C. pedunculatus to the same extent as homologous zooxanthellae.

Cost-effective endo-mannanase from Bacillus sp. CFR1601 and its application in generation of oligosaccharides from guar gum and as detergent additive.

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Srivastava, Praveen Kumar; Kapoor, Mukesh

2014-01-01

The indigenous bacteria Bacillus sp. CFR1601 produced significant levels of endo-mannanase when grown on agro-wastes, namely, green gram husk and sunflower oil cake (25.6 IU/mL), used as sole carbon and nitrogen sources, respectively. Under immobilized cell system, synthetic supports (polyurethane foam, scotch brite, polyester; up to 33.2 IU/mL) were found marginally superior as compared to natural supports (cotton and silk; up to 28.2 IU/mL) for endo-mannanase production. Cooperative interactions between L-lysine HCl (0.3% w/v), Tween 60 (0.3% v/v), and sunflower oil cake (3.0% w/v) in central composite design response surface methodology ameliorated (1.61-fold) endo-mannanase titers to 48.0 IU/mL. Partially purified endo-mannanase was tested for its ability to produce oligosaccharides from guar gum. These oligosaccharides were tested in vitro for their ability to promote growth of Lactobacillus plantarum MTCC 5422 and Lactobacillus salivarius CHS 1E. Results indicated that low-molecular-weight degraded products from guar gum were (1) able to support the growth of tested strains [increased O.D600nm up to 2.3-fold and decrease in pH (production of short chain fatty acid (SCFA)] when used as sole carbon source; and (2) after purification and analysis by electron spray ionization-mass spectrometry (ESI-MS) were found to be composed of mainly disaccharide and tetrasaccharide. The compatibility of endo-mannanase with various detergents together with wash performance test confirmed its potential applicability for laundry industry.

Utilization of Bagasse Cellulose for Ethanol Production through Simultaneous Saccharification and Fermentation by Xylanase

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M Samsuri

2010-10-01

Full Text Available Bagasse is a solid residue from sugar cane process, which is not many use it for some product which have more added value. Bagasse, which is a lignosellulosic material, be able to be use for alternative energy resources like bioethanol or biogas. With renewable energy resources a crisis of energy in Republic of Indonesia could be solved, especially in oil and gas. This research has done the conversion of bagasse to bioethanol with xylanase enzyme. The result show that bagasse contains of 52,7% cellulose, 20% hemicelluloses, and 24,2% lignin. Xylanase enzyme and Saccharomyces cerevisiae was used to hydrolyse and fermentation in SSF process. Variation in this research use pH (4, 4,5, and 5, for increasing ethanol quantity, SSF process was done by added chloride acid (HCl with concentration 0.5% and 1% (v/v and also pre-treatment with white rot fungi such as Lentinus edodes (L.edodes as long 4 weeks. The SSF process was done with 24, 48, 72, and 96 hour's incubation time for fermentation. Variation of pH 4, 4,5, and 5 can produce ethanol with concentrations 2,357 g/L, 2,451 g/L, 2,709 g/L. The added chloride acid (HCl with concentration 0.5% and 1% (v/v and L. edodes can increase ethanol yield, The highest ethanol concentration with added chloride acid (HCl concentration 0.5% and 1% consecutively is 2,967 g/L, 3,249 g/L. The highest ethanol concentration with pre-treatment by L. edodes is 3,202 g/L.

A case series associated with different kinds of endo-perio lesions

OpenAIRE

Aksel, Hacer; Serper, Ahmet

2014-01-01

Pulpal and periodontal problems are responsible for more than half of the tooth mortality. There are some articles published in the literature about this issue. Many of them are quite old. There has been also lack of knowledge about the effect of endodontic treatment on the periodontal tissue healing and suitable treatment interval between endodontic and periodontal treatments. In this case report, different kinds of endo-perio lesion were treated with sequential endodontic and periodontal tr...

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Directory of Open Access Journals (Sweden)

Qinghua Cao

2016-01-01

Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

Science.gov (United States)

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Cellulase and xylanase activity during the decomposition of three aquatic macrophytes in a tropical oxbow lagoon

Directory of Open Access Journals (Sweden)

L Sciessere

2011-09-01

Full Text Available Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91 and xylanase (EC 3.2.1.8 during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus on the surface and water-sediment interface (w-s interface of an oxbow lagoon (Óleo lagoon within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality.

Treatment of oligodontia with endo-osseous fixtures: experience in eight consecutive patients at the end of dental growth.

Science.gov (United States)

Becelli, Roberto; Morello, Roberto; Renzi, Giancarlo; Dominici, Chiara

2007-11-01

Clinical manifestations of oligodontia consist in agenesia of multiple teeth eventually with deciduous retained teeth, atrophy of alveolar ridge, aberrations of teeth dimension, and shape, with consequent aesthetic and functional defects. The first choice treatment is based on a team collaboration of maxillo-facial surgeon, orthodontist, and prosthodontist, and is conditioned by various clinical parameters as number and site of lacking teeth, age and dental development of patients, eventual alveolar ridge atrophy. Treatment planning should be individualized for each patient. In our experience, based on 8 consecutive patients at the end of dental growth affected by oligodontia, endo-osseous fixtures positioning was carried out in consideration of long-lasting stability and optimal aesthetical characteristics. In 5 patients rehabilitative preprosthetic surgical procedures were performed, consisting in 2 sinus lift with immediate positioning of 3 fixtures in both cases, 4 heterologous bone graft in postextractive sites with retained ankylotic deciduous teeth and 1 positioning of reabsorbable biomembrane. A temporary removable denture was positioned immediately after surgery in order to obtain a prompt aesthetical and psychosocial restore. Osseointegration ratio as observed at 8.5 years follow-up was analyzed according to surgical variables and differences in prosthetic rehabilitation (fixtures supporting single crown versus multiple crowns). Successful osteointegration was observed at 8.5 years mean follow-up in 58 fixtures, corresponding to a 96.6% ratio. Failure of integration was encountered in fixtures immediately positioned in postextractive sites having a mild grade of bone atrophy, supporting single crown. A rate of success of 100% was observed in cases of immediate or delayed positioning in postextractive or traditional sites.Fixtures positioning in patients affected by isolated oligodontia, without malformative syndromes, and at the end of dental development

Structural effects on the chemical shift of exo - endo and exo - exo tetra cyclo dodecane; Efeitos estruturais sobre o deslocamento quimico de exo-endo e exo-exo tetraciclododecanos

Energy Technology Data Exchange (ETDEWEB)

Leal, Katia Zaccur [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Dept. de Fisico-Quimica; Costa, Valentim E.U. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Dept. de Quimica Organica; Seidl, Peter Rudolf [Instituto Militar de Engenharia (IME), Rio de Janeiro, RJ (Brazil). Secao de Quimica

1992-12-31

The systematic investigation of rigid cyclic molecules has provided a basis for a number of concepts presently employed in NMR spectroscopy. Bicyclic systems of norbornyl family have been extensively studied in this respect but their tetracyclic analogs have received much less attention. The large number of peaks that into narrow chemical shift ranges and the presence of intramolecular affects that arise from groups brought into close proximity have probably been responsible for this state of affairs. As in the case of other polycyclic rings of interest, more data from model systems would be desirable. We have analysed the proton and carbon-13 spectra of endo-endo tetra cyclo [6.1.1{sup 3,6}.0{sup 2,7}] dodeca-4-en-10-ol (1A) and its analogs and found interactions between atoms that are four or more bonds away. Although these upfield {delta} and {delta}{sub E} effects could be accounted for by invoking steric interactions similar to those that give to the well-know {gamma}gauche effect, there are other discrepancies between carbon-13 chemical shifts of tetracyclic dodecanes and their bicyclic analogs that are not amenable to simple rationalizations and a look other situations where such effects may be present would be recommendable. (author) 19 refs., 8 figs., 8 tabs.

Feasibility Study of EndoTAG-1, a Tumor Endothelial Targeting Agent, in Combination with Paclitaxel followed by FEC as Induction Therapy in HER2-Negative Breast Cancer.

Directory of Open Access Journals (Sweden)

Michail Ignatiadis

Full Text Available EndoTAG-1, a tumor endothelial targeting agent has shown activity in metastatic triple-negative breast cancer (BC in combination with paclitaxel.HER2-negative BC patients candidates for neoadjuvant chemotherapy were scheduled to receive 12 cycles of weekly EndoTAG-1 22mg/m2 plus paclitaxel 70mg/m2 followed by 3 cycles of FEC (Fluorouracil 500mg/m2, Epirubicin 100mg/m2, Cyclophosphamide 500mg/m2 every 3 weeks followed by surgery. Primary endpoint was percent (% reduction in Magnetic Resonance Imaging (MRI estimated Gadolinium (Gd enhancing tumor volume at the end of EndoTAG-1 plus paclitaxel administration as compared to baseline. Safety, pathological complete response (pCR defined as no residual tumor in breast and axillary nodes at surgery and correlation between % reduction in MRI estimated tumor volume and pCR were also evaluated.Fifteen out of 20 scheduled patients were included: Six patients with estrogen receptor (ER-negative/HER2-negative and 9 with ER-positive/HER2-negative BC. Nine patients completed treatment as per protocol. Despite premedication and slow infusion rates, grade 3 hypersensitivity reactions to EndoTAG-1 were observed during the 1st, 2nd, 3rd and 6th weekly infusion in 4 patients, respectively, and required permanent discontinuation of the EndoTAG-1. Moreover, two additional patients stopped EndoTAG-1 plus paclitaxel after 8 and 9 weeks due to clinical disease progression. Two patients had grade 3 increases in transaminases and 1 patient grade 4 neutropenia. pCR was achieved in 5 of the 6 ER-/HER2- and in none of the 9 ER+/HER2- BC patients. The mean % reduction in MRI estimated tumor volume at the end of EndoTAG-1 plus paclitaxel treatment was 81% (95% CI, 66% to 96%, p<0.001 for the 15 patients that underwent surgery; 96% for patients with pCR and 73% for patients with no pCR (p = 0.04.The EndoTAG-1 and paclitaxel combination showed promising preliminary activity as preoperative treatment, especially in ER-/HER2

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

Energy Technology Data Exchange (ETDEWEB)

Manikandan, K. [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bhardwaj, Amit [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ghosh, Amit [Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 (India); Reddy, V. S. [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ramakumar, S., E-mail: ramak@physics.iisc.ernet.in [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 (India)

2005-08-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution.

Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

International Nuclear Information System (INIS)

Manikandan, K.; Bhardwaj, Amit; Ghosh, Amit; Reddy, V. S.; Ramakumar, S.

2005-01-01

A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution

A Sequential Approach in Treatment of Endo-Perio Lesion A Case Report

OpenAIRE

Kambale, Sharanappa; Aspalli, Nagaveni; Munavalli, Anil; Ajgaonkar, Nishant; Babannavar, Roopa

2014-01-01

Endo-perio lesions primarily occur by way of the intimate anatomic and vascular connections between the pulp and the periodontium. Endodontic-periodontal combined lesion is a clinical dilemma because making a differential diagnosis and deciding a prognosis are difficult. An untreated primary endodontic lesion may become secondarily involved with periodontal breakdown, which clinically present unusual signs and symptoms. This may delay the diagnosis and hence the correct treatment. This case r...

Degradation of polysaccharides by endo- and exoenzymes: dextran--dextranase model systems

Energy Technology Data Exchange (ETDEWEB)

Wheatley, M A; Moo-Young, M

1977-02-01

Experiments were carried out on dextran-dextranase systems to test the prediction of a mechanistic model recently proposed by us, for the synergistic effect of combined exo/endo enzymic action in the degradation of polymeric substrates. Soluble forms of the substrate were used. Preliminary experiments with an insoluble form of the substrate were also carried out to demonstrate the applicability of the analytical techniques to these cases. Molecular weight distributions of the degradation products were determined (by gel-permeation chromatography) and the rates of production of glucose and of other reducing sugars were also measured. It was found that the exodextranase alone had very little effect on the molecular weight distributions compared to a significant shift towards lower molecular weights obtained with the endodextranase which was synergistically enhanced by the action of the combined enzymes. Glucose was produced more rapidly by the exoenzyme compared to the endoenzyme, but combinations of the two enzymes gave a rate enhancement greater than the linear sum of the effects of the two individual enzymes. In comparing the degradation indices and polydispersities of the various degradation products, similar synergistic effects of the combined enzymes in accordance with the theoretical predictions were observed. The practical implications of these findings to the design of fermentation processes which depend on the action of endo- and exoenzyme mixtures are noted.

Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

Science.gov (United States)

Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

2009-10-26

The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

Science.gov (United States)

Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

2001-01-01

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

Torsional resistance of XP-endo Shaper at body temperature compared with several nickel-titanium rotary instruments.

Science.gov (United States)

Elnaghy, A M; Elsaka, S E

2018-05-01

To compare the torsional resistance of XP-endo Shaper (XPS; size 30, .01 taper, FKG Dentaire, La Chaux-de-Fonds, Switzerland) instruments at body temperature with TRUShape (TRS; size 30, .06 taper, Dentsply Tulsa Dental Specialties, Tulsa, OK, USA), ProFile Vortex (PV; size 30, .04 taper, Dentsply Tulsa Dental Specialties) and FlexMaster (FM; size 30, .04 taper, VDW GmbH, Munich, Germany) nickel-titanium rotary instruments. A metal block with a square-shaped mould (5 mm × 5 mm × 5 mm) was positioned inside a glass container. Five millimetres of the tip of each instrument was held inside the metal block by filling the mould with a resin composite. The instruments were tested for torsional resistance in saline solution at 37 °C. Data were analysed using one-way analysis of variance (anova) and Tukey post hoc tests. The significance level was set at P instruments tested (P instruments (P = 0.211). The ranking for torsional resistance values was: FM > PV > TRS > XPS. FlexMaster and ProFile Vortex instruments were more resistant to torsional stress compared with TRUShape and XP-endo Shaper instruments. The manufacturing process used to produce XP-endo Shaper instruments did not enhance their resistance to torsional stress as compared with the other instruments. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Development of EndoTOFPET-US, a multi-modal endoscope for ultrasound and time of flight positron emission tomography

International Nuclear Information System (INIS)

Pizzichemi, M

2014-01-01

The EndoTOFPET-US project aims at delevoping a multi-modal imaging device that combines Ultrasound with Time-Of-Flight Positron Emission Tomography into an endoscopic imaging device. The goal is to obtain a coincidence time resolution of about 200 ps FWHM and sub-millimetric spatial resolution for the PET head, integrating the components in a very compact detector suitable for endoscopic use. The scanner will be exploited for the clinical test of new bio-markers especially targeted for prostate and pancreatic cancer as well as for diagnostic and surgical oncology. This paper focuses on the status of the Time-Of-Flight Positron Emission Tomograph under development for the EndoTOFPET-US project

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

OpenAIRE

Davoodi, J.; Wakarchuk, W. W.; Surewicz, W. K.; Carey, P. R.

1998-01-01

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second...

Heterologous humoral immune response in patients treated with human growth hormone from different sources

International Nuclear Information System (INIS)

Cardoso, A.I.; Llera, A.S.; Iacono, R.F.

1993-01-01

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [ 125 I]hGH or [ 125 I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[ 125 I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab

Heterologous humoral immune response in patients treated with human growth hormone from different sources

Energy Technology Data Exchange (ETDEWEB)

Cardoso, A.I.; Llera, A.S.; Iacono, R.F. (and others) (Inst. de Estudios de la Inmunidad Humoral, Buenos Aires (Argentina))

1993-07-01

The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [[sup 125]I]hGH or [[sup 125]I]bGH as tracers. 57 hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[[sup 125]I]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response. 16 refs., 4 figs., 1 tab.

Effects of diet acidification and xylanase supplementation on performance, nutrient digestibility, duodenal histology and gut microflora of broilers fed wheat based diet

NARCIS (Netherlands)

Esmaeilipour, O.; Moravej, H.; Shivazad, M.; Rezaian, M.; Aminzadeh, S.; Krimpen, van M.M.

2012-01-01

1. The objective of this experiment was to study the influences of xylanase and citric acid on the performance, nutrient digestibility, digesta viscosity, duodenal histology, and gut microflora of broilers fed on a wheat based diet. 2. The experiment was carried out as a 2 x 3 factorial arrangement

Structural basis and catalytic mechanism for the dual functional endo-beta-N-acetylglucosaminidase A.

Directory of Open Access Journals (Sweden)

Jie Yin

Full Text Available Endo-beta-N-acetylglucosaminidases (ENGases are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A in 3 forms, one in native form, one in complex with Man(3GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues - E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as "gate-keepers". Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.

Enzymatic hydrolysis of pretreated Alfa fibers (Stipa tenacissima) using β-d-glucosidase and xylanase of Talaromyces thermophilus from solid-state fermentation.

Science.gov (United States)

Mallek-Fakhfakh, Hanen; Fakhfakh, Jawhar; Walha, Kamel; Hassairi, Hajer; Gargouri, Ali; Belghith, Hafedh

2017-10-01

This work aims at realizing an optimal hydrolysis of pretreated Alfa fibers (Stipa tenacissima) through the use of enzymes produced from Talaromyces thermophilus AX4, namely β-d-glucosidase and xylanase, by a solid state fermentation process of an agro-industrial waste (wheat bran supplemented with lactose). The carbon source was firstly selected and the optimal values of three other parameters were determined: substrate loading (10g), moisture content (85%) and production time (10days); which led to an optimized enzymatic juice. The outcome was then supplemented with cellulases of T. reesei and used to optimize the enzymatic saccharification of alkali-pretreated Alfa fibers (PAF). The maximum saccharification yield of 83.23% was achieved under optimized conditions (substrate concentration 3.7% (w/v), time 144h and enzyme loading of 0.8 FPU, 15U CMCase, 60U β-d-glucosidase and 125U xylanase).The structural modification of PAF due to enzymatic saccharification was supported by the changes of morphologic and chemical composition observed through macroscopic representation, FTIR and X-Ray analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Fodder radish cake (Raphanus sativus L. as an alternative biomass for the production of cellulases and xylanases in solid-state cultivation

Directory of Open Access Journals (Sweden)

L. Zukovski

Full Text Available Abstract Fodder radish (FR is an oilseed crop with a high potential for biodiesel production due to its high productivity and the quality of its seed oil. FR oil extraction results in a residue that is rich in protein and fiber. In this study, FR cake (FRC was evaluated as carbon and nitrogen source for the production of cellulases and xylanases using Penicillium echinulatum S1M29 during solid-state cultivation. It was determined that it is possible to partially replace wheat bran (WB by FRC, resulting in 24.22 ± 0.25U/g Filter Paper Activity (144 hours, 210.5 ± 5.8U/g endoglucanase activity (144 hours, 22.62 ± 0.01U/g (-glucosidase activity (96 hours and 784.7 ± 70.19U/g xylanase activity (120 hours. These values are equal or higher than the enzymatic activity obtained using WB. These results may contribute to the reduction of the cost of enzymes used in the production of cellulosic ethanol or other biotechnological applications.

Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

DEFF Research Database (Denmark)

2016-01-01

A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

Longitudinal study of experimental induction of AA amyloidosis in mice seeded with homologous and heterologous AA fibrils.

Science.gov (United States)

Muhammad, Naeem; Murakami, Tomoaki; Inoshima, Yasuo; Ishiguro, Naotaka

2016-09-01

To investigate pathogenesis and kinetics of experimentally induced murine AA amyloidosis seeded with homologous (murine) and heterologous (bovine) AA fibrils. Experimental AA amyloidosis was induced by administration of inflammatory stimulus and preformed AA fibrils to a total of 111 female C57/Black mice. In this longitudinal study, heterologous (bovine) as well as homologous (murine) AA fibrils were injected intraperitoneally to mice in various combinations. Re-stimulation was done at 120 or 300 days post first inoculation. To analyze the intensity of amyloid depositions in mice organs, immunohistochemical techniques and image J software were used. Assessment of cytokines level in sera was done using a Mouse Th1/Th2/Th17 Cytokine CBA Kit. Incidence and severity of AA amyloidosis were quite low in mice inoculated with heterologous bovine AA fibrils than homologous murine one. Homologous AA fibrils administration at first and second inoculation caused maximum amount of amyloid depositions and severe systemic form of amyloidosis. Increase in the level of pro-inflammatory cytokine IL-6 was observed after first inoculation, while second inoculation caused a further increase in the level of anti-inflammatory cytokine IL-10. AA amyloidosis can be induced by heterologous as well as homologous AA fibrils. Severity of AA amyloidosis induced with homologous AA fibrils is higher compared to heterologous AA fibrils.

Glycan analysis of recombinant Aspergillus niger endo-polygalacturonase A.

Science.gov (United States)

Woosley, Bryan D; Kim, Young Hwan; Kumar Kolli, V S; Wells, Lance; King, Dan; Poe, Ryan; Orlando, Ron; Bergmann, Carl

2006-10-16

The enzyme endo-polygalacturonase A, or PGA, is produced by the fungus, Aspergillus niger, and appears to play a critical role during invasion of plant cell walls. The enzyme has been homologously overexpressed in order to provide sufficient quantities of purified enzyme for structural and biological studies. We have characterized this enzyme in terms of its post-translational modifications (PTMs) and found it to be both N- and O-glycosylated. Additionally, we have characterized the glycosyl moieties using MALDI-TOF and LC-ESI mass spectrometry. The characterization of all PTMs on PGA, along with molecular modeling, allows us to reveal potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

Science.gov (United States)

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-01-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

Heterologous surface display on lactic acid bacteria: non-GMO alternative?

Science.gov (United States)

Zadravec, Petra; Štrukelj, Borut; Berlec, Aleš

2015-01-01

Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

Purification and characterization of a novel endo-β-1,4-glucanases , AfEG22, from the giant snail, Achatina fulica frussac.

Science.gov (United States)

Teng, Yigang; Yin, Qiuyu; ding, Ming; Zhao, Fukun

2010-10-01

In this study, we confirmed that at least three endo-β-1,4-glucanases existed in the digestive juice of the giant snail, Achatina fulica ferussac, by Congo red staining assay. One of these enzymes, a novel endo-β-1,4-glucanase (AfEG22), was purified 29.5-fold by gel filtration, anion exchange, and hydrophobic interaction chromatography. The carboxymethyl cellulose (CMC) hydrolytic activity of the purified enzyme was 12.3 U/mg protein. The molecular mass of AfEG22 was 22081 Da determined by MALDI-TOF. N-terminal amino acid sequencing revealed a sequence of EQRCTNQGGILKYYNT, which did not have significant homology with any proteins in BLAST database. The optimal pH and temperature for hydrolytic activity toward CMC were pH 4.0 and 50°C, respectively. AfEG22 was stable between pH 3.0 and pH 12.0 when incubated at 4°C for 3 h or at 37°C for 1 h. The enzyme remained more than 80% activity between pH 4.5 and pH 7.0 after incubation at 50°C for 1 h. AfEG22 possessed excellent thermostability as more than 70% activity was remained after incubation at 60°C for 3 h. Substrate specific analysis revealed that AfEG22 was a typical endo-β-1,4-glucanase. This is the first time to report a novel endo-β-1,4-glucanase with high stability from the digestive juice of A. fulica.

Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

Science.gov (United States)

Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

2016-10-01

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

Gene cloning and enzymatic characterization of an alkali-tolerant endo-1,4-β-mannanase from Rhizomucor miehei.

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Katrolia, Priti; Yan, Qiaojuan; Zhang, Pan; Zhou, Peng; Yang, Shaoqing; Jiang, Zhengqiang

2013-01-16

An endo-1,4-β-mannanase gene (RmMan5A) was cloned from the thermophilic fungus Rhizomucor miehei for the first time and expressed in Escherichia coli . The gene had an open reading frame of 1330 bp encoding 378 amino acids and contained four introns. It displayed the highest amino acid sequence identity (42%) with the endo-1,4-β-mannanases from glycoside hydrolase family 5. The purified enzyme was a monomer of 43 kDa. RmMan5A displayed maximum activity at 55 °C and an optimal pH of 7.0. It was thermostable up to 55 °C and alkali-tolerant, displaying excellent stability over a broad pH range of 4.0-10.0, when incubated for 30 min without substrate. The enzyme displayed the highest specificity for locust bean gum (K(m) = 3.78 mg mL⁻¹), followed by guar gum (K(m) = 7.75 mg mL⁻¹) and konjac powder (K(m) = 22.7 mg mL⁻¹). RmMan5A hydrolyzed locust bean gum and konjac powder yielding mannobiose, mannotriose, and a mixture of various mannose-linked oligosaccharides. It was confirmed to be a true endo-acting β-1,4-mannanase, which showed requirement of four mannose residues for hydrolysis, and was also capable of catalyzing transglycosylation reactions. These properties make RmMan5A highly useful in the food/feed, paper and pulp, and detergent industries.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

Science.gov (United States)

Heshof, Ruud; van Schayck, J Paul; Tamayo-Ramos, Juan Antonio; de Graaff, Leo H

2014-01-01

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

Prediction of ingredient quality and the effect of a combination of xylanase, amylase, protease and phytase in the diets of broiler chicks. 2. Energy and nutrient utilisation.

Science.gov (United States)

Cowieson, A J; Singh, D N; Adeola, O

2006-08-01

1. In order to investigate the effects of xylanase, amylase, protease and phytase in the diets of broiler chickens containing graded concentrations of metabolisable energy (ME), two 42-d experiments were conducted using a total of 2208 broiler chicks (8 treatments with 12 replicate pens in each experiment). 2. Four diets including one positive and three negative control diets were used. Three maize/soybean meal-based negative control (NC) diets were formulated to be identical in available phosphorus (P), calcium (Ca) and amino acids but NC1 contained approximately 0.17 MJ/kg less ME than NC2 and approximately 0.34 MJ/kg less ME than NC3. A positive control (PC) was fed for comparison and was formulated to be adequate in all nutrients, providing approximately 0.63 MJ/kg ME, 0.13% available P, 0.12% Ca and 1 to 2% amino acids more than NC1. 3. The reduction in nutrient density between NC1 and PC was determined using ingredient quality models Avichecktrade mark Corn and Phychecktrade mark that can predict the response to exogenous enzymes in maize/soybean meal-based broiler diets. Supplementation of each diet with or without a cocktail of xylanase, amylase, protease and phytase gave a total of 8 dietary treatments in a 4 x 2 factorial arrangement. The same treatments and diet designs were used in both experiments but conducted in different locations using different batches of maize, soybean meal and minor ingredients. 4. In both experiments, digestibility was improved by the addition of exogenous enzymes, particularly those for P, Ca and certain amino acids. In addition, the supplementation of the PC with enzymes elicited a positive response indicating that over-the-top addition of xylanase, amylase, protease and phytase may offer a nutritionally and economically viable alternative to feed cost reduction. 5. It can be concluded that the digestibility of nutrients by broilers fed on maize/soybean meal-based diets can be improved by the use of a combination of xylanase

Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions.

Science.gov (United States)

Rucker, Joanna; Paul, Julie; Pfeifer, Blaine A; Lee, Kyongbum

2013-03-01

Triglycerides, traditionally sourced from plant oils, are heavily used in both industrial and healthcare applications. Commercially significant products produced from triglycerides include biodiesel, lubricants, moisturizers, and oils for cooking and dietary supplements. The need to rely upon plant-based production, however, raises concerns of increasing demand and sustainability. The reliance on crop yields and a strong demand for triglycerides provides motivation to engineer production from a robust microbial platform. In this study, Escherichia coli was engineered to synthesize and accumulate triglycerides. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). A liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze the production of triglycerides by the engineered E. coli strains. This proof-of-concept study demonstrated a yield of 1.1 mg/L triglycerides (2 g/L dry cell weight) in lysogeny broth medium containing 5 g/L glucose at 8 h following induction of PAP and DGAT expression. LC-MS results also demonstrated that the intracellular triglyceride composition of E. coli was highly conserved. Triglycerides containing the fatty acid distributions 16:0/16:0/16:1, 16:0/16:0/18:1, and 18:1/16:0/16:1 were found in highest concentrations and represent ∼70 % of triglycerides observed.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

Science.gov (United States)

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Endovascular Treatment of Descending Thoracic Aortic Aneurysms with the EndoFit Stent-Graft

International Nuclear Information System (INIS)

Saratzis, N.; Saratzis, Athanasios; Melas, N.; Ginis, G.; Lioupis, A.; Lykopoulos, D.; Lazaridis, J.; Kiskinis, Dimitrios

2007-01-01

Objective. To evaluate the mid-term feasibility, efficacy, and durability of descending thoracic aortic aneurysm (DTAA) exclusion using the EndoFit device (LeMaitre Vascular). Methods. Twenty-three (23) men (mean age 66 years) with a DTAA were admitted to our department for endovascular repair (21 were ASA III+ and 2 refused open repair) from January 2003 to July 2005. Results. Complete aneurysm exclusion was feasible in all subjects (100% technical success). The median follow-up was 18 months (range 8-40 months). A single stent-graft was used in 6 cases. The deployment of a second stent-graft was required in the remaining 17 patients. All endografts were attached proximally, beyond the left subclavian artery, leaving the aortic arch branches intact. No procedure-related deaths have occurred. A distal type I endoleak was detected in 2 cases on the 1 month follow-up CT scan, and was repaired with reintervention and deployment of an extension graft. A nonfatal acute myocardial infarction occurred in 1 patient in the sixth postoperative month. Graft migration, graft infection, paraplegia, cerebral or distal embolization, renal impairment or any other major complications were not observed. Conclusion. The treatment of DTAAs using the EndoFit stent-graft is technically feasible. Mid-term results in this series are promising

Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

Science.gov (United States)

Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2016-11-01

Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

endo-3,3-Dimethyl-4-oxobicyclo[3.1.0]hexan-2-yl methanesulfonate

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Adrian Kremer

2010-04-01

Full Text Available The relative configuration of the endo isomer of the title compound, C9H14O4S, has been established and the conformation of the diastereoisomer is discussed. The five-membered ring adopts an envelope conformation. The conformation of the methanesulfonate substituent is stabilized by intermolecular C—H...O hydrogen bonds. The crystal packing results in alternating layers of polar methanesulfonates and stacked bicyclohexanyl rings parallel to ab.

Heterologous expression of AtMYB12 in kale (Brassica oleracea var. acephala) leads to high flavonol accumulation.

Science.gov (United States)

Lännenpää, Mika

2014-08-01

Overexpression of Arabidopsis AtMYB12 transcription factor greatly increases the total phenolic and flavonol content in transgenic kale leaves. Flavonoids are a diverse group of plant secondary metabolites exhibiting a number of health-promoting effects. There has been a growing interest to develop biotechnological methods for the enhanced production of flavonoids in crop plants. AtMYB12 is an Arabidopsis transcription factor which specifically activates flavonol synthesis and its overexpression has led to increased flavonol accumulation in several transgenic plants. In the present study, AtMYB12 was overexpressed in a commercial cultivar of kale and the transgenic plants were tested both in in vitro and in semi-field conditions in cages under natural light. Using this method, a severalfold increase in both total phenolics content and flavonol accumulation was achieved. This study provides a reliable and efficient transformation protocol for kale and suggests the potential of this flavonol-enriched vegetable for the production of kaempferol.

Heterologous gene expression in filamentous fungi.

Science.gov (United States)

Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

2012-01-01

Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

DEFF Research Database (Denmark)

Murphy, Leigh; Cruys-Bagger, Nicolaj; Baumann, Martin J.

2012-01-01

by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B...

The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells.

Science.gov (United States)

Jeffery, N; Richardson, S; Beall, C; Harries, L W

2017-12-15

Interaction between islet cell subtypes and the extracellular matrix influences beta-cell function in mammals. The tissue architecture of rodent islets is very different to that of human islets; cell-to-cell communication and interaction with the extracellular matrix may vary between species. In this work, we have compared the responses of the human EndoC-βH1 cell line to non-human and human-derived growth matrices in terms of growth morphology, gene expression and glucose-stimulated insulin secretion (GSIS). EndoC-βH1 cells demonstrated a greater tendency to form cell clusters when cultured in a human microenvironment and exhibited reduced alpha cell markers at the mRNA level; mean expression difference - 0.23 and - 0.51; p = 0.009 and 0.002 for the Aristaless-related homeobox (ARX) and Glucagon (GCG) genes respectively. No differences were noted in the protein expression of mature beta cell markers such as Pdx1 and NeuroD1 were noted in EndoC-βH1 cells grown in a human microenvironment but cells were however more sensitive to glucose (4.3-fold increase in insulin secretion following glucose challenge compared with a 1.9-fold increase in cells grown in a non-human microenvironment; p = 0.0003). Our data suggests that the tissue origin of the cellular microenvironment has effects on the function of EndoC-βH1 cells in vitro, and the use of a more human-like culture microenvironment may bring benefits in terms of increased physiological relevance. Copyright © 2017 Elsevier Inc. All rights reserved.

A one-step bioprocess for production of high-content fructo-oligosaccharides from inulin by yeast.

Science.gov (United States)

Wang, Da; Li, Fu-Li; Wang, Shi-An

2016-10-20

Commercial fructo-oligosaccharides (FOS) are predominantly produced from sucrose by transfructosylation process that presents a maximum theoretical yield below 0.60gFOSgSucrose(-1). To obtain high-content FOS, costly purification is generally employed. Additionally, high-content FOS can be produced from inulin by using endo-inulinases. However, commercial endo-inulinases have not been extensively used in scale-up production of FOS. In the present study, a one-step bioprocess that integrated endo-inulinase production, FOS fermentation, and non-FOS sugars removal into one reactor was proposed to produce high-content FOS from inulin. The bioprocess was implemented by a recombinant yeast strain JZHΔS-TSC, in which a heterologous endo-inulinase gene was expressed and the inherent invertase gene SUC2 was disrupted. FOS fermentation at 40°C from 200g/L chicory inulin presented the maximun titer, yield, and productivity of 180.2±0.8g/L, 0.9gFOSgInulin(-1), and 7.51±0.03g/L/h, respectively. This study demonstrated that the one-step bioprocess was simple and highly efficient. Copyright © 2016 Elsevier Ltd. All rights reserved.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

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Olga A. Kladova

2018-03-01

Full Text Available Endonuclease III (Endo III or Nth is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO, a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C. Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

Science.gov (United States)

Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

2005-10-01

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Science.gov (United States)

Badino, Silke F; Christensen, Stefan J; Kari, Jeppe; Windahl, Michael S; Hvidt, Søren; Borch, Kim; Westh, Peter

2017-08-01

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 10 3 -10 4 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

Science.gov (United States)

Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

Science.gov (United States)

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Malignant phyllodes tumor of the breast with heterologous high-grade angiosarcoma

Directory of Open Access Journals (Sweden)

Ghassan Tranesh

2017-03-01

Full Text Available Phyllodes tumors (PTs account for <3% of fibroepithelial breast lesions and for 0.3% to 1.0% of primary breast tumors. They occur predominantly in middle-aged women (mean age range, 40–50 years. PTs can be categorized into benign, borderline, and malignant; the first 2 categories are distinguished only by degree of cellular atypia and mitotic activity. Malignant PTs are more frequent among persons of Hispanic ethnicity, especially those born in Central America or South America. Heterologous sarcomatous elements may be present in malignant PTs, predominantly liposarcoma and rarely fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma, and chondrosarcoma. Breast angiosarcoma (BA is a rare heterologous, sarcomatous element that may arise secondary to malignant PT. We report a 47-year-old woman with no history of previous surgery or radiation therapy who presented to the emergency department with a painful right breast mass. She admittedly noticed the right breast mass for many years; however, recently it increased in size. Mammography and ultrasonography identified a partially cystic mass. Core needle biopsy showed dense hyalinized fibrous tissue with old blood clots, suggestive of infarcted fibroadenoma. The patient received antibiotics and analgesics; however, she reported intractable pain and a worsening skin rash of her right breast. Chest computed tomography and magnetic resonance imaging showed a doubling in mass size, with pectoralis major muscle involvement. Incisional biopsy showed malignant PT with heterologous high-grade angiosarcoma. The diagnosis of angiosarcoma was confirmed through immunoreactivity for CD31, FLI1, and ERG immunostains.

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.

Science.gov (United States)

Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

2011-09-01

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

Science.gov (United States)

Katoch, Meenu; Mazmouz, Rabia; Chau, Rocky; Pearson, Leanne A.; Pickford, Russell

2016-01-01

ABSTRACT Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens. PMID:27520810

Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

Science.gov (United States)

Li, X Y; Liu, F; Hu, Y F; Xia, M; Cheng, B J; Zhu, S W; Ma, Q

2015-12-21

The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion.

Comparative evaluation of debris removal from root canal wall by using EndoVac and conventional needle irrigation: An in vitro study

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Vandana J Gade

2013-01-01

Full Text Available Context: Microbial control is of paramount importance in Clinical Endodontics. Therefore, cleaning and disinfection of root canals are essential to achieve endodontic success. Aims: The purpose of this study is to compare the efficacy of EndoVac irrigation system and conventional needle (30 gauges side venting needle irrigation for removal of debris from the root canal walls at coronal, middle and apical third by using the scanning electron microscopy (SEM. Settings and Design: An in vitro randomized control trial study. Materials and Methods: A total of 20 mandibular premolars with completely formed roots were selected and randomly divided into two groups - Group 1: Irrigation with the Conventional system and Group 2: EndoVac irrigation. After access opening and working length determination biomechanical preparation completed up to a rotary protaper F4 file. Groupwise irrigation with sodium hypochlorite and ethylenediaminetetraacetic acid was done with each canal in between instrumentation. Then, the teeth were sectioned in buccolingual direction and the halves were sputter-coated with gold palladium and coronal, middle and apical third were examined by SEM at x2000 magnification. Statistical Analysis: Mann-Whitney test for comparison between methods, Kruskal-Wallis test for comparison among thirds and Miller test for individual comparisons. Results: The apical, middle and cervical root canal thirds were evaluated and the results were analyzed statistically by the Mann-Whitney test for comparison between methods, Kruskal-Wallis test for comparison among thirds and Miller test for individual comparisons. Conclusions: EndoVac group resulted in significantly less debris at apical third compared with the conventional needle irrigation group. There was no statistical significant difference found in debris removal at coronal and middle third of root canal wall between the EndoVac group and conventional needle irrigation group.

Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

DEFF Research Database (Denmark)

Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe

2007-01-01

in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis......The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...

Effects of xylanase and citric acid on the performance, nutrient retention, and characteristics of gastrointestinal tract of broilers fed low-phosphorus wheat-based diets

NARCIS (Netherlands)

Esmaeilipour, O.; Shivazad, M.; Moravej, H.; Aminzadeh, S.; Rezaian, M.; Krimpen, van M.M.

2011-01-01

An experiment was conducted to study the effects of xylanase and citric acid on the performance, nutrient retention, jejunal viscosity, and size and pH of the gastrointestinal tract of broilers fed a low-P wheat-based diet. The experiment was conducted as a 2 × 3 factorial arrangement with 2 levels

Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophyll a-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, in Nicotiana sp.*

Science.gov (United States)

Pinnola, Alberta; Ghin, Leonardo; Gecchele, Elisa; Merlin, Matilde; Alboresi, Alessandro; Avesani, Linda; Pezzotti, Mario; Capaldi, Stefano; Cazzaniga, Stefano; Bassi, Roberto

2015-01-01

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called non-photochemical quenching, occurs within the photosystem II antenna system by the action of two essential light-harvesting complex (LHC)-like proteins, photosystem II subunit S (PSBS) in plants and light-harvesting complex stress-related (LHCSR) in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, which are present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems Nicotiana benthamiana and Nicotiana tabacum using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes, and is correctly folded with chlorophyll a and xanthophylls but without chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo, implying that the recombinant protein obtained is a good material for future structural and functional studies. PMID:26260788

THE INFLUENCE OF ASOCIATED SUPPLEMENT OF ALFA AMYLASE AND XYLANASE ON THE RHEOLOGY OF DOUGH CONCEARNING ITS CONSTITOGRAPHICAL PARAMETERS

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RODICA CHEREJI

2009-05-01

Full Text Available In this paper we determined the influence of associated supplement of alfa amylase and xylanase on the rheology of dough concearning its constitographical parameters : maximum pressure (Pr max, (mb and the absorbed water (Wa, %. The analysis on the consistograph were conducted for constant hydration at the consistency of 500 UF. Determinations were made on 4 types of flour and optimal dosages were found for each enzyme, after which we prepared the optimal dosage of the enzymes in the compund for flour F1 and F2 : P1-840000 U. SKB/100 kg flour +8100 U. FXU /100 kg flour, P2-840000 U. SKB/100 kg flour+16200 U. FXU, /100 kg flour , P3-840000 U. SKB/100 kg flour+24300 U. FXU/100 kg flour , and for F3 and F4 thus: P1-280000 U. SKB/100 kg flour +8100 U. FXU /100 kg flour, P2- 280000 U. SKB/100 kg flour+16200 U. FXU/100 kg flour, P3-280000 U. SKB/100 kg flour+24300 U. FXU/100 kg flour. Fungous α-amylase and xylanase were used in these concentrations to establish which one is more apropriate to be added in flour to obtain superior quality of bread: finer texture of the crumb, prolongation of the freashness of the bread, improvind the colour and flavour, emproving the slicing ability.

Midterm outcome of EndoAnchors for the prevention of endoleak and stent-graft migration in patients with challenging proximal aortic neck anatomy.

Science.gov (United States)

Jordan, William D; de Vries, Jean-Paul P M; Ouriel, Kenneth; Mehta, Manish; Varnagy, David; Moore, William M; Arko, Frank R; Joye, James; Henretta, John

2015-04-01

To explore the use of EndoAnchors as an adjunct to endovascular abdominal aortic aneurysm repair for prevention of proximal neck complications in patients with challenging neck anatomy. Over a 28-month period, 208 patients (159 men; mean age 72±8 years) were enrolled in the ANCHOR prospective, multicenter registry (ClinicalTrials.gov; identifier NCT01534819) for prophylaxis against proximal neck complications. Patients were eligible when, in the opinion of the investigators, they were at increased risk for type Ia endoleak or migration owing to a hostile neck (length 28 mm, angulation >60°, mural thrombus or calcium >2 mm in thickness or >180° in circumference, or conical shape). Overall, 123/157 (78.3%) patients met the criteria for a hostile neck according to core laboratory assessment of 157 adequate preoperative computed tomographic (CT) images. Implantation of EndoAnchors was technically successful in 204/208 (98.1%) patients. The frequency of fracture was 0.3% (3/1118); there were no clinical sequelae associated with the fractures. Over the mean 14-month follow-up, 95.2% of patients were alive, and no deaths were attributable to EndoAnchors. There were no ruptures, migrations, or open surgical conversions. Aneurysm-related reinterventions were performed in 8 (3.8%) patients. Among 130 patients with postprocedure contrast CT studies, core laboratory analysis identified 2 (1.5%) patients with type Ia endoleaks. Aneurysm sac diameter decreased >5 mm in 42.9% of patients with CT scans at or beyond 1 year; 1.6% of patients developed sac enlargement >5 mm. Prophylactic EndoAnchor use for challenging aortic neck anatomy was associated with satisfactory midterm results. © The Author(s) 2015.

New measures of upper esophageal sphincter distensibility and opening patterns during swallowing in healthy subjects using EndoFLIP®

LENUS (Irish Health Repository)

Regan, J

2013-01-01

Background  This paper aims to measure upper esophageal sphincter (UES) distensibility and extent and duration of UES opening during swallowing in healthy subjects using EndoFLIP(®) . Methods  Fourteen healthy subjects (20-50 years) were recruited. An EndoFLIP(®) probe was passed trans-orally and the probe balloon was positioned across the UES. Two 20-mL ramp distensions were completed and UES cross-sectional area (CSA) and intra-balloon pressure (IBP) were evaluated. At 12-mL balloon volume, subjects completed dry, 5- and 10-mL liquid swallows and extent (mm) and duration (s) of UES opening and minimum IBP (mmHg) were analyzed across swallows. Key Results  Thirteen subjects completed the study protocol. A significant change in UES CSA (P < .001) and IBP (P < .000) was observed during 20-mL distension. UES CSA increased up to 10-mL distension (P < .001), from which point IBP raised significantly (P = 0.004). There were significant changes in UES diameter (mm) (P < .000) and minimum IBP (mmHg) (P < .000) during swallowing events. Resting UES diameter (4.9 mm; IQR 0.02) and minimum IBP (18.8 mmHg; IQR 2.64) changed significantly during dry (9.6 mm; IQR 1.3: P < .001) (3.6 mmHg; IQR 4.1: P = 0.002); 5 mL (8.61 mm; IQR 2.7: P < .001) (4.8 mmHg; IQR 5.7: P < .001) and 10-mL swallows (8.3 mm; IQR 1.6: P < 0.001) (3 mmHg; 4.6: P < .001). Median duration of UES opening was 0.5 s across dry and liquid swallows (P = 0.91). Color contour plots of EndoFLIP(®) data capture novel information regarding pharyngo-esophageal events during swallowing. Conclusions & Inferences  Authors obtained three different types of quantitative data (CSA, IBP, and timing) regarding UES distensibility and UES opening patterns during swallowing in healthy adults using only one device (EndoFLIP(®) ). This new measure of swallowing offers fresh information regarding UES dynamics which may ultimately improve patient

Xylanase Production from Trichoderma harzianum 1073 D3 with Alternative Carbon and Nitrogen Sources

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Isil Seyis

2005-01-01

Full Text Available The effect of some natural wastes (orange pomace, orange peel, lemon pomace, lemon peel, apple pomace, pear peel, banana peel, melon peel and hazelnut shell on the production of xylanase from Trichoderma harzianum 1073 D3 has been studied and maximum activity has been observed on melon peel (26.5 U/mg of protein followed by apple pomace and hazelnut shell. Also, molasses could be used as an additional carbon source as it decreased the production time approximately by 50 %. Finally, potential alternatives of organic nitrogen source (cotton leaf and soybean residue wastes were analyzed and it was concluded that peptone could be replaced with these residues especially when economics of the process is the major objective.

Effect of penicillium mutation by UV and gamma radiation on xylanase production

International Nuclear Information System (INIS)

Bakri, Y.; Shamma, M.; Hammoudeh, A.; Sharabi, N.

2007-07-01

Many microorganisms produce enzymes which have importance in industrial processes. Usually this production, is not sufficient for these needs at economical level. The bioindustry concentrates on increasing the production of these enzymes. This leads to the progress of this kind of industry, which use different biotechnology means, for example mutation and screening to choice more potent strain. In this study Ultra Violet and Gamma irradiation conducted on Penicillium canescen in order to produce new mutant strains, have the ability to produce more xylanase enzyme for industrial uses. Ultra Violet irradiation enable to select five mutant strains having more enzyme production ability. The best mutant strain PCUV12 (159 unit/ml) was 40% higher than the mother strain, at the dose 150.72 j/cm 2 . Gamma radiation produced new mutant strain PCGR6 which produced 26% more enzyme than the mother strain at dose 250 Gy.(author)

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates

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Violeta Georgieva Tsonkova

2018-02-01

Full Text Available Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins.Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate.By screening of various proteins and peptides, we found Bombesin (BB receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation.ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated

Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy

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Song Letian

2012-01-01

Full Text Available Abstract Background Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second-generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn. Results Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilized arabinoxylans that were not modified by the parental enzyme. The most active mutants, S27T and Y111T, increased the solubilization of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold, respectively, in comparison to the wild-type enzyme. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%tot. xyl (wild-type Tx-Xyn to 18.6% to 20.4%tot. xyl. Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500, thus procuring increases in overall wheat straw hydrolysis. Conclusions Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to (i improved ligand binding in a putative secondary binding site, (ii the diminution of surface hydrophobicity, and/or (iii the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organization of the plant cell wall and the lignin barrier.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production

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Paul N. Schwarz

2018-03-01

Full Text Available The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

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Alderete JF

2005-03-01

Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Surgical planning of total hip arthroplasty: accuracy of computer-assisted EndoMap software in predicting component size

International Nuclear Information System (INIS)

Davila, Jesse A.; Kransdorf, Mark J.; Duffy, Gavan P.

2006-01-01

The purpose of our study was to assess the accuracy of a computer-assisted templating in the surgical planning of patients undergoing total hip arthroplasty utilizing EndoMap software (Siemans AG, Medical Solutions, Erlangen, Germany). Endomap Software is an electronic program that uses DICOM images to analyze standard anteroposterior radiographs for determination of optimal prosthesis component size. We retrospectively reviewed the preoperative radiographs of 36 patients undergoing uncomplicated primary total hip arthroplasty, utilizing EndoMap software, Version VA20. DICOM anteroposterior radiographs were analyzed using standard manufacturer supplied electronic templates to determine acetabular and femoral component sizes. No additional clinical information was reviewed. Acetabular and femoral component sizes were assessed by an orthopedic surgeon and two radiologists. Mean and estimated component size was compared with component size as documented in operative reports. The mean estimated acetabular component size was 53 mm (range 48-60 mm), 1 mm larger than the mean implanted size of 52 mm (range 48-62 mm). Thirty-one of 36 acetabular component sizes (86%) were accurate within one size. The mean calculated femoral component size was 4 (range 2-7), 1 size smaller than the actual mean component size of 5 (range 2-9). Twenty-six of 36 femoral component sizes (72%) were accurate within one size, and accurate within two sizes in all but four cases (94%). EndoMap Software predicted femoral component size well, with 72% within one component size of that used, and 94% within two sizes. Acetabular component size was predicted slightly better with 86% within one component size and 94% within two component sizes. (orig.)

Management of an endo-perio lesion in an immature tooth using autologous platelet-rich fibrin: a case report.

Science.gov (United States)

Nagaveni, N B; Kumari, K Nandini; Poornima, P; Reddy, V V Subba

2015-01-01

Treatment of an endo-perio lesion involving a non-vital young permanent tooth is a highly challenging task to Pediatric Dentists. There is a quest for the newer biological approach to management of these lesions as traditional methods have various disadvantages. Recently, platelet-rich fibrin (PRF), a second-generation platelet concentrate, is rich in growth factors have been used in the periodontal regeneration procedure. The purpose of this paper is to describe the efficacy of PRF in the treatment of a deep intra bony defect associated with an endo-perio lesion in an immature right mandibular first premolar of 12-year-old female patient. A freshly prepared autologous PRF membrane was placed in the bony defect following debridement. Clinical and radiographic follow-up were performed at regular intervals that revealed absence of pain, gain in clinical attachment level, reduction in probing depth, and excellent bone regeneration indicating successful outcome.

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification.

Science.gov (United States)

de Cassia Pereira, J; Paganini Marques, N; Rodrigues, A; Brito de Oliveira, T; Boscolo, M; da Silva, R; Gomes, E; Bocchini Martins, D A

2015-04-01

To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification. Thirty-two heat-tolerant fungi were isolated from the environment, identified (morphological/molecular tools) and the production of the enzymes was evaluated by solid state fermentation using lignocellulosic materials as substrates. Myceliophthora thermophila JCP 1-4 was the best producer of endoglucanase (357·51 U g(-1) ), β-glucosidase (45·42 U g(-1) ), xylanase (931·11 U g(-1) ) and avicelase (3·58 U g(-1) ). These enzymes were most active at 55-70°C and stable at 30-60°C. Using crude enzymatic extract from M. thermophila JCP 1-4 to saccharify sugarcane bagasse pretreated with microwaves and glycerol, glucose and xylose yields obtained were 15·6 and 35·13% (2·2 and 1·95 g l(-1) ), respectively. All isolated fungi have potential to produce the enzymes; M. thermophila JCP 1-4 enzymatic extract have potential to be better explored in saccharification experiments. Pretreatment improved enzymatic saccharification, as sugar yields were much higher than those obtained from in natura bagasse. Myceliophthora thermophila JCP 1-4 produces avicelase (not commonly found among fungi; important to hydrolyse crystalline cellulose) and a β-glucosidase resistant to glucose inhibition, interesting characteristics for saccharification experiments. © 2015 The Society for Applied Microbiology.

Structure-based domain assignment in Leishmania infantum EndoG: characterization of a pH-dependent regulatory switch and a C-terminal extension that largely dictates DNA substrate preferences.

Science.gov (United States)

Oliva, Cristina; Sánchez-Murcia, Pedro A; Rico, Eva; Bravo, Ana; Menéndez, Margarita; Gago, Federico; Jiménez-Ruiz, Antonio

2017-09-06

Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145-180 is indeed far more active than its wild-type counterpart at pH values >7.0. In addition, we discovered that (i) LiEndoG exists as a homodimer, (ii) replacement of Ser211 in the active-site SRGH motif with the canonical aspartate from the DRGH motif of other nucleases leads to a catalytically deficient enzyme, (iii) the activity of the S211D variant can be restored upon the concomitant replacement of Ala247 with Arg and (iv) a C-terminal extension is responsible for the observed preferential cleavage of single-stranded DNA (ssDNA) and ssDNA-dsDNA junctions. Taken together, our results support the view that LiEndoG is a multidomain molecular machine whose nuclease activity can be subtly modulated or even abrogated through architectural changes brought about by environmental conditions and interaction with other binding partners. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

Science.gov (United States)

Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

2015-03-27

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

Heterologous Prime-Boost Immunizations with a Virosomal and an Alphavirus Replicon Vaccine

NARCIS (Netherlands)

Walczak, Mateusz; de Mare, Arjan; Riezebos-Brilman, Annelies; Regts, Joke; Hoogeboom, Baukje-Nynke; Visser, Jeroen T.; Fiedler, Marc; Jansen-Duerr, Pidder; van der Zee, Ate G. J.; Nijman, Hans W.; Wilschut, Jan; Daemen, Toos

2011-01-01

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of

Genetic toolbox for controlled expression of functional proteins in Geobacillus spp.

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Ivan Pogrebnyakov

Full Text Available Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins.

Heterologous Prime-Boost HIV-1 Vaccination Regimens in Pre-Clinical and Clinical Trials

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Julia L. Hurwitz

2010-02-01

Full Text Available Currently, there are more than 30 million people infected with HIV-1 and thousands more are infected each day. Vaccination is the single most effective mechanism for prevention of viral disease, and after more than 25 years of research, one vaccine has shown somewhat encouraging results in an advanced clinical efficacy trial. A modified intent-to-treat analysis of trial results showed that infection was approximately 30% lower in the vaccine group compared to the placebo group. The vaccine was administered using a heterologous prime-boost regimen in which both target antigens and delivery vehicles were changed during the course of inoculations. Here we examine the complexity of heterologous prime-boost immunizations. We show that the use of different delivery vehicles in prime and boost inoculations can help to avert the inhibitory effects caused by vector-specific immune responses. We also show that the introduction of new antigens into boost inoculations can be advantageous, demonstrating that the effect of ‘original antigenic sin’ is not absolute. Pre-clinical and clinical studies are reviewed, including our own work with a three-vector vaccination regimen using recombinant DNA, virus (Sendai virus or vaccinia virus and protein. Promising preliminary results suggest that the heterologous prime-boost strategy may possibly provide a foundation for the future prevention of HIV-1 infections in humans.

Concomitant production of cellulase and xylanase by thermophilic mould Sporotrichum thermophile in solid state fermentation and their applicability in bread making.

Science.gov (United States)

Bala, Anju; Singh, Bijender

2017-06-01

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45 °C, pH 5.0 after 72 h inoculated with 2.9 × 10 7  CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

Energy Technology Data Exchange (ETDEWEB)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui; Mansfield, Elisabeth; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd

2017-04-24

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific

Endo-laparoscopic rendezvous approach for pericardia with gastric posterior wall of gastrointestinal stromal tumor: Analysis of 52 consecutive cases

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Po Ding

2014-01-01

Conclusions: Endo-laparoscopic rendezvous approach is considered to represent the next revolution in surgery. The new technique is reliable and effective in clinical application, due to the advantages of accurate and quick localization for pericardial GIST within gastric posterior wall.

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

Science.gov (United States)

Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

2018-01-01

Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Intra-annual variation in responses by flying southern pine beetles (Coleoptera: Curculionidae: Scolytinae) to pheromone component endo-brevicomin

Science.gov (United States)

Brian T. Sullivan; Cavell Brownie; JoAnne P. Barrett

2016-01-01

The southern pine beetle Dendroctonus frontalis Zimmermann (Coleoptera: Curculionidae: Scolytinae) is attracted to an aggregation pheromone that includes the multifunctional pheromone component endobrevicomin. The effect of endo-brevicomin on attractive lures varies from strong enhancement to reduction of beetle attraction depending upon release rate, lure component...

A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean.

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Seong-Jin Jang

Full Text Available Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L Merr., we developed a near-isogenic line (NIL of a permeable (soft-seeded cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3(1,4-glucan that reinforce the impermeability of seed coats in soybean.

Y682 mutation of amyloid precursor protein promotes endo-lysosomal dysfunction by disrupting APP-SorLA interaction

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Luca Rosario La Rosa

2015-04-01

Full Text Available The intracellular transport and localization of amyloid precursor protein (APP are critical determinants of APP processing and β-amyloid peptide production, thus crucially important for the pathophysiology of Alzheimer’s disease (AD. Notably, the C-terminal Y682ENPTY687 domain of APP binds to specific adaptors controlling APP trafficking and sorting in neurons. Mutation on the Y682 residue to glycine (Y682G leads to altered APP sorting in hippocampal neurons that favors its accumulation in intracellular compartments and the release of soluble APPα. Such alterations induce premature aging and learning and cognitive deficits in APP Y682G mutant mice (APPYG/YG. Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA, resulting in endo-lysosomal dysfunctions and neuronal degeneration. Moreover, disruption of the APP/SorLA complex changes the trafficking pathway of SorLA, with its consequent increase in secretion outside neurons. Mutations in the SorLA gene are a prognostic factor in AD, and increases in SorLA levels in cerebrospinal fluid are predictive of AD in humans. These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration. In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

Management of an endo-perio lesion in an immature tooth using autologous platelet-rich fibrin: A case report

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N B Nagaveni

2015-01-01

Full Text Available Treatment of an endo-perio lesion involving a non-vital young permanent tooth is a highly challenging task to Pediatric Dentists. There is a quest for the newer biological approach to management of these lesions as traditional methods have various disadvantages. Recently, platelet-rich fibrin (PRF, a second-generation platelet concentrate, is rich in growth factors have been used in the periodontal regeneration procedure. The purpose of this paper is to describe the efficacy of PRF in the treatment of a deep intra bony defect associated with an endo-perio lesion in an immature right mandibular first premolar of 12-year-old female patient. A freshly prepared autologous PRF membrane was placed in the bony defect following debridement. Clinical and radiographic follow-up were performed at regular intervals that revealed absence of pain, gain in clinical attachment level, reduction in probing depth, and excellent bone regeneration indicating successful outcome.

The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

DEFF Research Database (Denmark)

Engelholm, L H; Nielsen, B S; Netzel-Arnett, Sarah

2001-01-01

), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify...... intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues...

Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

Science.gov (United States)

Yin, Shouliang; Li, Zilong; Wang, Xuefeng; Wang, Huizhuan; Jia, Xiaole; Ai, Guomin; Bai, Zishang; Shi, Mingxin; Yuan, Fang; Liu, Tiejun; Wang, Weishan; Yang, Keqian

2016-12-01

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

Long-Lasting Effects of BCG Vaccination on Both Heterologous Th1/Th17 Responses and Innate Trained Immunity

DEFF Research Database (Denmark)

Kleinnijenhuis, Johanneke; Quintin, Jessica; Preijers, Frank

2013-01-01

'. In the present study we assessed whether BCG was able to induce long-lasting effects on both trained immunity and heterologous T helper 1 (Th1) and Th17 immune responses 1 year after vaccination. The production of TNFα and IL-1β to mycobacteria or unrelated pathogens was higher after 2 weeks and 3 months...... in proinflammatory cytokine production after stimulation with the TLR4 ligand lipopolysaccharide. The heterologous production of Th1 (IFN-γ) and Th17 (IL-17 and IL-22) immune responses to nonmycobacterial stimulation remained strongly elevated even 1 year after BCG vaccination. In conclusion, BCG induces sustained...... changes in the immune system associated with a nonspecific response to infections both at the level of innate trained immunity and at the level of heterologous Th1/Th17 responses. © 2013 S. Karger AG, Basel....

Adaptation in the innate immune system and heterologous innate immunity.

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Martin, Stefan F

2014-11-01

The innate immune system recognizes deviation from homeostasis caused by infectious or non-infectious assaults. The threshold for its activation seems to be established by a calibration process that includes sensing of microbial molecular patterns from commensal bacteria and of endogenous signals. It is becoming increasingly clear that adaptive features, a hallmark of the adaptive immune system, can also be identified in the innate immune system. Such adaptations can result in the manifestation of a primed state of immune and tissue cells with a decreased activation threshold. This keeps the system poised to react quickly. Moreover, the fact that the innate immune system recognizes a wide variety of danger signals via pattern recognition receptors that often activate the same signaling pathways allows for heterologous innate immune stimulation. This implies that, for example, the innate immune response to an infection can be modified by co-infections or other innate stimuli. This "design feature" of the innate immune system has many implications for our understanding of individual susceptibility to diseases or responsiveness to therapies and vaccinations. In this article, adaptive features of the innate immune system as well as heterologous innate immunity and their implications are discussed.

A Distinct Class of Genome Rearrangements Driven by Heterologous Recombination.

Science.gov (United States)

León-Ortiz, Ana María; Panier, Stephanie; Sarek, Grzegorz; Vannier, Jean-Baptiste; Patel, Harshil; Campbell, Peter J; Boulton, Simon J

2018-01-18

Erroneous DNA repair by heterologous recombination (Ht-REC) is a potential threat to genome stability, but evidence supporting its prevalence is lacking. Here we demonstrate that recombination is possible between heterologous sequences and that it is a source of chromosomal alterations in mitotic and meiotic cells. Mechanistically, we find that the RTEL1 and HIM-6/BLM helicases and the BRCA1 homolog BRC-1 counteract Ht-REC in Caenorhabditis elegans, whereas mismatch repair does not. Instead, MSH-2/6 drives Ht-REC events in rtel-1 and brc-1 mutants and excessive crossovers in rtel-1 mutant meioses. Loss of vertebrate Rtel1 also causes a variety of unusually large and complex structural variations, including chromothripsis, breakage-fusion-bridge events, and tandem duplications with distant intra-chromosomal insertions, whose structure are consistent with a role for RTEL1 in preventing Ht-REC during break-induced replication. Our data establish Ht-REC as an unappreciated source of genome instability that underpins a novel class of complex genome rearrangements that likely arise during replication stress. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Diels-Alder Synthesis of endo-cis-N-phenylbicyclo[2.2.2]oct-5-en-2,3-dicarboximide

Science.gov (United States)

Baar, Marsha R.; Wustholz, Kristin

2005-01-01

A study investigated the Diels-Alder synthesis of endo-cis-N-phenylbicyclo [2.2.2]oct-5-en-2,3-dicarboximide. The amount of time taken by a reaction between the 1,3-cyclohexadiene and N-phenylmaleimide at room temperature and also whether the desired cycloadduct would precipitate directly from the reaction mixture was examined.

Increased expression of the collagen internalization receptor uPARAP/Endo180 in the stroma of head and neck cancer

DEFF Research Database (Denmark)

Sulek, Jay; Wagenaar-Miller, Rebecca A; Shireman, Jessica

2007-01-01

Local growth, invasion, and metastasis of malignancies of the head and neck involve extensive degradation and remodeling of the underlying, collagen-rich connective tissue. Urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 is an endocytic receptor recently shown to play...

The binding of zinc ions to Emericella nidulans endo-β-1,4-galactanase is essential for crystal formation

DEFF Research Database (Denmark)

Otten, Harm; Michalak, Malwina; Mikkelsen, Jørn Dalgaard

2013-01-01

A novel Emericella nidulans endo-β-1,4-galactanase (EnGAL) demonstrates a strong capacity to generate high levels of very potent prebiotic oligosaccharides from potato pulp, a by-product of the agricultural potato-starch industry. EnGAL belongs to glycoside hydrolase family 53 and shows high (72...

Cyclic fatigue resistance of XP-endo Shaper compared with different nickel-titanium alloy instruments.

Science.gov (United States)

Elnaghy, Amr; Elsaka, Shaymaa

2018-04-01

The aims of this study were to assess and compare the resistance to cyclic fatigue of XP-endo Shaper (XPS; FKG Dentaire, La Chaux-de-Fonds, Switzerland) instruments with TRUShape (TRS; Dentsply Tulsa Dental Specialties, Tulsa, OK, USA), HyFlex CM (HCM; Coltene, Cuyahoga Falls, OH, USA), Vortex Blue (VB; Dentsply Tulsa Dental Specialties), and iRace (iR; FKG Dentaire) nickel-titanium rotary instruments at body temperature. Size 30, 0.01 taper of XPS, size 30, 0.04 taper of HCM, VB, iR, and size 30, 0.06 taper of TRS instruments were immersed in saline at 37 ± 1 °C during cyclic fatigue testing. The instruments were tested with 60° angle of curvature and a 3-mm radius of curvature. The number of cycles to failure (NCF) was calculated and the length of the fractured segment was measured. Fractographic examination of the fractured surface was performed using a scanning electron microscope. The data were analyzed statistically using Kruskal-Wallis H test and Mann-Whitney U tests. Statistical significance was set at P ductile fracture of cyclic fatigue failure. XPS instruments exhibited greater cyclic fatigue resistance compared with the other tested instruments. XP-endo Shaper instruments could be used more safely in curved canals due to their higher fatigue resistance.

Controlling carrageenan structure using a novel formylglycine-dependent sulfatase, an endo-4S-iota-carrageenan sulfatase.

Science.gov (United States)

Préchoux, Aurélie; Genicot, Sabine; Rogniaux, Hélène; Helbert, William

2013-06-01

Carrageenans are sulfated polysaccharides that are found in the cell walls of red algae. These polysaccharides have gelling and texturizing properties that are widely appreciated in industrial applications. However, these functional properties depend strongly on the sulfation of the moieties of the carrabiose repetition unit. Here we aimed to monitor the sulfate composition of gelling carrageenan. To do so, we screened and purified from Pseudoalteromonas atlantica a 4S-iota carrageenan sulfatase that converts ι-carrabiose into α-carrabiose units. The sequence of this protein matched the annotated Q15XH3 (Uniprot databank) formylglycine-dependent sulfatase found in the P. atlantica genome. With pure enzyme, ι-carrageenan could be transformed into a hybrid ι-/α-carrageenan or pure α-carrageenan. Analysis of the distribution of the carrabiose moieties in hybrid carrageenan chain using enzymatic degradation with Alteromonas fortis ι-carrageenase, coupled with chromatography and NMR spectroscopy experiments, showed that the sulfatase has an endo mode of action. The endo-character and the specificity of the sulfatase made it possible to prepare hybrid κ-/ι-/α-carrageenan and κ-/α-carrageenan starting from κ-/ι-carrageenan.

Palato-gingival groove: An innocuous culprit for endo-perio lesion

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Rafeza Sultana

2016-09-01

Full Text Available This case report represents the clinical management of tooth with palato-gingival groove in a right maxillary lateral incisor with endo-perio lesion leading to dento-alveolar abscess and sinus tract. The right maxillary lateral incisor was examined clinico-radiographically. On clinical examination, the offending tooth revealed localized swelling and an intraoral draining sinus pointing on the labial gingiva without any evidence of caries, discoloration and trauma. The palatal surface of lateral incisor showed a groove with mild calculus embedded in it. The radiographic examination revealed periapical radiolucency. This case provides an evidence of morphological defect of tooth. Complete clinical and radiological examination and adequate knowledge of such morphological/ developmental defects of teeth are necessary for recognition and identification especially because of their diagnostic complexity and further consequences. 

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

NARCIS (Netherlands)

Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

2015-01-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential

Production of xylooligosaccharides from forest waste by membrane separation and Paenibacillus xylanase hydrolysis

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Chun-Han Ko

2013-02-01

Full Text Available Xylooligosaccharides (XO, derived from the alkaline (NaOH extractant of Mikania micrantha, were produced using multiple staged membrane separation and enzymatic xylanolysis. Staged nanofiltration (NMX, ultrafiltration (EUMX, and centrifugation (EMX processes for the ethanol precipitates were conducted. NMX recovered 97.26% of total xylose and removed 73.18% of sodium ions. Concentrations of total xylose were raised from 10.98 to 51.85 mg/mL by the NMX process. Recovered xylan-containing solids were hydrolyzed by the recombinant Paenibacillus xylanase. 68% XO conversions from total xylose of NMX was achieved in 24 hours. Xylopentaose (DP 5 was the major product from NMX and EMX hydrolysis. Xylohexaose (DP 6 was the major product from EUMX hydrolysis. Results of the present study suggest the applicability for XO production by nanofiltration, as NMX gave higher XO yields compared to those from a conventional ethanol-related lignocellulosic waste conversion process.

Biological synthesis of Au nanoparticles using liquefied mash of cassava starch and their functionalization for enhanced hydrolysis of xylan by recombinant xylanase.

Science.gov (United States)

Zeng, Sumei; Du, Liangwei; Huang, Meiying; Feng, Jia-Xun

2016-05-01

Au nanoparticles (AuNPs) have shown the potential for a variety of applications due to their unique physical and chemical properties. In this study, a facile and affordable method for the synthesis of AuNPs via the liquefied mash of cassava starch has been described and the functionalized AuNPs by L-cysteine improved activity of recombinant xylanase was demonstrated. UV-Vis absorption spectroscopy, transmission electron microscopy, and zeta potential measurements were performed to characterize the AuNPs and monitor their synthesis. The presence of Au was confirmed by energy-dispersive X-ray spectroscopy (EDX) and the X-ray diffraction patterns showed that Au nanocrystals were face-centered cubic. The C=O stretching vibration in the Fourier transform infrared spectrum of AuNPs suggested that the hemiacetal C-OH of sugar molecules performed the reduction of Au³⁺ to Au⁰. The presence of C and O in the EDX spectrum and the negative zeta potential of AuNPs suggested that the biomolecules present in liquefied cassava mash were responsible for the stabilization of AuNPs. The surface of AuNPs was easily functionalized by L-cysteine, which improved the stability of AuNPs. Moreover, cysteine-functionalized AuNPs could significantly improve recombinant xylanase efficiency and stability.

Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes.

Science.gov (United States)

Huitron, C; Perez, R; Sanchez, A E; Lappe, P; Rocha Zavaleta, L

2008-01-01

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.

[Endo-exo prostheses : Osseointegrated percutaneously channeled implants for rehabilitation after limb amputation].

Science.gov (United States)

Aschoff, H-H; Juhnke, D-L

2016-05-01

In 1999 the first endo-exo femoral prosthesis (EEFP) was implanted in Germany in a patient who had suffered a traumatic above-knee amputation. This procedure involves a skeletally anchored exoprosthetic device that is inserted into the residual femur. The distal part of the implant protrudes transcutaneously and allows attachment to a prosthetic limb which provides direct force transmission to the external prosthetic components. The technique originated from dental implantology and helps to avoid possible problems resulting from treatment of amputated limbs using socket prostheses. In the meantime, durability times of over 10 years have now helped to invalidate the initially well-founded reservations held against the procedure. What advantages can be achieved by osseointegrated and percutaneously channeled prostheses and which problems had to be overcome for treatment. Critical evaluation of data from patients operated on in Lübeck, Germany from January 2003 to December 2014. With osseointegrated and percutaneously channeled prostheses permanent durability times can be achieved. Infection-associated soft tissue problems at the site of skin protrusion (stoma) can be successfully prevented. The creation of this so-called stoma means acceptance of a possible bacterial portal of entry into the body. Patient satisfaction has so far been high, postoperative rehabilitation is simplified and the technique could possibly lower the costs of medical treatment. Endo-exo prostheses have proved to be successful for more than 15 years. A critical appraisal of the indications as well as a close cooperation between the surgeon, orthopedic technician and the associated rehabilitation facilities with the patient are the basis for the long-term success of this relatively new treatment approach.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Science.gov (United States)

Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function

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Phillips R

2006-04-01

Full Text Available Abstract Background Dendritic cells (DCs are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host's systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusion Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

Functional food properties of non-digestible oligosaccharides : a consensus report from the ENDO project (DGXII AIRII-CT94-1095)

NARCIS (Netherlands)

Loo, J. van; Cummings, J.; Delzenne, N.; Englyst, H.; Franck, A.; Hopkins, M.; Kok, N.; Macfarlane, G.; Newton, D.; Quigley, M.; Roberfroid, M.; Vliet, T. van; Heuvel, E. van den

1999-01-01

This paper results from the final phase of the ENDO project (DGXII AIRII-CT94-1095), a European Commission-funded project on non-digestible oligosaccharides (NDO). All participants in the programme met to perform a consensus exercise on the possible functional food properties of NDO. Topics studied

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

DEFF Research Database (Denmark)

Jørgensen, Mikael Skaanning; Skovlund, Dominique Aubert; Johannesen, Pia Francke

2014-01-01

ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools...

Apical extrusion of debris during the preparation of oval root canals: a comparative study between a full-sequence SAF system and a rotary file system supplemented by XP-endo finisher file.

Science.gov (United States)

Kfir, Anda; Moza-Levi, Rotem; Herteanu, Moran; Weissman, Amir; Wigler, Ronald

2018-03-01

The purpose of this study was to assess the amount of apically extruded debris during the preparation of oval canals with either a rotary file system supplemented by the XP-endo Finisher file or a full-sequence self-adjusting file (SAF) system. Sixty mandibular incisors were randomly assigned to two groups: group A: stage 1-glide path preparation with Pre-SAF instruments. Stage 2-cleaning and shaping with SAF. Group B: stage 1-glide path preparation with ProGlider file. Stage 2-cleaning and shaping with ProTaper Next system. Stage 3-Final cleaning with XP-endo Finisher file. The debris extruded during each of the stages was collected, and the debris weights were compared between the groups and between the stages within the groups using t tests with a significance level set at P file followed by XP-endo Finisher file extruded significantly more debris than a full-sequence SAF system. Each stage, in either procedure, had its own contribution to the extrusion of debris. Final preparation with XP-endo Finisher file contributes to the total amount of extruded debris, but the clinical relevance of the relative difference in the amount of apically extruded debris remains unclear.

Heterologous expression of cellobiohydrolases in filamentous fungi

DEFF Research Database (Denmark)

Zoglowek, Marta; Lübeck, Peter S.; Ahring, Birgitte K.

2015-01-01

Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well...... into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering...... promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation...

Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-β-1,4-mannanase

DEFF Research Database (Denmark)

Hekmat, Omid; Lo Leggio, Leila; Rosengren, Anna

2010-01-01

To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant...

The K Domain Mediates Homologous and Heterologous Interactions Between FLC and SVP Proteins of Brassica juncea

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Ma Guanpeng

2015-07-01

Full Text Available The transcription factors FLOWERING LOCUS C (FLC and SHORT VEGETATIVE PHASE (SVP can interact to form homologous and heterologous protein complexes that regulate flowering time in Brassica juncea Coss. (Mustard.Previous studies showed that protein interactions were mediated by the K domain, which contains the subdomains K1, K2 and K3. However, it remains unknown how the subdomains mediate the interactions between FLC and SVP. In the present study, we constructed several mutants of subdomains K1–K3 and investigated the mechanisms involved in the heterologous interaction of BjFLC/BjSVP and in the homologous interaction of BjFLC/BjFLC or BjSVP/BjSVP. Yeast two-hybrid and β-Galactosidase activity assays showed that the 19 amino acids of the K1 subdomain in BjSVP and the 17 amino acids of the K1 subdomain in BjFLC were functional subdomains that interact with each other to mediate hetero-dimerization. The heterologous interaction was enhanced by the K2 subdomain of BjSVP protein, but weakened by its interhelical domain L2. The heterologous interaction was also enhanced by the K2 subdomain of BjFLC protein, but weakened by its K3 subdomain. The homologous interaction of BjSVP was mediated by the full K-domain. However, the homologous interaction of BjFLC was regulated only by its K1 and weakened by its K2 and K3 subdomains. The results provided new insights into the interactions between FLC and SVP, which will be valuable for further studies on the molecular regulation mechanisms of the regulation of flowering time in B. juncea and other Brassicaceae.

An Aspergillus nidulans GH26 endo-β-mannanase with a novel degradation pattern on highly substituted galactomannans

DEFF Research Database (Denmark)

von Freiesleben, Pernille; Spodsberg, Nikolaj; Holberg Blicher, Thomas

2016-01-01

The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-β-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podosp...

Ex vivo analysis of root canal cleaning using Endo-PTC associated to NaOCl and different irrigant solutions

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Andrea Kanako Yamazaki

2010-03-01

Full Text Available The aim of this study was to assess qualitatively, by means of SEM images, the cleaning of the dentin walls of root canals after chemical-surgical preparation using Endo-PTC cream with 0.5% and 1% sodium hypochlorite and different final irrigating solutions. Seventy-two single-rooted human teeth were divided into eight groups and prepared using Endo-PTC cream with sodium hypochlorite (NaOCl at different concentrations, and irrigated with NaOCl at different concentrations. Final irrigation was performed with either EDTA-T or EDTA-C. The best results were obtained with Group 1, followed by Groups 5, 2, 7, 8, 3, 6 and 4. We can conclude that the use of 0.5% NaOCl during instrumentation and final flush of the root canals was more efficient in cleaning than was 1% sodium hypochlorite. EDTA-T was more efficient in removing smear layer than EDTA-C, and the cervical third presented better cleaning of the root canal walls than did the middle third, which showed cleaner dentin walls than the apical third.

Response surface methodology optimization of partitioning of xylanase form Aspergillus Niger by metal affinity polymer-salt aqueous two-phase systems.

Science.gov (United States)

Fakhari, Mohamad Ali; Rahimpour, Farshad; Taran, Mojtaba

2017-09-15

Aqueous two phase affinity partitioning system using metal ligands was applied for partitioning and purification of xylanase produced by Aspergillus Niger. To minimization the number of experiments for the design parameters and develop predictive models for optimization of the purification process, response surface methodology (RSM) with a face-centered central composite design (CCF) has been used. Polyethylene glycol (PEG) 6000 was activated using epichlorohydrin, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu was attached to the polyethylene glycol-iminodiacetic acid (PEG-IDA). The influence of some experimental variables such as PEG (10-18%w/w), sodium sulfate (8-12%), PEG-IDA-Cu 2+ concentration (0-50% w/w of total PEG), pH of system (4-8) and crude enzyme loading (6-18%w/w) on xylanase and total protein partitioning coefficient, enzyme yield and enzyme specific activity were systematically evaluated. Two optimal point with high enzyme partitioning factor 10.97 and yield 79.95 (including 10% PEG, 12% Na 2 SO 4 , 50% ligand, pH 8 and 6% crude enzyme loading) and high specific activity in top phase 42.21 (including 14.73% PEG, 8.02% Na 2 SO 4 , 28.43% ligand, pH 7.7 and 6.08% crude enzyme loading) were attained. The adequacy of the RSM models was verified by a good agreement between experimental and predicted results. Copyright © 2017 Elsevier B.V. All rights reserved.

Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair.

Science.gov (United States)

Mozafari, Roghayeh; Kyrylenko, Sergiy; Castro, Mateus Vidigal; Ferreira, Rui Seabra; Barraviera, Benedito; Oliveira, Alexandre Leite Rodrigues

2018-01-01

Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F + T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. The experiments indicated that

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

OpenAIRE

Sigoillot Jean-Claude; Kim-Anh To; Haltrich Dietmar; Berrin Jean-Guy; Thi-Thu Dang; Bien-Cuong Do; Yamabhai Montarop

2009-01-01

Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pret...

Non-granulomatous prostatitis: MR appearance with an endorectal surface coil; Nichtgranulomatoese Prostatitis: Erscheinungsbild im MRT mit endorektaler Oberflaechenspule (``Endo-MRT``)

Energy Technology Data Exchange (ETDEWEB)

Szolar, D.H.M. [Magnetresonanztomographie-Zentrum, Univ. Graz (Austria); Ranner, G. [Magnetresonanztomographie-Zentrum, Univ. Graz (Austria); Preidler, K.W. [Magnetresonanztomographie-Zentrum, Univ. Graz (Austria); Lax, S. [Inst. fuer Pathologische Anatomie, Univ. Graz (Austria)

1995-01-01

Inflammatory conditions of the prostate are often idfficult to distinguish from early stages of prostate cancer with imaging techniques. The use of an endorectal surface coil in MRI of the prostate gland has been reported to provide superior resolution and better imaging of details than MRI with a body coil in the diagnosis of early prostate cancer. We report a 34-year-old patient with nonspecific non-granlomatous prostatitis in whom T{sub 2}-weighted endorectal surface coil magnetic resonance imaging (ESCMRI) showed a region of markedly decreased signal intensity in the periphery of the gland. The low signal intensity of the lesion, its sharp demarcation from the normal part of the peripheral zone of the prostate and the marked bulge of the surface contour without capsular breach of the organ were interpreted as evidence of a bioptically proven benign inflammatory condition. (orig.) [Deutsch] Die Unterscheidung entzuendlicher Prozesse von Fruehstadien maligner Neoplasien der Prostata mittels Magnetresonanztomographie bereitet immer wieder Schwierigkeiten. Die Anwendung einer Oberflaechenspule erlaubt durch hoehere Aufloesung eine exaktere Beurteilung der Prostatakapsel, was eine hoehere Treffsicherheit beim Nachweis moeglicher organueberschreitender Infiltrationen bedeutet. Wir praesentieren den Fall eines 34jaehrigen Mannes mit unspezifischer, nichtgranulomatoeser Prostatitis, bei dem das MR-Tomogramm mit endorektal plazierter Oberflaechenspule (Endo-MRT) in der Peripherzone ein Areal deutlich herabgesetzter Signalintensitaet auf T{sub 2}-gewichteten Aufnahmen zeigte. Die scharfe Begrenzung der ausgedehnten signalarmen Laesion gegenueber dem nichtbefallenen Anteil der Peripherzone beim im Endo-MRT identifizierbarer intakter Kapsel liess ein organueberschreitendes Malignom ausschliessen und bestaetigte durch histologische Aufarbeitung der Bioptate den Endo-MRT-Verdachtsbefund einer Prostatitis. (orig.)

Potential application of waste from castor bean (Ricinus communis L.) for production for xylanase of interest in the industry.

Science.gov (United States)

Herculano, Polyanna Nunes; Moreira, Keila Aparecida; Bezerra, Raquel Pedrosa; Porto, Tatiana Souza; de Souza-Motta, Cristina Maria; Porto, Ana Lúcia Figueiredo

2016-12-01

Xylanases activity (XY) from Aspergillus japonicus URM5620 produced by Solid-State Fermentation (SSF) of castor press cake (Ricinus communis) on different conditions of production and extraction by PEG/citrate aqueous two-phase system (ATPS) were investigated. XY production was influenced by substrate amount (5-10 g), initial moisture (15-35 %), pH (4.0-6.0) and temperature (25-35 °C), obtaining the maximum activity of 29,085 ± 1808 U g ds -1 using 5.0 g of substrate with initial moisture of 15 % at 25 °C and pH 6.0, after 120 h of fermentation. The influence of PEG molar mass (1000-8000 g mol -1 ), phase concentrations (PEG 20.0-24.0 % w/w and sodium citrate 15-20 % w/w) and pH (6.0-8.0) on partition coefficient, purification factor, yield and selectivity of XY were determinate. Enzyme partitioning into the PEG rich phase was favored by M PEG 8000 (g mol -1 ), C PEG 24 % (w/w), C C 20 % (w/w) and pH 8.0, resulting in partition coefficient of 50.78, activity yield of 268 %, 7.20-fold purification factor and selectivity of 293. A. japonicus URM5620 has a potential role in the development of a bioprocess for the XY production using low-cost media. In addition, the present study proved it is feasible to extract xylanase from SSF by adopting the one step ATPS consisting of PEG/citrate.

[Implementation of the EndoCert system for certification of arthroplasty centers. Experiences from the pilot phase].

Science.gov (United States)

Haas, H; Mittelmeier, W

2014-06-01

EndoCert is an initiative of the Deutschen Gesellschaft für Orthopädie und Orthopädische Chirurgie (DGOOC, German Society for Orthopedics and Orthopedic Surgery) which has been available since October 2012 and is the first system worldwide for certification of specialized arthroplasty centers. Before implementation of this certification concept two sequential pilot phases were carried out with representative treatment institutions. The results from these pilot clinics are presented with respect to quality improvement effects. Early effects on the quality of treatment have been achieved by rectification of nonconformities determined in the audit with respect to structural and process quality. A total of 172 nonconformities found in the 23 participating pilot clinics could be rectified. Long-term effects on the quality of results will in future be analyzed in cooperation with the German endoprosthesis register (EPRD) and by accompanying evaluations. A close feedback of the collated experiences and results to the certification committee, which is responsible for the procedure together with the DGOOC, allows continuous further development of the system EndoCert represents a substantial step towards a nationwide safety and improvement of the quality in arthroplasty treatment within the preoperative, perioperative and postoperative framework and can in future represent a decisive tool together with the EPRD in quality management.

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.

Science.gov (United States)

Tsonkova, Violeta Georgieva; Sand, Fredrik Wolfhagen; Wolf, Xenia Asbæk; Grunnet, Lars Groth; Kirstine Ringgaard, Anna; Ingvorsen, Camilla; Winkel, Louise; Kalisz, Mark; Dalgaard, Kevin; Bruun, Christine; Fels, Johannes Josef; Helgstrand, Charlotte; Hastrup, Sven; Öberg, Fredrik Kryh; Vernet, Erik; Sandrini, Michael Paolo Bastner; Shaw, Allan Christian; Jessen, Carsten; Grønborg, Mads; Hald, Jacob; Willenbrock, Hanni; Madsen, Dennis; Wernersson, Rasmus; Hansson, Lena; Jensen, Jan Nygaard; Plesner, Annette; Alanentalo, Tomas; Petersen, Maja Borup Kjær; Grapin-Botton, Anne; Honoré, Christian; Ahnfelt-Rønne, Jonas; Hecksher-Sørensen, Jacob; Ravassard, Philippe; Madsen, Ole D; Rescan, Claude; Frogne, Thomas

2018-02-01

To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

Science.gov (United States)

Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

2014-10-01

Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Substance P and Calcitonin gene-related peptide expression in human periodontal ligament after root canal preparation with Reciproc Blue, WaveOne Gold, XP EndoShaper and hand files.

Science.gov (United States)

Caviedes-Bucheli, J; Rios-Osorio, N; Rey-Rojas, M; Laguna-Rivero, F; Azuero-Holguin, M M; Diaz, L E; Curtidor, H; Castaneda-Ramirez, J J; Munoz, H R

2018-05-17

To quantify the Substance P (SP) and Calcitonin gene-related peptide (CGRP) expression in healthy human periodontal ligament from premolars after root canal preparation with Reciproc Blue, WaveOne Gold, XP EndoShaper and hand files. Fifty human periodontal ligament samples were obtained from healthy mandibular premolars where extraction was indicated for orthodontic reasons. Prior to extraction, 40 of these premolars were equally divided into four groups, and root canals were prepared using four different systems: Reciproc Blue, WaveOne Gold, XP EndoShaper, and a hand instrumentation technique. The remaining 10 healthy premolars were extracted without treatment and served as a negative control group. All periodontal ligament samples were processed, and SP and CGRP were measured by radioimmunoassay. The Kruskal-Wallis test was used to establish significant differences between groups and LSD post hoc comparisons were also performed. Greater SP and CGRP values were found in the hand instrumentation group, followed by the XP EndoShaper, WaveOne Gold and the Reciproc groups. The lower SP and CGRP values were for the healthy periodontal ligament group. The Kruskal-Wallis test revealed significant differences between groups (p 0.05). All the root canal preparation techniques tested increased SP and CGRP expression in human periodontal ligament, with hand files and XP EndoShaper instruments being associated with greater neuropeptide release compared to Reciproc Blue and WaveOne Gold files. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

Directory of Open Access Journals (Sweden)

Zi Yang

Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

EFSA CEF Panel (EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids), 2014. Scientific Opinion on xylanase from a genetically modified strain of Aspergillus oryzae (strain NZYM-FB)

DEFF Research Database (Denmark)

Poulsen, Morten; Binderup, Mona-Lise; Hallas-Møller, Torben

. The xylanase is intended to be used in a number of food manufacturing processes, such as starch processing, beverage alcohol (distilling), brewing, baking and other cereal based processes. The dietary exposure was assessed according to the Budget method. The food enzyme did not induce gene mutations...

Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

DEFF Research Database (Denmark)

Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

2014-01-01

Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as stre...

Functional diversity for biomass deconstruction in family 5 subfamily 5 (GH5_5) of fungal endo-β1,4-glucanases.

Science.gov (United States)

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.

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion

DEFF Research Database (Denmark)

Engelholm, Lars H; List, Karin; Netzel-Arnett, Sarah

2003-01-01

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (u......, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions....

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

Science.gov (United States)

Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

2017-03-01

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

On the sensitivity of protein data bank normal mode analysis: an application to GH10 xylanases

Science.gov (United States)

Tirion, Monique M.

2015-12-01

Protein data bank entries obtain distinct, reproducible flexibility characteristics determined by normal mode analyses of their three dimensional coordinate files. We study the effectiveness and sensitivity of this technique by analyzing the results on one class of glycosidases: family 10 xylanases. A conserved tryptophan that appears to affect access to the active site can be in one of two conformations according to x-ray crystallographic electron density data. The two alternate orientations of this active site tryptophan lead to distinct flexibility spectra, with one orientation thwarting the oscillations seen in the other. The particular orientation of this sidechain furthermore affects the appearance of the motility of a distant, C terminal region we term the mallet. The mallet region is known to separate members of this family of enzymes into two classes.