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Sample records for chrysanthemi endoglucanase cely

  1. Adolfo Celi, um olhar para dois mundos

    Directory of Open Access Journals (Sweden)

    Alessandra Vanucci

    2002-11-01

    Full Text Available O ensaio resgata a vivência brasileira de Adolfo Celi, ator e diretor italiano ativo em cinema e teatro (1922-1986, inserido no laboratório paulista da renovação, na década de 1950. Sua atuação impôs, no palco, o legado texto-cêntrico de Sílvio d'Amico e a utopia do ator plástico de Copeau e, na tela, a estética neo-realista associada a uma atuação fortemente marcada pelo ideário teatral moderno. As intuições, a herança, as oportunidades e as invenções perdidas desta experiência transcultural sugerem um quadro de realização somente parcial do projeto modernizador. Ao entrar na década de 60, é a sensação de ter esgotado um ciclo existencial que fornece a Celi um válido álibi para o retorno. Álibi (1969, seu terceiro e último filme como diretor, realizado na Itália com Gassmann e Lucignani, é um testemunho de vida e memórias, de raiva e de saudade do Brasil; e um precioso guia para entender sua extraordinária ‘aventura americana’.

  2. Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

    Science.gov (United States)

    Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

    2000-07-01

    A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

  3. Thermostability in endoglucanases is fold-specific

    Science.gov (United States)

    2011-01-01

    Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

  4. Thermostability in endoglucanases is fold-specific

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    Wolt Jeffrey D

    2011-02-01

    Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

  5. [The analyses and identification of Flos rhododendri mollis and Flos chrysanthemi indici via infrared spectroscopy].

    Science.gov (United States)

    Jin, Zhe-Xiong; Wang, Yue; Zhou, Qun; Chen, Jian-Bo; Ma, Fang; Sun, Su-Qin

    2014-09-01

    In this study, major chemical components of Flos rhododendri mollis and Flos chrysanthemi indici were characterized using Fourier transform infrared spectroscopy (FTIR). For Flos rhododendri mollis, the bands at 1,648 and 1,543 cm(-1) were attributed to amide I and amide II , respectively, indicating that it contained proteins probably resulting in immunization. In case of Flos chrysanthemi indici, stretching vibration of C==O function group was responsible for the bands at 1,734 and 1,515 cm(-1), as a result of essential oils, lipids, etc. Since FTIR spectra of Flos rhododendri mollis and Flos chrysanthemi indici are almost identical and it is difficult to discriminate them, two-step identification was investigated via secondary derivative of the FTIR spectra. The bands at 1,656 and 1,515 cm(-1) corresponds to flavonoides in Flos rhododendri mollis and Flos chrysanthemi indici. In the secondary derivative of the FTIR spectrum of Flos chrysanthemi indici, characteristic bands of inulin were present at 1,163, 1,077, 1,026, 986 and 869 cm(-1), and therefore Flos chrysanthemi indici contained inulin as well. Tri-step identification was carried out for Flos rhododendri mollis and Flos chrysanthemi indici by means of comparing their 2D-IR correlation spectra in different wave number range. In the characteristic range of flavonoides (1,700-1,400 cm(-1)), Flos rhododendri mollis exhibited 3 obvious autopeaks, while 10 autopeaks were visualized in the 2D-IR correlation spectrum of Flos chrysanthemi indici Moreover, in the characteristic range of glucoside (1,250-900 cm(-1)), 10 and 9 autopeaks were present in the 2D-IR correlation spectra of Flos rhododendri mollis and Flos chrysanthemi indici, respectively. Therefore, the tri-step identification of FTIR is a time-saving; accurate, cost-saving and convenient method to effectively distinguish traditional Chinese medicines.

  6. High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

    DEFF Research Database (Denmark)

    Øbro, Jens; Sørensen, Iben; Derkx, Patrick

    2009-01-01

    the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime...

  7. Kinetic Properties of α-Galactosidase and the Localization of Total Proteins in Erwinia chrysanthemi

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    John Morgan Brand

    2004-01-01

    Full Text Available Erwinia chrysanthemi is an enterobacterium that causes soft-rot in plants in general, resulting in enormous economic losses annually. For the pathogen to survive in the host plant, it has to use the readily assimilable compounds from the host fluids and degrade the host tissue. To accomplish this, E. chrysanthemi produces several extracellular and intracellular enzymes. Among the intracellular enzymes there is a special digestive class, the galactosidases, which can be either periplasmic or cytoplasmic. α-Galactosidase is known to degrade melibiose and raffinose into glucose and galactose, and into galactose and sucrose respectively. The aim of the present study was to investigate the kinetic properties of α-galactosidase in E. chrysanthemi, and the localization of total proteins, after culturing it in the presence of raffinose and melibiose. The α-galactosidase that degrades melibiose seems to be the same enzyme that is also responsible for the breakdown of raffinose in E. chrysanthemi. It is localized mainly in the cytoplasm with a fraction of between 2.4 and 5.4 % localized in the periplasm. The majority of E. chrysanthemi proteins have cytoplasmic localization.

  8. Erwinia chrysanthemi ook bij ploffers in Dahlia boosdoener

    NARCIS (Netherlands)

    Leeuwen, van P.J.; Trompert, J.P.T.

    2006-01-01

    Sinds een aantal jaren komt bij de stekproduktie van Dahlia veel uitval voor door ploffers. Na het verhogen van de kastemperatuur vallen de knollen natrot weg. Bovendien kan verdere besmetting snel om zich heen grijpen. Onderzoek heeft aangetoond dat de bacterie Erwinia chrysanthemi de veroorzaker

  9. Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

    Science.gov (United States)

    He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

    1991-02-01

    The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

  10. Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

    OpenAIRE

    He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

    1991-01-01

    The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-c...

  11. Biosynthesis, purification and characterization of endoglucanase from a xylanase producing strain Aspergillus niger B03

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    Georgi Todorov Dobrev

    2012-03-01

    Full Text Available An extracellular endoglucanase was isolated from the culture liquid of xylanase producing strain Aspergillus niger B03. The enzyme was purified to a homogenous form, using consecutive ultrafiltration, anion exchange chromatography, and gel filtration. Endoglucanase was a monomer protein with a molecular weight of 26,900 Da determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 28,800 Da determined by gel filtration. The optimal pH and temperature values for the enzyme action were 3.5 and 65ºC respectively. Endoglucanase was stable at 40ºC, pH 3.0 for 210 min. The substrate specificity of the enzyme was determined with carboxymethyl cellulose, filter paper, and different glycosides. Endoglucanase displayed maximum activity in the case of carboxymethyl cellulose, with a Km value of 21.01 mg/mL. The substrate specificity and the pattern of substrate degradation suggested that the enzyme is an endoglucanase. Endoglucanase showed a synergism with endoxylanase in corn cobs hydrolysis.

  12. Endogenous cellulases in animals: Isolation of β-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes

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    Smant, Geert; Stokkermans, Jack P. W. G.; Yan, Yitang; de Boer, Jan M.; Baum, Thomas J.; Wang, Xiaohong; Hussey, Richard S.; Gommers, Fred J.; Henrissat, Bernard; Davis, Eric L.; Helder, Johannes; Schots, Arjen; Bakker, Jaap

    1998-01-01

    β-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing β-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37–44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3′ end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation. PMID:9560201

  13. PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937.

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    Nasser, William; Reverchon, Sylvie; Vedel, Regine; Boccara, Martine

    2005-11-01

    Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.

  14. Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry.

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    Shimonaka, Atsushi; Koga, Jinichiro; Baba, Yuko; Nishimura, Tomoko; Murashima, Koichiro; Kubota, Hidetoshi; Kono, Toshiaki

    2006-04-01

    We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.

  15. Production of thermophilic and acidophilic endoglucanases by ...

    African Journals Online (AJOL)

    Production of thermophilic and acidophilic endoglucanases by mutant Trichoderma atroviride 102C1 using agro-industrial by-products. ... The effect of the carbon (sugarcane bagasse: SCB) and nitrogen (corn steep liquor: CSL) sources on ...

  16. PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937.

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    Shevchik, Vladimir E; Hugouvieux-Cotte-Pattat, Nicole

    2003-05-01

    Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are

  17. Effects of nitrogen ion irradiation on endoglucanase activity and gene mutation of Bacillus subtilis Bac01

    International Nuclear Information System (INIS)

    Lv Jie; Mao Peihong; Jin Xiang; Yu Long; Ying Hanjie

    2009-01-01

    Bacillus subtilis Bac01 was mutated by 15 keV N + ions of 1.5xl0 16 cm -2 . The mutant strain Bac11 with high yield of endoglucanase was isolated using carboxymethylcellulose sodium and congo red indicative plates. It exhibited higher endoglucanase activity (381.89IU) than the original strain Bac01 (93.33IU). Two 1,500 bp endoglucanase gene fragments were obtained with PCR amplification from B. subtilis Bac01 and mutant strain Bac11. BLAST comparison result indicated that 10 nucleotides mutated. Bioinformatics methods were used to analyze the two predicted amino acid sequences, and it was found that 5 amino acid residues changed, being all in the cellulose-binding domain of endoglucanase. (authors)

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

    Science.gov (United States)

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

    1997-10-01

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

  19. Production of cellulose nanofibrils from bleached eucalyptus fibers by hyperthermostable endoglucanase treatment and subsequent microfluidization

    Science.gov (United States)

    Wangxia Wang; Michael D. Mozuch; Ronald C. Sabo; Phil Kersten; J.Y. Zhu; Yongcan Jin

    2015-01-01

    A GH5 hyperthermostable endoglucanase from the archaeon Pyrococcus honkoshii (ph-GH5) and a commercial endoglucanase FR were used to treat bleached eucalyptus pulp (BEP) fibers to produce cellulose nanofibrils (CNFs) through subsequent microfluidization Enzymatic treatments facilitated CNF production due to the reduced degree of polymerization (DP)...

  20. EGVII endoglucanase and nucleic acids encoding the same

    Science.gov (United States)

    Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

    2009-05-05

    The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  1. Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

    Directory of Open Access Journals (Sweden)

    Gheysen Godelieve

    2008-11-01

    Full Text Available Abstract Background Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5 have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN. The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Results Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea; all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida. Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. Conclusion We conclude that the ancestral

  2. Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event.

    Science.gov (United States)

    Kyndt, Tina; Haegeman, Annelies; Gheysen, Godelieve

    2008-11-03

    Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of

  3. Carboxymethyl-cellulase from Erwina chrysanthemi. I. Production and regulation of extracellular carboxymethyl-cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, M.H.; Chambost, J.P.; Magnan, M.; Cattaneo, J.

    1984-01-01

    Erwinia chrysanthemi strain 3665 growing aerobically in a mineral salts medium containing various carbon sources constitutively secreted low levels of carboxymethyl-cellulase activity. Increased production of this activity was triggered by conditions which reduced the growth rate. The results obtained with continuous culture suggested that this production was controlled by a mechanism similar to catabolite repression. However, other factors might be implicated in the regulation of cellulase production.

  4. Chemical modification of β -endoglucanase from Trichoderma viridin ...

    African Journals Online (AJOL)

    β-Endoglucanase from Trichoderma viride was modified by methanol to explore the catalytic functional groups of cellulase. Crude cellulase was produced, and the conditions of saturation and pH by salting out with ammonium sulfate were optimized. Under optimal conditions, crude cellulase was isolated and purified.

  5. Endoglucanase post-milling treatment for producing cellulose nanofibers from bleached eucalyptus fibers by a supermasscolloider

    Science.gov (United States)

    Wangxia Wang; Michael D. Mozuch; Ronald C. Sabo; Philip Kersten; Junyong Zhu; Yongcan Jin

    2016-01-01

    Three recombinant GH5 endoglucanases chosen for their contrasting hydrolytic activities, and a commercial endoglucanase were used to treat cellulose nanofibers (CNFs) after they were milled from bleached eucalyptus pulp with a supermasscolloider. This enzyme ‘‘post-treatment’’ resulted in different properties for the CNFs depending on enzyme treatment. The degree of...

  6. Purification and characterization of an endoglucanase from a newly isolated thermophilic anaerobic bacterium

    Energy Technology Data Exchange (ETDEWEB)

    Creuzet, N; Frixon, C [Laboratoire de Chimie Bacterienne, C.N.R.S., 13 - Marseille (France)

    1983-02-01

    An endoglucanase (1,4-..beta..-D-glucan glucanohydrolase, EC 3.2.1.4) from a new cellulotytic thermophilic bacterium was purified to apparent homogeneity after being separated from a xylanase. Little carbohydrate was associated with the endoglucanase. A molecular weight of 91,000 and 99,000 was determined by SDS-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme on Ultrogel ACA 34. The optimal pH was approximately 6.4 and the enzyme was isoelectric at pH 3.85. The enzyme was found highly thermostable: it retained 50% of its activity after 1 hour at 85/sup 0/C. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating to hydrolyze highly ordered cellulose. Cellobiose inhibited the activity of the endoglucanase. None of the metal ions tested stimulated the activity. The enzyme was completely inactivated by 1 mM Hg/sup 2 +/ and was inhibited by thiol reagents.

  7. The Good News of the prophet-woman: identity and political culture in the writings of Fourierists Mª Josefa Zapata and Margarita Pérez de Celis | La Buena Nueva de la mujer profeta: identidad y cultura política en las fourieristas Mª Josefa Zapata y Margarita Pérez de Celis

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    Gloria Espigado Tocino

    2008-12-01

    Full Text Available This article describes how the Fourierists Josefa Zapata and Margarita Pérez de Celis constructed a liberating identity for women through their writings in their own newspapers. Followers of the theories of the French utopian socialist Charles Fourier, they used a prophetic form of discourse to announce the advent of a new era for mankind, in which women would be redeemed. Advocates of a humanitarian Christianity which sought to correct fundamental social inequalities, they were, however, subject to censorship which was always on the alert for the threat of subversion that their discourse entailed. | Este artículo describe cómo las fourieristas Josefa Zapata y Margarita Pérez de Celis construyeron una identidad emancipadora para la mujer, a través de sus escritos en sus propios periódicos. Seguidoras de las teorías del socialista utópico francés Charles Fourier, adoptaron la expresión profética para anunciar el advenimiento de una nueva era para la humanidad, donde la mujer sería igualmente redimida. Seguidoras de un cristianismo humanitarista que resolviera los desequilibrios sociales fundamentales fueron, sin embargo, objeto de la censura que estuvo atenta a la amenaza de subversión que su discurso encerraba.

  8. Karakterisasi Erwinia Chrysanthemi Penyebab Penyakit Busuk Bakteri Pada Daun Lidah Buaya (Aloe Vera)

    OpenAIRE

    SUPRIADI,; IBRAHIM, NILDAR; TARYONO,

    2002-01-01

    Characteristics o/Envinia chysanthemi causing bacterial soft rot ofAloe (Aloe VeraJThe bacterial sot rot of aloe, caused by Erwinia chrysanthemi, was first identified in Caibbean Island in 1992. In early 2001, similar symptoms were found on the aloe plants grown in Semplak, Bogor, West Java. Based on its symptom and progressively spread, especially on the leaf and basal stem, it appeared that the disease was serious and therefore threatened the current development of die plants. This study wa...

  9. The Phytopathogen Dickeya dadantii (Erwinia chrysanthemi 3937) Is a Pathogen of the Pea Aphid†

    OpenAIRE

    Grenier, Anne-Marie; Duport, Gabrielle; Pagès, Sylvie; Condemine, Guy; Rahbé, Yvan

    2006-01-01

    Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, eit...

  10. Erwinia chrysanthemi: pectolytic bacterium causing soft rot outbreaks of arracacha in Brazil Erwinia chrysanthemi: bactéria pectolítica envolvida na "mela" da mandioquinha-salsa no Brasil

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    Gilmar Paulo Henz

    2006-10-01

    Full Text Available The objetive of this work was to identify the pectolytic bacteria associated with soft rot of arracacha roots in Brazil. From 1998 to 2001, 227 isolates of Erwinia spp. were obtained from arracacha roots and identified by biochemical and physiological tests (pectolytic activity, lecithinase, a-methyl glucoside, phosphatase, erythromycin sensivity, growth at 37ºC. Of these isolates, 89.9% were identified as E. chrysanthemi (Ech, 9.7% as E. carotovora subsp. carotovora (Ecc and 0.5% as E. carotovora subsp. atroseptica. The identity of seventeen out of twenty representative isolates of Ech and Ecc was confirmed by PCR (primers '149f', 'L1r', 'ADE1', 'ADE2'.O objetivo deste trabalho foi identificar as bactérias pectolíticas envolvidas na podridão-mole de raízes de mandioquinha-salsa no Brasil. De 1998 a 2001, 227 isolados de Erwinia spp. foram obtidos de raízes de mandioquinha-salsa e identificados por testes bioquímicos e fisiológicos (atividade pectolítica, lecitinase, a-methyl glucosídeo, fosfatase, sensibilidade à eritromicina, crescimento a 37ºC. Destes isolados, 89,9% foram identificados como E. chrysanthemi (Ech, 9,7% como E. carotovora subsp. carotovora (Ecc e somente 0,5% como E. carotovora subsp. atroseptica. A identidade de 20 isolados representativos de Ech e Ecc foi confirmada por PCR (primers '149f', 'L1r', 'ADE1', 'ADE2', com exceção de dois isolados de Ech e um de Ecc.

  11. Physical and Mechanical Properties of Cellulose Nanofibril Films from Bleached Eucalyptus Pulp by Endoglucanase Treatment and Microfluidization

    Science.gov (United States)

    Wangxia Wang; Ronald C. Sabo; Michael D. Mozuch; Phil Kersten; J. Y. Zhu; Yongcan Jin

    2015-01-01

    A GH5 hyperthermostable endoglucanase (Ph-GH5) from the archaeon Pyrococcus horikoshii and a commercial endoglucanase (FR) were used to treat bleached eucalyptus pulp (BEP) fibers to produce cellulose nanofibrils (CNF) and subsequently to CNF films. TEM imaging indicated that Ph-GH5 produced longer and more entangled CNF than FR with the same number...

  12. Regulation of expression of pectate lyase genes pelA, pelD, and pelE in Erwinia chrysanthemi.

    Science.gov (United States)

    Reverchon, S; Robert-Baudouy, J

    1987-06-01

    The regulation of pelA, pelD, and pelE genes encoding three of the five major pectate lyase isoenzymes (PLa, PLd, and PLe) in Erwinia chrysanthemi B374 was analyzed by using genetic fusions to lacZ. These three genes are clustered on a 5-kilobase DNA fragment in the order pelD-pelE-pelA and constitute three independent transcriptional units. We localized the pelDEA cluster near the pro-1 marker on the genetic map of B374 by chromosomal mobilization with RP4::mini-Mu plasmid pULB110. Three classes of regulatory mutations responsible for constitutive pectate lyase synthesis have been described (kdgR, gpiR, and cri). We studied the effects of each mutation on pelE, pelD, and pelA expression independently. The mutations kdgR and gpiR mainly affect the expression of pelE and pelD, although PLa synthesis is slightly increased. The cri mutation results in a low level of constitutive expression of the three pel genes, but it is a pleiotropic mutation since other genes not involved in pectinolysis are also affected. In addition, we demonstrated that exuR, a negative regulatory gene governing the catabolism of hexuronates, does not modify the expression of pel genes. The frequency of gpiR or cri mutations (about 10(-8)) and the resulting constitutivity of pectate lyase synthesis suggest that these genes act as negative regulatory genes in addition to kdgR, which is already known to encode a repressor. Moreover, we found that expression of pel-lac fusions carried on pBR322 derivatives was higher in E. chrysanthemi than in Escherichia coli; this fact suggests the existence of positive regulation of pectate lyase synthesis in E. chrysanthemi.

  13. Effects of Pretreatment of Single and Mixed Lignocellulosic Substrates on Production of Endoglucanase by Bacillus aerius S5.2

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    Mushafau Adebayo Oke

    2016-06-01

    Full Text Available A mixed substrate (MS comprising oil palm empty fruit bunch (EFB, oil palm frond (OPF, and rice husk (RH was evaluated for endoglucanase production by Bacillus aerius S5.2. Effects of sulphuric acid, sodium hydroxide, N-methylmorpholine-N-oxide (NMMO, and hydrothermal pretreatments on endoglucanase production were investigated. Endoglucanase production by B. aerius on the untreated (0.677 U/mL and pretreated MS (0.305 – 0.630 U/mL was generally similar, except that the acid (0.305 U/mL and hydrothermal (0.549 U/mL pretreatments that were more severe consequently produced significantly lower titres. Alkali pretreatment supported the highest enzyme production (0.630 U/mL among all pretreatments that were studied. When endoglucanase production on the alkali-pretreated MS and single substrates (SS was compared, alkali-pretreated EFB produced a titre (0.655 U/mL similar to the MS, and this was significantly higher than titres recorded on OPF (0.504 U/mL and RH (0.525 U/mL. Lower enzyme production was found to be consistent with higher pretreatment severity and greater removal of amorphous regions in all the pretreatments. Furthermore, combining the SS showed no adverse effects on endoglucanase production.

  14. Kinetics, improved activity and thermostability of endoglucanase and beta glucosidase from a mutant-derivative of aspergillus niger ms82

    International Nuclear Information System (INIS)

    Sohail, M.; Ahmad, A.; Khan, S.A.; Uddin, F.

    2013-01-01

    A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and beta-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (Tm and T1/2) showed that the wild type and mutant endoglucanase was more heat-resistant compared to beta-glucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and beta-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant beta-glucosidase was substantially lower than the parent enzyme while Ea of mutant endoglucanase was slightly less than the parent. The lowered Ea values can be attributed to the improved beta-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82. (author)

  15. Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity.

    Science.gov (United States)

    Reverchon, Sylvie; Rouanet, Carine; Expert, Dominique; Nasser, William

    2002-02-01

    In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha

  16. Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi.

    Science.gov (United States)

    Avrova, Anna O; Hyman, Lizbeth J; Toth, Rachel L; Toth, Ian K

    2002-04-01

    The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.

  17. Environmentally friendly ionic liquid-in-water microemulsions for extraction of hydrophilic and lipophilic components from Flos Chrysanthemi.

    Science.gov (United States)

    Chen, Jue; Cao, Jun; Gao, Wen; Qi, Lian-Wen; Li, Ping

    2013-10-21

    Ionic liquids (ILs) have numerous chemical applications as environmentally green solvents that are extending into microemulsion applications. In this work, a novel benign IL-in-water microemulsion system modified by an IL surfactant has been proposed for simultaneous extraction of hydrophilic and lipophilic constituents from Flos Chrysanthemi (Chrysanthemum morifolium). Constituents were analyzed by rapid-resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. A mixture-design approach was used to optimize the IL surfactant and the IL oil phase in the microemulsion system. Microemulsions consisting of 6.0% 1-dodecyl-3-methylimidazolium hydrogen sulfate, 0.1% 1-vinyl-3-methylimidazolium hexafluorophosphate and 93.9% water offered the acceptable extract efficiency that are comparable to or even better than conventional volatile organic solvents. This assay was fully validated with respect to the linearity of response (r(2) > 0.999 over two orders of magnitude), precision (intra-RSD < 0.49 and inter-day RSD < 2.21), and accuracy (recoveries ranging from 93.73% to 101.84%). The proposed IL-in-water microemulsion method provided an environmentally friendly alternative for efficient extraction of compounds from Flos Chrysanthemi and could be extended to complex environmental and pharmaceutical samples.

  18. Isolation and characterization of a novel endoglucanase from a Bursaphelenchus xylophilus metagenomic library.

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    Lin Zhang

    Full Text Available A novel gene (designated as cen219 encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50 °C and 6.0. It was stable from 30 to 50 °C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn(2+ and dramatically reduced by detergent SDS and metals Fe(3+, Cu(2+ or Hg(2+. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The Km and Vmax of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host-parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.

  19. Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system.

    Science.gov (United States)

    Ham, Jong Hyun; Cui, Yaya; Alfano, James R; Rodríguez-Palenzuela, Pablo; Rojas, Clemencia M; Chatterjee, Arun K; Collmer, Alan

    2004-02-01

    The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an

  20. Inhibition of Erwinia chrysanthemi growth to different concentrations of folic acid: possible use of folic acid as bacteriostatic agent and fortifying of Solanum tuberosum potato

    Directory of Open Access Journals (Sweden)

    Andrea Marcelo Correa

    2017-05-01

    Full Text Available Introduction:The enterobacteria of the Erwinia spp genus produce disease in potatoes, which is a tuber of mass consumption. The regulation of DNA methylation can regulate the proliferation of Erwinia in such a way that the concentrations of folic acid may have an effect on the microorganism pathogenic ability. On the other hand, the folic acid prevents the appearance of neural tube defects in humans. Objective: To evaluate folic acid as a bacteriostatic agent of Erwinia and, at the same time, as part of the fortification of mass consumption food such as the potatoes. Materials and methods: The biochemical characterization of the Erwinia chrysanthemi was carried out and its growth compared to different concentrations of folic acid was studied. Results: When increasing the concentrations of the vitamin from 0.3 µg/L up to 6.8 µg/L, the bacterial growth of Erwinia chrysanthemi is inhibited. The vitamin inhibits the growth in cultivation of Erwinia chrysanthemi and acts as a bacteriostatic agent. This aspect is of great importance given that, theoretically, if potatoes were fortified with micro-nutrient, this would act against the infectious agent and, at the same time, contribute to the adequate intake of the vitamin in the general population.

  1. Streptomyces misionensis PESB-25 Produces a Thermoacidophilic Endoglucanase Using Sugarcane Bagasse and Corn Steep Liquor as the Sole Organic Substrates

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    Marcella Novaes Franco-Cirigliano

    2013-01-01

    Full Text Available Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB as carbon source and corn steep liquor (CSL as nitrogen source, whose concentrations were optimized using response surface methodology (RSM. A peak of endoglucanase accumulation (1.01 U·mL−1 was observed in a medium with SCB 1.0% (w/v and CSL 1.2% (w/v within three days of cultivation. S. misionensis PESB-25 endoglucanase activity was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62° to 70°C, respectively. In these conditions, values of 1.54 U mL−1 of endoglucanase activity were observed. Moreover, Mn2+ was demonstrated to have a hyperactivating effect on the enzyme. In the presence of MnSO4 (8 mM, the enzyme activity increased threefold, up to 4.34 U·mL−1. Mn2+ also improved endoglucanase stability as the catalyst retained almost full activity upon incubation at 50°C for 4 h, while in the absence of Mn2+, enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.

  2. 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.

  3. Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation

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    P. Chellapandi

    2008-03-01

    Full Text Available Cellulase is a complex enzyme system, commercially produced by filamentous fungi under solid-state and submerged cultivation. It has wide applicability in textile, food and beverage industry for effective saccharification process. In this study, cellulolytic enzyme activity, particularly endoglucanase of 26 Streptomyces strains isolated from garden soil was examined, including two isolates selected on the basis of potential cellulolytic activity on Bennett's agar medium. To enhance the endoglucanase formation in broth culture, different conditions including carbon and nitrogen sources, and growth conditions were tested. The maximum endoglucanase activity (11.25-11.90 U/mL was achieved within 72-88 h in fermentation medium containing Tween-80, followed by phosphate sources. Both cellulolytic Streptomyces isolates gave almost equal quantity of enzyme in all trials. However the effect of medium ingredients on endoglucanase induction diverged with strains in some extent.A celulase é um sistema enzimático complexo, produzido comercialmente a partir de fungos filamentosos através de cultivo em estádio sólido e submerso. Tem uma grande aplicação na indústria têxtil e de alimentos e bebidas no processo de sacarificação. Nesse estudo, examinou-se a atividade celulolítica, especialmente de englucanase, de 26 cepas de Streptomyces isoladas de solo, incluindo duas cepas selecionadas por sua atividade celulolítica no ágar Bennett. Para estimular a produção de englucanase em meio de cultura, diferentes condições de cultivo, incluindo fonte de carbono e nitrogênio e condições de crescimento, foram avaliadas. A atividade máxima de glucanase (11,25 a 11,90 U/mL foi obtida em 72-88h em meio de cultura contendo Tween-80, seguido por fontes de fosfato. Ambas as cepas celulolíticas de Streptomyces produziram quase a mesma quantidade de enzima em todos os experimentos. Entretanto, o efeito dos ingredientes do meio na indução da glucanase

  4. Bioprocessing of some agro-industrial residues for endoglucanase production by the new subsp.; Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J

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    Noura El-Ahmady El-Naggar

    2014-06-01

    Full Text Available The use of low cost agro-industrial residues for the production of industrial enzymes is one of the ways to reduce significantly production costs. Cellulase producing actinomycetes were isolated from soil and decayed agricultural wastes. Among them, a potential culture, strain NEAE-J, was selected and identified on the basis of morphological, cultural, physiological and chemotaxonomic properties, together with 16S rDNA sequence. It is proposed that strain NEAE-J should be included in the species Streptomyces albogriseolus as a representative of a novel sub-species, Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J and sequencing product was deposited in the GenBank database under accession number JN229412. This organism was tested for its ability to produce endoglucanase and release reducing sugars from agro-industrial residues as substrates. Sugarcane bagasse was the most suitable substrate for endoglucanase production. Effects of process variables, namely incubation time, temperature, initial pH and nitrogen source on production of endoglucanase by submerged fermentation using Streptomyces albogriseolus subsp. cellulolyticus have been studied. Accordingly optimum conditions have been determined. Incubation temperature of 30 ºC after 6 days, pH of 6.5, 1% sugarcane bagasse as carbon source and peptone as nitrogen source were found to be the optimum for endoglucanase production. Optimization of the process parameters resulted in about 2.6 fold increase in the endoglucanase activity. Therefore, Streptomyces albogriseolus subsp. cellulolyticus coud be potential microorganism for the intended application.

  5. Bioprocessing of some agro-industrial residues for endoglucanase production by the new subsp.; Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J

    Science.gov (United States)

    El-Naggar, Noura El-Ahmady; Abdelwahed, Nayera A.M.; Saber, Wesam I.A.; Mohamed, Asem A.

    2014-01-01

    The use of low cost agro-industrial residues for the production of industrial enzymes is one of the ways to reduce significantly production costs. Cellulase producing actinomycetes were isolated from soil and decayed agricultural wastes. Among them, a potential culture, strain NEAE-J, was selected and identified on the basis of morphological, cultural, physiological and chemotaxonomic properties, together with 16S rDNA sequence. It is proposed that strain NEAE-J should be included in the species Streptomyces albogriseolus as a representative of a novel sub-species, Streptomyces albogriseolus subsp. cellulolyticus strain NEAE-J and sequencing product was deposited in the GenBank database under accession number JN229412. This organism was tested for its ability to produce endoglucanase and release reducing sugars from agro-industrial residues as substrates. Sugarcane bagasse was the most suitable substrate for endoglucanase production. Effects of process variables, namely incubation time, temperature, initial pH and nitrogen source on production of endoglucanase by submerged fermentation using Streptomyces albogriseolus subsp. cellulolyticus have been studied. Accordingly optimum conditions have been determined. Incubation temperature of 30 °C after 6 days, pH of 6.5, 1% sugarcane bagasse as carbon source and peptone as nitrogen source were found to be the optimum for endoglucanase production. Optimization of the process parameters resulted in about 2.6 fold increase in the endoglucanase activity. Therefore, Streptomyces albogriseolus subsp. cellulolyticus coud be potential microorganism for the intended application. PMID:25242966

  6. Identification of an Extracellular Endoglucanase That Is Required for Full Virulence in Xanthomonas citri subsp. citri.

    Directory of Open Access Journals (Sweden)

    Tian Xia

    Full Text Available Xanthomonas citri subsp. citri causes citrus canker disease, which is characterized by the formation of water-soaked lesions, white or yellow spongy pustules and brown corky canker. In this work, we report the contribution of extracellular endoglucanase to canker development during infection. The ectopic expression of nine putative cellulases in Escherichia coli indicated that two endoglucanases, BglC3 and EngXCA, show carboxymethyl cellulase activity. Both bglC3 and engXCA genes were transcribed in X. citri subsp. citri, however, only BglC3 protein was detected outside the cell in western blot analysis. The deletion of bglC3 gene resulted in complete loss of extracellular carboxymethyl cellulase activity and delayed the onset of canker symptoms in both infiltration- and wound-inoculation assays. When growing in plant tissue, the cell density of bglC3 mutant was lower than that of the wild type. Our data demonstrated that BglC3 is an extracellular endoglucanase required for the full virulence of X. citri subsp. citri.

  7. Functional and modular analyses of diverse endoglucanases from Ruminococcus albus 8, a specialist plant cell wall degrading bacterium.

    Science.gov (United States)

    Iakiviak, Michael; Devendran, Saravanan; Skorupski, Anna; Moon, Young Hwan; Mackie, Roderick I; Cann, Isaac

    2016-07-21

    Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases, and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli, and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461, and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.

  8. Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

    Energy Technology Data Exchange (ETDEWEB)

    Kojima, Motoki; Akahoshi, Tomohiro; Okamoto, Kenji; Yanase, Hideshi [Tottori Univ. (Japan). Dept. of Chemistry and Biotechnology

    2012-11-15

    In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae. (orig.)

  9. Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: involvement of the main transcriptional factors.

    Science.gov (United States)

    Rouanet, C; Nomura, K; Tsuyumu, S; Nasser, W

    1999-10-01

    The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the pelD and pelE genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a lacZ reporter gene allowed us to quantify the repression of E. chrysanthemi 3937 pelD expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the pelD and pelE wild-type or pelE mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the pelD and pelE genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the pelD and pelE genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates pelD and pelE expression rather than acting as a true repressor like KdgR. Overall, control of the pelD and pelE genes of E. chrysanthemi appears to be both complex and novel.

  10. Two tomato endoglucanases have a function during syncytium development

    Directory of Open Access Journals (Sweden)

    Małgorzata Lichocka

    2011-01-01

    Full Text Available Globodera rostochiensis, as well as other cyst nematodes, induces formation of a multinucleate feeding site, called syncytium, in host roots. In tomato roots infected with a potato cyst nematode, the syncytium is initiated in the cortex or pericycle. Progressive cell wall dissolution and subsequent fusion of protoplasts of newly incorporated cells lead to syncytium formation. Expansion and development of a syncytium strongly depends on modifications of a cell wall, including its degradation, elongation, thickening, and formation of ingrowths within it in close contact with tracheary elements. Recent reports have demonstrated that during formation of syncytium, numerous genes of plant origin, coding for cell wall-modifying enzymes are up-re-gulated. In this research, we studied a detailed distribution and function of two tomato 1,4-β-endoglucanases in developing feeding sites induced by G. rostochiensis. In situ localization of tomato LeCel7 and LeCel8 transcripts and proteins demonstrated that these enzymes were specifically up-regulated within syncytium and in the cells adjacent to the syncytium. In non-infected roots an expression of LeCel7 and LeCel8 was observed in the root cap and lateral root primordia. Our data confirm that cell wall-modifying enzymes of plant origin have a role in a modification of cell wall within syncytia, and demonstrate that plant endoglucanases are involved in syncytia formation.

  11. Screening of endoglucanase-producing bacteria in the saline rhizosphere of Rhizophora mangle

    Science.gov (United States)

    Sá, André Luís Braghini; Dias, Armando Cavalcante Franco; Quecine, Maria Carolina; Cotta, Simone Raposo; Fasanella, Cristiane Cipola; Andreote, Fernando Dini; de Melo, Itamar Soares

    2014-01-01

    In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity. PMID:24948930

  12. Screening of endoglucanase-producing bacteria in the saline rhizosphere of Rhizophora mangle

    Directory of Open Access Journals (Sweden)

    André Luís Braghini Sá

    2014-01-01

    Full Text Available In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity.

  13. Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase.

    Science.gov (United States)

    Kotaka, Atsushi; Bando, Hiroki; Kaya, Masahiko; Kato-Murai, Michiko; Kuroda, Kouichi; Sahara, Hiroshi; Hata, Yoji; Kondo, Akihiko; Ueda, Mitsuyoshi

    2008-06-01

    Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.

  14. Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

    2014-10-01

    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  15. Reaction of arracacha genotypes to the root soft rot caused by Pectobacterium chrysanthemi Reação de genótipos de mandioquinha-salsa à podridão-mole das raízes causada por Pectobacterium chrysanthemi

    Directory of Open Access Journals (Sweden)

    Gilmar Paulo Henz

    2005-01-01

    Full Text Available The purpose of this paper was to screen thirty-two arracacha genotypes for their reaction to root soft rot. Twenty roots of each genotype were inoculated with two Pectobacterium chrysanthemi isolates in a randomized experiment (10 roots/isolate. After inoculation, roots were individually wrapped with PVC film and kept at 26ºC in closed plastic bags. Soft rot lesions were recorded after 36 hours and genotypes were grouped in four classes of susceptibility by cluster analysis: 10 were less susceptible, 16 intermediate, 3 susceptible and 3 very susceptible. All the tested arracacha genotypes showed only variation in the degree of susceptibility.O objetivo deste trabalho foi avaliar a reação de 32 genótipos de mandioquinha-salsa à podridão-mole das raízes. Vinte raízes de cada genótipo foram inoculadas com dois isolados de Pectobacterium chrysanthemi em um experimento casualizado (10 raízes/isolado. Após a inoculação, as raízes foram embaladas com filmes de PVC e mantidas a 26ºC em sacos de plástico. As lesões de podridão-mole foram avaliadas após 36 horas e os genótipos agrupados em quatro classes de suscetibilidade por análise de agrupamento: 10 foram menos suscetíveis, 16 intermediários, 3 suscetíveis e 3 muito suscetíveis. Todos os genótipos avaliados demonstraram apenas variação no grau de suscetibilidade.

  16. PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937▿ †

    OpenAIRE

    Hommais, Florence; Oger-Desfeux, Christine; Van Gijsegem, Frédérique; Castang, Sandra; Ligori, Sandrine; Expert, Dominique; Nasser, William; Reverchon, Sylvie

    2008-01-01

    Pathogenicity of the enterobacterium Erwinia chrysanthemi (Dickeya dadantii), the causative agent of soft-rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. PecS is a transcriptional regulator that controls production of various virulence factors. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS notably regulates a wide range of genes that could be linked to path...

  17. The extracellular β-1,3-endoglucanase EngA is involved in autolysis of Aspergillus nidulans.

    Science.gov (United States)

    Szilágyi, M; Kwon, N-J; Dorogi, C; Pócsi, I; Yu, J-H; Emri, T

    2010-11-01

    To elucidate the roles of the β-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. A β-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. The β-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall-degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  18. Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124.

    Science.gov (United States)

    de Brito, Aila Riany; Santos Reis, Nadabe Dos; Silva, Tatielle Pereira; Ferreira Bonomo, Renata Cristina; Trovatti Uetanabaro, Ana Paula; de Assis, Sandra Aparecida; da Silva, Erik Galvão Paranhos; Aguiar-Oliveira, Elizama; Oliveira, Julieta Rangel; Franco, Marcelo

    2017-11-26

    Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8 hr (rice husks) and 7.3 hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5-7 (rice hulls) and 6-9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.

  19. Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

    OpenAIRE

    Avrova, Anna O.; Hyman, Lizbeth J.; Toth, Rachel L.; Toth, Ian K.

    2002-01-01

    The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (c...

  20. Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kelvin Swee Chuan Wei

    2015-03-01

    Conclusions: The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.

  1. Multivariable parameter optimization for the endoglucanase production by Trichoderma reesei Rut C30 from Ocimum gratissimum seed

    Directory of Open Access Journals (Sweden)

    Mithu Das

    2008-02-01

    Full Text Available The aim of this study was to evaluate the interaction effects of the physico-chemical parameters on the endoglucanase (CMCase production by Trichoderma reesei Rut C30 on a cellulosic agro-residue by the solid-state fermentation (SSF and to determine their optimum values by the EVOP factorial design technique. The best combination of physical parameters for the maximum production of the endoglucanase (CMCase was 28ºC temperature, 79% relative humidity and 4.8 pH of the medium. The best combination of the chemical parameters was (mg/L nicotinic acid 15, naphthalene acetic acid 7, ferric chloride 5 and Tween-80 6. With the application of this technique, the yield of the CMCase increased by ~ 2.3 fold.

  2. Effect of pH, Temperature, and Chemicals on the Endoglucanases and β-Glucosidases from the Thermophilic Fungus Myceliophthora heterothallica F.2.1.4. Obtained by Solid-State and Submerged Cultivation

    Directory of Open Access Journals (Sweden)

    Vanessa de Cássia Teixeira da Silva

    2016-01-01

    Full Text Available This work reports endoglucanase and beta-glucosidase production by the thermophilic fungus Myceliophthora heterothallica in solid-state (SSC and submerged (SmC cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. For β-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSC β-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl3 exerted the strongest inhibition for endoglucanases and β-glucosidases.

  3. Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid Hh, and regulation of pectolytic enzymes.

    Science.gov (United States)

    Llama-Palacios, Arancha; López-Solanilla, Emilia; Rodríguez-Palenzuela, Pablo

    2005-03-01

    Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad number of crops. The PhoP-PhoQ system is a key factor in pathogenicity of several bacteria and is involved in the bacterial resistance to different factors, including acid stress. Since E. chrysanthemi is confronted by acid pH during pathogenesis, we have studied the role of this system in the virulence of this bacterium. In this work, we have isolated and characterized the phoP and phoQ mutants of E. chrysanthemi strain 3937. It was found that: (i) they were not altered in their growth at acid pH; (ii) the phoQ mutant showed diminished ability to survive at acid pH; (iii) susceptibility to the antimicrobial peptide thionin was increased; (iv) the virulence of the phoQ mutant was diminished at low and high magnesium concentrations, whereas the virulence of the phoP was diminished only at low magnesium concentrations; (v) in planta Pel activity of both mutant strains was drastically reduced; and (vi) both mutants lagged behind the wild type in their capacity to change the apoplastic pH. These results suggest that the PhoP-PhoQ system plays a role in the virulence of this bacterium in plant tissues, although it does not contribute to bacterial growth at acid pH.

  4. Analysis of the pelE promoter in Erwinia chrysanthemi EC16.

    Science.gov (United States)

    Gold, S; Nishio, S; Tsuyumu, S; Keen, N T

    1992-01-01

    The pelE gene of Erwinia chrysanthemi strain EC16 encodes an extracellular pectate lyase protein that is important in virulence on plants. Control of pelE expression is complex, because the gene is regulated by catabolite repression, substrate induction, and growth-phase inhibition. A Tn7-lux reporter gene system was employed to define DNA sequences comprising the pelE promoter. When EC16 cells were grown on medium containing sodium polypectate, pelE transcriptional start sites were observed only at 95 and 96 bases upstream of the translational start site. However, DNA sequences required for pelE expression were also shown by deletion analysis to reside between 196 and 215 base pairs upstream of the translational start site. In addition to these upstream elements, two putative operator sequences that interact with negative regulatory factors occurred downstream of the transcriptional start. Finally, deletion of three bases from a putative catabolite gene activator protein binding site in the pelE promoter eliminated activity. The data demonstrate that the pelE promoter is complex and suggest that it interacts with several regulatory proteins.

  5. Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins

    DEFF Research Database (Denmark)

    Krogh, Kristian Bertel Rømer; Kastberg, H.; Jørgensen, C. I.

    2009-01-01

    The cel5C gene, coding for an endoglucanase (Cel5C) of Penicillium brasilianum was cloned and heterologously expressed in Aspergillus oryzae. This is only the second GH5 EG from the genus penicillium reported in the CAZy database. The promoter region of the gene has I)putative binding sites...

  6. Espaços e equipamentos para a educação física escolar e não-escolar: entrevista com Celi Nelza Zulke Taffarel

    Directory of Open Access Journals (Sweden)

    Christiane Garcia Macedo

    2012-12-01

    Full Text Available http://dx.doi.org/10.5007/2175-8042.2012v24n39p66 Entrevista com a Professora Celi Neuza Taffarel sobre espaços e equipamentos para a Educação Física escolar e não escolar. Na entrevista são apresentados as relações deste tema com as políticas públicas e a formação de professores. A professora destaca que é necessário que a comunidade acadêmica se debruce sobre o tema, buscando mais pesquisas e com a finalidade de garantir acesso público aos trabalhadores. Essa entrevista está integrada ao projeto Garimpando Memórias (CEME/ESEF/UFRGS.

  7. Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β-endoglucanase.

    Science.gov (United States)

    Yan, Junjie; Liu, Weidong; Li, Yujie; Lai, Hui-Lin; Zheng, Yingying; Huang, Jian-Wen; Chen, Chun-Chi; Chen, Yun; Jin, Jian; Li, Huazhong; Guo, Rey-Ting

    2016-06-17

    Eukaryotic 1,4-β-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-β-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (β/α)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to -4 - -1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. 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...

  9. Nucleotide sequences of the Erwinia chrysanthemi ogl and pelE genes negatively regulated by the kdgR gene product.

    Science.gov (United States)

    Reverchon, S; Huang, Y; Bourson, C; Robert-Baudouy, J

    1989-12-21

    The nucleotide sequences of the coding and regulatory regions of the genes encoding oligoglacturonate lyase (OGL) and pectate lyase e isoenzyme (PLe) from Erwinia chrysanthemi 3937 were determined. The ogl sequence contains an open reading frame (ORF) of 1164 bp coding for a 388-amino acid (aa) polypeptide with a predicted Mr of 44,124. A possible transcriptional start signal showing homology with the Escherichia coli promoter consensus sequence was detected. In addition, a sequence 3' to the coding region was found to be able to form a secondary structure which may function as an Rho-independent transcriptional termination signal. For the pelE sequence, a long ORF of 1212 bp coding for a 404-aa polypeptide was detected. PLe is secreted into the external medium by E. chrysanthemi, and a potential signal peptide sequence was identified in the pelE gene. In the 5' upstream pelE coding region, a putative promoter resembling E. coli promoter consensus sequences was detected. Furthermore, the region immediately 3' to the pelE translational stop codon may function as an Rho-independent translational termination signal. In strain 3937, the synthesis of OGL and PLe, as well as the other enzymes involved in the pectin-degradative pathway (particularly the kdgT product), are known to be regulated by the KdgR repressor, which mediates galacturonate and polygalacturonate induction. Synthesis of these enzymes is also regulated by the CRP-cAMP complex which mediates catabolite repression. Analysis of the regulatory regions of ogl and pelE allowed us to identify possible CRP-binding sites for these two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Optimization of extraction of linarin from Flos chrysanthemi indici by response surface methodology and artificial neural network.

    Science.gov (United States)

    Pan, Hongye; Zhang, Qing; Cui, Keke; Chen, Guoquan; Liu, Xuesong; Wang, Longhu

    2017-05-01

    The extraction of linarin from Flos chrysanthemi indici by ethanol was investigated. Two modeling techniques, response surface methodology and artificial neural network, were adopted to optimize the process parameters, such as, ethanol concentration, extraction period, extraction frequency, and solvent to material ratio. We showed that both methods provided good predictions, but artificial neural network provided a better and more accurate result. The optimum process parameters include, ethanol concentration of 74%, extraction period of 2 h, extraction three times, solvent to material ratio of 12 mL/g. The experiment yield of linarin was 90.5% that deviated less than 1.6% from that obtained by predicted result. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Coexpression and Secretion of Endoglucanase and Phytase Genes in Lactobacillus reuteri

    Science.gov (United States)

    Wang, Lei; Yang, Yuxin; Cai, Bei; Cao, Pinghua; Yang, Mingming; Chen, Yulin

    2014-01-01

    A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade β-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21. PMID:25050780

  12. Plant Phenolics Extraction from Flos Chrysanthemi: Response Surface Methodology Based Optimization and the Correlation Between Extracts and Free Radical Scavenging Activity.

    Science.gov (United States)

    Wu, Yanfang; Wang, Xinsheng; Xue, Jintao; Fan, Enguo

    2017-11-01

    Huaiju is one of the most famous and widely used Flos Chrysanthemi (FC) for medicinal purposes in China. Although various investigations aimed at phenolics extraction from other FC have been reported, a thorough optimization of the phenolics extraction conditions from Huaiju has not been achieved. This work applied the widely used response surface methodology (RSM) to investigate the effects of 3 independent variables including ethanol concentration (%), extraction time (min), and solvent-to-material ratio (mL/g) on the ultrasound-assisted extraction (UAE) of phenolics from FC. The data suggested the optimal UAE condition was an ethanol concentration of 75.3% and extraction time of 43.5 min, whereas the ratio of solvent to material has no significant effect. When the free radical scavenging ability was used as an indicator for a successful extraction, a similar optimal extraction was achieved with an ethanol concentration of 72.8%, extraction time of 44.3 min, and the ratio of solvent to material was 29.5 mL/g. Furthermore, a moderate correlation between the antioxidant activity of TP extract and the content of extracted phenolic compounds was observed. Moreover, a well consistent of the experimental values under optimal conditions with those predicted values suggests RSM successfully optimized the UAE conditions for phenolics extraction from FC. The work of the research investigated the plant phenolics in Flos Chrysanthemi and antioxidant capacities. These results of this study can support the development of antioxidant additive and relative food. © 2017 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.

  13. Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene ( from sp. isolated from Holstein steers’ rumen

    Directory of Open Access Journals (Sweden)

    Tansol Park

    2018-04-01

    Full Text Available Objective This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A from the isolated microorganism. Methods To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens Ce2 (Accession number: AB163733. The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. Results The maximum activity of recombinant Cel5A (rCel5A was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 μmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s−1 and 6.88 (mL/mg/s, respectively. Conclusion Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

  14. 19 July 2013 - Chairman of the Policy Committee, European Cancer Organisation, President, European Association for Cancer Research E. Celis visiting the ATLAS experimental cavern with ATLAS Collaboration Deputy Spokesperson, B. Heinemann and signing the Guest Book with Director for Accelerators and Technology S. Myers. Life Sciences Adviser M. Dosanjh present.

    CERN Multimedia

    Anna Pantelia

    2013-01-01

    19 July 2013 - Chairman of the Policy Committee, European Cancer Organisation, President, European Association for Cancer Research E. Celis visiting the ATLAS experimental cavern with ATLAS Collaboration Deputy Spokesperson, B. Heinemann and signing the Guest Book with Director for Accelerators and Technology S. Myers. Life Sciences Adviser M. Dosanjh present.

  15. A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures: an anomaly of truly cold-adapted evolution in a mesophilic environment.

    Science.gov (United States)

    Khalili Ghadikolaei, Kamran; Gharechahi, Javad; Haghbeen, Kamahldin; Akbari Noghabi, Kambiz; Hosseini Salekdeh, Ghasem; Shahbani Zahiri, Hossein

    2018-03-01

    Endoglucanases are important enzymes in plant biomass degradation. They have current and potential applications in various industrial sectors including human and animal food processing, textile, paper, and renewable biofuel production. It is assumed that the cold-active endoglucanases, with high catalytic rates in moderate and cold temperatures, can improve the cost-effectiveness of industrial processes by lowering the need for heating and, thus, energy consumption. In this study, the endoglucanase CelCM3 was procured from a camel rumen metagenome via gene cloning and expression in Escherichia coli BL21 (DE3). The maximum activity of the enzyme on carboxymethyl cellulose (CMC) was obtained at pH 5 and 30 °C with a V max and K m of 339 U/mg and 2.57 mg/ml, respectively. The enzyme with an estimated low melting temperature of 45 °C and about 50% activity at 4 °C was identified to be cold-adapted. A thermodynamic analysis corroborated that CelCM3 with an activation energy (E a ), enthalpy of activation (ΔH), and Gibb's free energy (ΔG) of, respectively, 18.47 kJ mol -1 , 16.12 kJ mol -1 , and 56.09 kJ mol -1 is a cold-active endoglucanase. In addition, CelCM3 was tolerant of metal ions, non-ionic detergents, urea, and organic solvents. Given these interesting characteristics, CelCM3 shows promise to meet the requirements of industrial applications.

  16. 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.

  17. An endoglucanase is involved in infection of rice roots by the not-cellulose-metabolizing endophyte Azoarcus sp. strain BH72.

    Science.gov (United States)

    Reinhold-Hurek, Barbara; Maes, Tamara; Gemmer, Sabrina; Van Montagu, Marc; Hurek, Thomas

    2006-02-01

    The nitrogen-fixing endophyte Azoarcus sp. strain BH72 infects roots of Kallar grass and rice inter- and intra-cellularly and can spread systemically into shoots without causing symptoms of plant disease. Although cellulose or its breakdown products do not support growth, this strain expresses an endoglucanase, which might be involved in infection. Sequence analysis of eglA places the secreted 34-kDa protein into the glycosyl hydrolases family 5, with highest relatedness (40% identity) to endoglucanases of the phytopathogenic bacteria Xanthomonas campestris and Ralstonia solanacearum. Transcriptional regulation studied by eglA:: gusA fusion was not significantly affected by cellulose or its breakdown products or by microaerobiosis. Strongest induction (threefold) was obtained in bacteria grown in close vicinity to rice roots. Visible sites of expression were the emergence points of lateral roots and root tips, which are the primary regions of ingress into the root. To study the role in endophytic colonization, eglA was inactivated by transposon mutagenesis. Systemic spreading of the eglA mutant and of a pilAB mutant into the rice shoot could no longer be detected by polymerase chain reaction. Microscopic inspection of infection revealed that the intracellular colonization of root epidermis cells was significantly reduced in the eglA- mutant BHE6 compared with the wild type and partially restored in the complementation mutant BHRE2 expressing eglA. This provides evidence that Azoarcus sp. endoglucanase is an important determinant for successful endophytic colonization of rice roots, suggesting an active bacterial colonization process.

  18. The genomic organization of four b-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications.

    NARCIS (Netherlands)

    Yan, Y.; Smant, G.; Stokkermans, J.P.W.G.; Qin Ling,; Baum, T.J.; Schots, A.; Davis, E.L.

    1998-01-01

    The genomic organization of genes encoding -1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of

  19. Genetic analysis of the pelA-pelE cluster encoding the acidic and basic pectate lyases in Erwinia chrysanthemi EC16.

    Science.gov (United States)

    Barras, F; Chatterjee, A K

    1987-10-01

    In Erwinia chrysanthemi (EC16) the clustered pelA and pelE genes encode an acidic (pI 4.2) and a basic (pI 10.0) pectate lyase (Pel), respectively. The pelA gene has been isolated on a 1.2 kb restriction fragment and the direction of transcription determined. DNA hybridization analysis showed that the pelE sequence shares DNA homology with pelA but not with pelB or pelC, two genes encoding other Pel species in EC16. Since Pel A and Pel E enzymes showed little similarity in terms of catalytic properties, it is proposed that pelA and pelE are duplicates which have highly diverged.

  20. Effect of multiple short highly energetic X-ray pulses on the synthesis of endoglucanase by a mutant strain of Trichoderma reesei-M7

    International Nuclear Information System (INIS)

    Gemishev, Orlin; Markova, Maya; Savov, Valentin; Zapryanov, Stanislav; Blagoev, Alexander

    2014-01-01

    Bioconversion of cellulose-containing substrate to glucose represents an important area of modern biotechnology. Enzymes for the degradation of the polysaccharide part of biomass have been produced, mostly by fungi belonging to genus Trichoderma. Studies were carried out with the mutant strain Trichoderma reesei-M7, a cellulase producer. Spores of the enzyme producer were irradiated with different doses of characteristic X-ray radiation from metallic tungsten (mainly the W Ka1 and Ka2 lines) with a high dose rate. The latter is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles. In this case, we focused our study on the influence of hard X-rays. The doses of X-rays absorbed by the spores varied in the range of approximately 5-11,000 mSv measured with thermoluminescent dosimeters (TLD). The influence of the applied doses in combination with exceptionally high dose rates (in the order of tens of millisieverts per microsecond) on the activity of the produced endoglucanase, amount of biomass and extra-cellular protein, was studied in batch cultivation conditions. In the dose range of 200-1200 mSv, some enhancement of endoglucanase activity was obtained: around 18%-32%, despite the drop of the biomass amount, compared with the untreated material. Keywords: endoglucanase; X-ray pulses; thermoluminescent dosimeters (TLD); dense plasma focus (DPF); Trichoderma reesei

  1. Carboxymethyl-cellulase from Erwinia chrysanthemi. II. Purification and partial characterization of an endo-. beta. -1,4-glucanase

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, M.H.; Chambost, J.P.; Magnan, M.; Cattaneo, J.

    1984-01-01

    The extracellular carboxymethyl-cellulase of Erwinia chrysanthemi, strain 3665, had a marked tendency to form aggregates when concentration and/or storage time of culture supernatant were increased. In submitting an unconcentrated glycerol culture supernatant to ion exchange chromatography, one major endo-..beta..-1,4,-glucanase could be isolated with a high degree of purity and partially characterized. The molecular size was 45 kd. The pI was 4.3. The enzyme rapidly decreased the viscosity of carboxymethyl-cellulose with a slow increase in the reducing sugars produced. It displayed its highest activity towards carboxymethyl-cellulose at a pH between 6.2 and 7.5. It had a significant capacity to hydrolyze amorphous cellulose such as phosphoric acid-swollen cellulose. The major products of this degradation were cellobiose and cellotriose. It exhibited a very low activity on microcrystalline cellulose. Glucose and cellobiose did not affect significantly its activity against carboxymethyl-cellulose. 21 references.

  2. Characteristics of a β-1,4-D endoglucanase from Trichoderma virens wholly applied in a palm-fruit husk-based diet for poultry layers

    Directory of Open Access Journals (Sweden)

    Olubusola A. Odeniyi

    2012-12-01

    Full Text Available The characteristics of an endoglucanase produced by a Trichoderma virens strain T9 newly isolated from a palm-fruit husk dump site, its physiological characteristics and enzyme production were studied. Whole cells of the depolymerizing-enzyme producing T. virens were applied to palm-fruit husk and bird performance characteristics when employed as poultry diet additive were considered. Endoglucanase activity in submerged fermentation was 1.6 nkat. Optimum activity was recorded at pH 6.0 and 55ºC. The enzyme retained 50% residual glucanase activity at 70ºC for 10 minutes. 1.0% Tween-80 and SDS yielded endoglucanase activity 2.15 times higher than the control. Activity wasboosted by 20mM Ca2+ (115.0%; 10mM K+ (106.5%; and was totally inhibited by 1mM Hg2+. The addition of T. virens -fermented palm-fruit husk with other layer feed components on the bird characteristics showed that change in bird weight between the control and test birds were not significantly different (p>0.05 but differed in terms of daily feed ingested (p0.05. The shell thickness (0.64mm and yolk content (23.61% were highest in the microbially-modified husk diet. The alternative to maize based diets proffered by the application of T. virens -modified palm-fruit husk in poultry nutrition in terms of bird weight and feed to weight-gain ratio affords the poultry farmer an economic advantage and allows for a greater utilization of the maize in human diets.

  3. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Directory of Open Access Journals (Sweden)

    Klose Holger

    2012-08-01

    Full Text Available Abstract Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354 isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

  4. Cellobiohydrolase and endoglucanase respond differently to surfactants during the hydrolysis of cellulose

    DEFF Research Database (Denmark)

    Hsieh, Chia-wen C.; Cannella, David; Jørgensen, Henning

    2015-01-01

    Background: Non-ionic surfactants such as polyethylene glycol (PEG) can increase the glucose yield obtained from enzymatic saccharification of lignocellulosic substrates. Various explanations behind this effect include the ability of PEG to increase the stability of the cellulases, decrease non......-productive cellulase adsorption to the substrate, and increase the desorption of enzymes from the substrate. Here, using lignin-free model substrates, we propose that PEG also alters the solvent properties, for example, water, leading the cellulases to increase hydrolysis yields.Results: The effect of PEG differs...... for the individual cellulases. During hydrolysis of Avicel and PASC with a processive monocomponent exo-cellulase cellobiohydrolase (CBH) I, the presence of PEG leads to an increase in the final glucose concentration, while PEG caused no change in glucose production with a non-processive endoglucanase (EG). Also...

  5. OPTIMIZATION OF FERMENTATION PARAMETERS FOR THE PRODUCTION OF EXTRACELLULAR ENDOGLUCANASE, β –GLUCOSIDASE AND ENDOXYLANASE BY A CHROMIUM RESISTANT STRAIN OF TRICHODERMA PSEUDOKONINGII

    Directory of Open Access Journals (Sweden)

    Rina Rani Ray

    2013-08-01

    Full Text Available Trichoderma pseudokoningii, a chromate reducing fungal strain, was isolated from the tannery-effluents. The present Cr (VI resistant strain was found to produce good amount of various extracellular enzymes that included cellulases (endoglucanase and β–glucosidase and hemicellulase (endoxylanase in submerged fermentation (SmF. The titre of β–glucosidase was found to be higher than that of endoglucanase. Cellulases were best induced in presence of 1% of respective substrates whereas only 0.5% xylan could induce endoxylanase production in this strain. Although the optimum temperature for all three enzymes was found to be 27oC, the pH optimum of cellulases (pH 5 were different from that of endoxylanase (pH 6. Under optimized conditions, maximum of production of all these enzymes was achieved within 48 hours of cultivation. Among nitrogen sources tested, potassium nitrate was found to be the most effective followed by gelatin.

  6. The role of the β-1,6-endoglucanase gene vegB in physiology and virulence of Verticillium dahliae

    Directory of Open Access Journals (Sweden)

    Lugard EBOIGBE

    2014-05-01

    Full Text Available The β-1,6-endoglucanase gene (vegB of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute, but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type.

  7. Purification, molecular cloning, and enzymatic properties of a family 12 endoglucanase (EG-II) from fomitopsis palustris: role of EG-II in larch holocellulose hydrolysis.

    Science.gov (United States)

    Shimokawa, Tomoko; Shibuya, Hajime; Nojiri, Masanobu; Yoshida, Shigeki; Ishihara, Mitsuro

    2008-09-01

    A family 12 endoglucanase with a molecular mass of 23,926 Da (EG-II) from the brown-rot basidiomycete Fomitopsis palustris was purified and characterized. One of the roles of EG-II in wood degradation is thought to be to loosen the polysaccharide network in cell walls by disentangling hemicelluloses that are associated with cellulose.

  8. Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members

    DEFF Research Database (Denmark)

    Godoy, Andre S.; Pereira, Caroline S.; Ramia, Marina Paglione

    2018-01-01

    The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general a...

  9. A Path to Freedom——Life of black women in The Color Purple

    Institute of Scientific and Technical Information of China (English)

    袁丽丹

    2005-01-01

    Alice Walker sThe Color Purple,published in 1982,tells the story of Celie,ablack woman in the South.Celie was a blackwoman growing up in the South.She mustovercome masogeny,racism and poverty toestablish herself as an independent person.Thenovel also follows the maturation of her sisterNettie and the lives of Shug,Albert,and muchof his extended family.Celie writes letters toGodin which she tells about her life-her roles asdaughter,wife,sister,and mother.In thecourse of her story,Celie meets a series of othe...

  10. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  11. Endoglucanase production with the newly isolated Myceliophtora sp. i-1d3b in a packed bed solid state fermentor

    Directory of Open Access Journals (Sweden)

    A. I. Zanelato

    2012-12-01

    Full Text Available This work is aimed to produce endoglucanase through solid state fermentation in a packed bed bioreactor with the use of the fungus Myceliophtora sp. I-1D3busing a mixture of wheat bran (WB and sugar cane bagasse (SCB as culture medium. Preliminary tests were performed in polypropylene plastic bags, controlling the variables temperature (40, 45, and 50ºC, initial moisture content (75, 80, and 85%, w.b., and weight proportion SCB/WB (1:1, 7:3, and 9:1. The highest enzyme activities in plastic bags were obtained using the substrate proportion of 7:3, 50ºC temperature, and 80% initial moisture content (878 U/grams of dry solid. High activities of filter-paper cellulase and xylanase were also obtained in plastic bags and some results are reported. For the packed bed experiments, the temperature (45 and 50ºC and the air flow rate (80, 100 and 120L/h were the controlled variables. Activity of endoglucanase was similar to plastic bag tests. A longitudinal gradient of moisture content, was observed increasing from the bottom to the top of the reactor, even though the longitudinal enzyme activity profile was flat for almost the whole bed. Air flow rate did not affect enzyme activity, while experiments carried out at 50ºC showed higher enzyme activities. The maximum temperature peak observed was at about 6ºC above the process temperature.

  12. The phytopathogen Dickeya dadantii (Erwinia chrysanthemi 3937) is a pathogen of the pea aphid.

    Science.gov (United States)

    Grenier, Anne-Marie; Duport, Gabrielle; Pagès, Sylvie; Condemine, Guy; Rahbé, Yvan

    2006-03-01

    Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.

  13. Variabilidade genética na região its do rDNA de isolados de trichoderma spp. (Biocontrolador e Fusarium oxysporum f. sp. Chrysanthemi Genetic variability in rDNA ITS region of Trichoderma spp. (biocontrole agent and Fusarium oxysporum f. sp. chrysanthemi isolates

    Directory of Open Access Journals (Sweden)

    Josiane Pacheco Menezes

    2010-02-01

    Full Text Available A análise de características morfológicas e culturais podem não ser suficientes para uma caracterização precisa das espécies de Trichoderma e Fusarium. Objetivou-se, neste trabalho, caracterizar a região do Espaço Interno Transcrito (ITS do rDNA dos isolados UFSMT15.1, UFSMT16 e UFSMT17 de Trichoderma spp. utilizados no biocontrole de Fusarium oxysporum f. sp. chrysanthemi (isolado UFSMF6. A extração de DNA de cada isolado foi realizada a partir de micélio produzido em meio líquido Batata-Dextrose. As amostras de DNA genômico foram submetidas à Reação em Cadeia da Polimerase (PCR com os oligonucleotídeos iniciadores universais ITS1 e ITS4 e o produto gerado foi sequenciado. Os fragmentos gerados pela amplificação da PCR foram tratados com as enzimas de restrição HaeIII, HinfI e MboI. As regiões ITS1, ITS2 e 5.8S do rDNA desses isolados fúngicos foram amplificadas com sucesso. A região ITS dos isolados UFSMT15.1, UFSMT16 e UFSMT17 de Trichoderma e o isolado UFSMF6 de Fusarium apresentaram uma banda simples com um fragmento de aproximadamente 600 pares de base (pb. As enzimas de restrição HaeIII, HinfI e MboI geraram polimorfismo de bandas entre os isolados. Com base nas análises da sequência de DNA, os isolados UFSMT15.1, UFSMT16, UFSMT17 e UFSMF6 apresentaram maior similaridade com as espécies Trichoderma koningiopsis, Hypocrea virens, Hypocrea lixii e Fusarium oxysporum, respectivamente.The analysis of morphological and cultural characteristics may not enough for the characterization of the species of Trichoderma and Fusarium. The aim of this work was to characterize the Internal Transcribed Spacer (ITS region of the rDNA of UFSMT15.1, UFSMT16 and UFSMT17 isolates of Trichoderma spp. used in the biocontrol of Fusarium oxysporum f. sp. chrysanthemi UFSMF6. DNA extraction of each isolate was accomplished starting from hyphae produced in liquid medium Potato-Dextrose-Agar. The samples of genomic DNA were submitted to

  14. Direct detection of the plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae in infected rice seedlings using matrix assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Kajiwara, Hideyuki

    2016-01-01

    The plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae were directly detected in extracts from infected rice seedlings by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method did not require culturing of the pathogens on artificial medium. In the MALDI-TOF MS analysis, peaks originating from bacteria were found in extracts from infected rice seedlings. The spectral peaks showed significantly high scores, in spite of minor differences in spectra. The spectral peaks originating from host plant tissues did not affect this direct MALDI-TOF MS analysis for the rapid identification of plant pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Role of motility and chemotaxis in the pathogenesis of Dickeya dadantii 3937 (ex Erwinia chrysanthemi 3937).

    Science.gov (United States)

    Antúnez-Lamas, María; Cabrera-Ordóñez, Ezequiel; López-Solanilla, Emilia; Raposo, Rosa; Trelles-Salazar, Oswaldo; Rodríguez-Moreno, Andrés; Rodríguez-Palenzuela, Pablo

    2009-02-01

    Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94%), cheY (80%), cheW (74%), cheB (54%) and cheZ (48%). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA

  16. Improved cellulolytic efficacy in Penicilium decumbens via heterologous expression of Hypocrea jecorina endoglucanase II

    Directory of Open Access Journals (Sweden)

    Qin Yuqi

    2013-01-01

    Full Text Available Hypocrea jecorina endoglucanase II (Hjegl2 was heterologously expressed in Penicillium decumbens (yielding strain Pd::Hjegl2. After induction in cellulose containing media, strain Pd::Hjeg2 displayed increased carboxymethylcellulase activity (CMCase, 5.77 IU/ml, representing a 21% increase and cellulose degradation determined with a filter paper assay (FPA, 0.40 IU/ml, 67% increase, as compared to the parent strain. In media supplemented with glucose (2%, Pd::Hjegl2, displayed 51.2-fold and 3-fold higher CMCase and FPA activities, respectively, as compared to the parent strain. No changes in the expression levels of the four main native cellulase genes of P. decumbens (Pdegl1, Pdegl2, Pdcbh1, and Pdcbh2 were noted between the transformant and wild-type strains. These data support the idea that Hjegl2 cleaves both internal and terminal glycosidic residues, in a relatively random and processive manner. In situ polyacrylamide gelactivity staining of extracts derived from wild-type and Pd::Hjegl2 revealed two additional active fractions in the latter strain; one with a molecular mass ~50-65 KDa and another ~80-116 kDa.

  17. EVALUATION OF ENDOGLUCANASE, EXOGLUCANASE, LACCASE, AND LIGNIN PEROXIDASE ACTIVITIES ON TEN WHITE-ROT FUNGI

    Directory of Open Access Journals (Sweden)

    Sandra Montoya B

    2014-12-01

    Full Text Available This paper presents a way of tracking the production of lignocellulolytic enzymes in ten species of white rot fungi: Lentinula edodes, Schizophyllum commune, Trametes trogii, Coriolus versicolor, Pycnoporus sanguineus, Ganoderma applanatum, Ganoderma lucidum, Grifola frondosa, Pleurotus ostreatus and Auricularia delicata. These species were first screened on solid culture media containing carboxymethyl cellulose, crystalline cellulose, ABTS (2,2´-azino-bis(3-ethylbenzothiazoline-6-sulphonate and azure B, which showed the production of endoglucanase, exoglucanase, laccase and lignin peroxidase (LiP enzymes. Cellulolytic activities were detected after five days of incubation with congo red indicator, forming a clear-white halo in areas where cellulose was degraded. For ligninases, the tracking consisted of the monitoring in the formation of green halos due to ABTS oxidation for laccase, and decolorization halos on azure B for LiP during 14 days of incubation. From this qualitative screening, four strains were selected (G. lucidum, L. edodes, C. versicolor and T. trogii as the best producers of cellulolytic and ligninolytic enzymes. These four species were inoculated on a substrate of sawdust oak, yielding 51,8% of lignin degraded by L. edodes and 22% of cellulose degraded by C. versicolor.

  18. Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

    Directory of Open Access Journals (Sweden)

    Siika-aho Matti

    2011-01-01

    Full Text Available Abstract Background Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases, resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties connected with high initial solid loadings in the lignocellulose to ethanol process. Results The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A. Conclusions Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

  19. [Determination of chlorogenic acid, caffeic acid and linarin in Flos Chrysanthemi Indici from different places by RP-hPLC].

    Science.gov (United States)

    Guo, Qiaosheng; Fang, Hailing; Shen, Haijin

    2010-05-01

    To evaluate the quality of Flos Chrysanthemi Indici which produced in twenty-two different producing places. Chlorogenic acid and caffeic acid were analyzed on a Shim-pack C8 colunm (4.6 mm x 250 mm, 5 microm) eluted with the mobile phase consisted of acetonitrile-0.5% phosphoric acid( 19:81). The detection wavelength was set at 326 nm. Linarin were eluted with the mobile phase consisted of methanol-water-acetic acid(26: 23: 1). The detection wavelength was set at 334 nm. The column temperature was 25 degrees C. The flow rate was 1.0 mL x min . The linear response ranged within 2.5-50 microg for chlorogenic acid (r = 0.998), 2.5-25 microg for caffeic acid (r = 0.998) and 4.97-41.47 microg for linarin (r = 0.999), respectively. Recoveries were 100.8% with RSD 2.1% for chlorogenic acid, 96.2% with RSD 2.3% for caffeic acid and 103.7% with RSD 1.8% for linarin. There was a significant difference in the content of chlorogenic acid, caffeic acid, linarin among the samples. The content of chlorogenic in the sample from Fengdou Chongqing city was the highest in those from other places. The content of caffeic acid in the all samples is very low. The content of linarin in the samples from Jiangsu province and Anhui province almost reached the national standard in pharmacopoeia.

  20. PecS is a global regulator of the symptomatic phase in the phytopathogenic bacterium Erwinia chrysanthemi 3937.

    Science.gov (United States)

    Hommais, Florence; Oger-Desfeux, Christine; Van Gijsegem, Frédérique; Castang, Sandra; Ligori, Sandrine; Expert, Dominique; Nasser, William; Reverchon, Sylvie

    2008-11-01

    Pathogenicity of the enterobacterium Erwinia chrysanthemi (Dickeya dadantii), the causative agent of soft-rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. PecS is a transcriptional regulator that controls production of various virulence factors. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS notably regulates a wide range of genes that could be linked to pathogenicity and to a group of genes concerned with evading host defenses. Among the targets are the genes encoding plant cell wall-degrading enzymes and secretion systems and the genes involved in flagellar biosynthesis, biosurfactant production, and the oxidative stress response, as well as genes encoding toxin-like factors such as NipE and hemolysin-coregulated proteins. In vitro experiments demonstrated that PecS interacts with the regulatory regions of five new targets: an oxidative stress response gene (ahpC), a biosurfactant synthesis gene (rhlA), and genes encoding exported proteins related to other plant-associated bacterial proteins (nipE, virK, and avrL). The pecS mutant provokes symptoms more rapidly and with more efficiency than the wild-type strain, indicating that PecS plays a critical role in the switch from the asymptomatic phase to the symptomatic phase. Based on this, we propose that the temporal regulation of the different groups of genes required for the asymptomatic phase and the symptomatic phase is, in part, the result of a gradual modulation of PecS activity triggered during infection in response to changes in environmental conditions emerging from the interaction between both partners.

  1. Thermodynamics and kinetic properties of halostable endoglucanase from Aspergillus fumigatus ABK9.

    Science.gov (United States)

    Das, Arpan; Jana, Arijit; Paul, Tanmay; Halder, Suman Kumar; Ghosh, Kuntal; Maity, Chiranjit; Mohapatra, Pradeep Kumar Das; Pati, Bikas Ranjan; Mondal, Keshab Chandra

    2014-07-01

    An endoglucanase from Aspergillus fumigatus ABK9 was purified from the culture extract of solid-state fermentation and its some characteristics were evaluated. The molecular weight of the purified enzyme (56.3 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymogram analysis and confirmed by MALDI-TOF mass spectrometry. The enzyme was active optimally at 50 °C, pH 5.0 and stable over a broad range of pH (4.0-7.0) and NaCl concentration of 0-3.0 M. The pKa1 and pKa2 of the ionizable groups of the active sites were 2.94 and 6.53, respectively. The apparent Km , Vmax , and Kcat values for carboxymethyl cellulose were 6.7 mg ml(-1), 775.4 µmol min(-1) , and 42.84 × 10(4)  s(-1), respectively. Thermostability of the enzyme was evidenced by the high activation energy (91.45 kJ mol(-1)), large enthalpy for activation of denaturation (88.77 kJ mol(-1)), longer half-life (T1/2) (433 min at 50 °C), higher melting temperature (Tm ) (73.5 °C), and Q10 (1.3) values. All the characteristics favors its suitability as halotolerant and thermostable enzyme during bioprocessing of lignocellulosic materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Definition of a consensus DNA-binding site for PecS, a global regulator of virulence gene expression in Erwinia chrysanthemi and identification of new members of the PecS regulon.

    Science.gov (United States)

    Rouanet, Carine; Reverchon, Sylvie; Rodionov, Dmitry A; Nasser, William

    2004-07-16

    In Erwinia chrysanthemi, production of pectic enzymes is modulated by a complex network involving several regulators. One of them, PecS, which belongs to the MarR family, also controls the synthesis of various other virulence factors, such as cellulases and indigoidine. Here, the PecS consensus-binding site is defined by combining a systematic evolution of ligands by an exponential enrichment approach and mutational analyses. The consensus consists of a 23-base pair palindromic-like sequence (C(-11)G(-10)A(-9)N(-8)W(-7)T(-6)C(-5)G(-4)T(-3)A(-2))T(-1)A(0)T(1)(T(2)A(3)C(4)G(5)A(6)N(7)N(8)N(9)C(10)G(11)). Mutational experiments revealed that (i) the palindromic organization is required for the binding of PecS, (ii) the very conserved part of the consensus (-6 to 6) allows for a specific interaction with PecS, but the presence of the relatively degenerated bases located apart significantly increases PecS affinity, (iii) the four bases G, A, T, and C are required for efficient binding of PecS, and (iv) the presence of several binding sites on the same promoter increases the affinity of PecS. This consensus is detected in the regions involved in PecS binding on the previously characterized target genes. This variable consensus is in agreement with the observation that the members of the MarR family are able to bind various DNA targets as dimers by means of a winged helix DNA-binding motif. Binding of PecS on a promoter region containing the defined consensus results in a repression of gene transcription in vitro. Preliminary scanning of the E. chrysanthemi genome sequence with the consensus revealed the presence of strong PecS-binding sites in the intergenic region between fliE and fliFGHIJKLMNOPQR which encode proteins involved in the biogenesis of flagellum. Accordingly, PecS directly represses fliE expression. Thus, PecS seems to control the synthesis of virulence factors required for the key steps of plant infection.

  3. Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols.

    Science.gov (United States)

    Chong, Barrie Fong; Harrison, Mark D; O'Hara, Ian M

    2014-01-01

    Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Deteksi dan Identifikasi Dickeya sp. sebagai Organisme Pengganggu Tumbuhan Karantina A2 pada Tanaman Kentang di Jawa

    Directory of Open Access Journals (Sweden)

    Haerani Haerani

    2015-09-01

    Full Text Available Erwinia chrysanthemi (currently Dickeya sp. is one of the A2 quarantine pest that must be concerned of its distribution on potato in Indonesia. The aim of this study is to detect and identify E. chrysanthemi from potato in Java. A total of 400 samples of potato plants showing symptoms of soft rot were obtained from several potato areas in Pangalengan and Garut (West Java, Dieng (Central Java, and Batu-Malang (East Java. Disease incidence was determined by indirect enzyme-linked immunosorbent assay (I-ELISA using polyclonal antiserum. E.chrysanthemi was isolated from plant samples with positive ELISA results. Furthermore, bacterial isolates were characterized by GEN III OmniLog ID System and PCR using specific primers Ec3F/Ec4R, as well as the universal 16S rRNA primer pair of 27F/1429R. The incidence of E. chrysanthemi based on ELISA was obtained. Based on physiological characters; Gram, catalase, oxidase, and oxidation-fermentation, there were 4 isolates similar to the genus of Erwinia. However, the results of Gen III OmniLog System, PCR, and nucleotide sequences analysis of 16S rRNA confirmed that none of the isolates were identified as E.chrysanthemi. Otherwise, those 4 isolates were identified as Pseudomonas oryzihabitans, Pantoea agglomerans, and Pseudomonas viridiflava. The result of this study indicated that the existence of E. chrysanthemi as an A2 quarantine pest on potato in Java can not be confirmed and remains as an A1 quarantine pest.

  5. Participants in a medical applications meeting hosted by CERN Head of Medical Applications S. Myers with J. E. Celis, Chairman of the Policy Committee, European Cancer Organisation, President, European Association for Cancer Research and Prof. JM. Gago, President of Laboratório de Instrumentação e Física Experimental de Partículas (LIP) and Former Minister for Science, Technology and Higher Education of Portugal of the XVII Governo. Were also participating: CERN Life Science Adviser M. Dosanjh with U. Ringborg R. Lewensohn, A. Brahme, R. Moeckli, O. Jäkel and S. Pieck.

    CERN Multimedia

    Anna Pantelia

    2013-01-01

    Participants in a medical applications meeting hosted by CERN Head of Medical Applications S. Myers with J. E. Celis, Chairman of the Policy Committee, European Cancer Organisation, President, European Association for Cancer Research and Prof. JM. Gago, President of Laboratório de Instrumentação e Física Experimental de Partículas (LIP) and Former Minister for Science, Technology and Higher Education of Portugal of the XVII Governo. Were also participating: CERN Life Science Adviser M. Dosanjh with U. Ringborg R. Lewensohn, A. Brahme, R. Moeckli, O. Jäkel and S. Pieck.

  6. Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme.

    Science.gov (United States)

    Gervais, David; Foote, Nicholas

    2014-10-01

    The enzyme Erwinia chrysanthemi L-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s(-1)) when compared to the WT (k cat 565 s(-1)), which was further increased in the double mutant, with a k cat of 798 s(-1). Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.

  7. Optimization of endoglucanase production from thermophilic strain of Bacillus licheniformis RT-17 and its application for saccharification of sugarcane bagasse

    International Nuclear Information System (INIS)

    Tariq, R.; Qadir, F.; Ahmed, A.; Shariq, M.; Zafar, U.; Khan, S.A.

    2018-01-01

    Thermostable cellulases are required for a variety of commercial processes. Bacillus is a house of thermostable proteins. Screening of indigenously isolated strains of bacteria revealed the promising production of cellulase by a strain, RT-17, at 50 degree C. The strain was identified on the basis of biochemical and molecular characteristics as B. licheniformis. The factors affecting cellulase production from B. licheniformis RT-17 were evaluated for their significant effect using Plackett Burman Design and were optimized by employing Box-Behnken Design. The model predicted 9.808 IU/ml of endoglucanase (EG) under optimum conditions of 50 degree C; 10% inoculum size; pH 5; and 1% peptone in fermentation medium. Practically, a titer of 9.128 IU/ml was obtained, showed the validity of the model. The enzyme preparation from B. licheniformis RT-17 was applied in combination with xylanase and pectinase preparations from indigenous yeasts for the hydrolysis of sugarcane bagasse (SCB). A higher degree of synergy (7.1 folds) was observed when yeast pectinase was used with bacterial cellulase for the hydrolysis of alkali treated SCB. Whereas, the degree of synergy was lower when bacterial cellulase was mixed with yeast xylanase. The study revealed the possibility of utilization of combination of yeast and bacterial enzymes for biomass saccharification. (author)

  8. Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E. carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Laing, E; Pretorius, I S

    1993-05-01

    A pectate lyase (PL)-encoding gene (pelE) from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E. carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). A pectinase cassette comprising ADC1P-MF alpha 1s-pelE-TRP5T and ADC1P-MF alpha 1s-peh1-TRP5T was subcloned into YCp50, generating plasmid pAMS14. Subsequently, the dominant selectable Geneticin G418-resistance (GtR) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10P) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3T), was cloned into pAMS14, resulting in plasmid pAMS15. Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains. DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively. A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S. cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation.

  9. Analysis of the Black Female Initiation Characteristics in The Color Purple%《紫色》中的黑人女性成长特点

    Institute of Scientific and Technical Information of China (English)

    冯丽君; 鱼戏溪

    2012-01-01

    美国传统成长小说的主人公大都是男性,艾丽斯·沃克在其代表作《紫色》中则成功塑造了一位黑人女性成长主人公——西丽。由于深受种族主义和父权主义的双重压迫,西丽被剥夺了受教育的权利和追求幸福的自由,但是在黑人女性同胞的指引下,西丽以其顽强的毅力克服了种种困难,成功地实现了从无知到成熟的转变。西丽艰辛的成长过程正是黑人女性寻求解放的过程,西丽用自己成功的成长经历,为黑人女性树立了榜样,为黑人妇女的解放指明了方向。%The heroes of the traditional American Initiation Stories generally are male, while in her masterpiece The Color Purple, Alice Walker successfully creats a black-female Initiation hero Celie. Suffering from the double oppressions of racism and sexism, Celie is deprived of her right of formal education and pursuit of happiness. Nevertheless, under the guide the black-female sisters, Celie manages to overcome all the difficulties and successfully grow from innocence to maturity. The hardness of Celie's growing process is just like the process of black-females' pursuing emancipation. Using her own experiences, Celie sets a good example for all the black women and a right direction for black women's liberation.

  10. Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

    Directory of Open Access Journals (Sweden)

    Jäger Gernot

    2010-08-01

    Full Text Available Abstract Background It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH and endoglucanases (EG. Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose. Results First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir and kinetics (pseudo-first-order. Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured. It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose. Conclusions α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation

  11. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    Science.gov (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  12. 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.

  13. Optimization of ruminococcus albus endoglucanase cel5-cbm6 production in plants by incorporating an elp tag and targeting to different subcellular compartments

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, E.O.; Menassa, R. [Western Ontario Univ., London, ON (Canada). Dept. of Biology; Agriculture and Agri-Food Canada, London, ON (Canada); Kolotilin, I. [Agriculture and Agri-Food Canada, London, ON (Canada)

    2009-07-01

    The production of biomass-based biofuel such as ethanol depends on the deconstruction of a cellulosic matrix and requires a variety of enzymes that hydrolyze glycosidic bonds to release fermentable sugars. Endoglucanases are one of most important groups of natural cellulosic hydrolytic enzymes that act on cellulose. In order to decrease ethanol production costs, the cost of producing cellulases must also be reduced. Genetically engineered transgenic plants are among the most economical systems for large scale production of recombinant proteins because of the large amount of enzymes that can be produced with minimal input. Cellulases present different levels of expression in different subcellular compartments. Cel5-CBM6 is a fused protein containing an endocellulase from Ruminococus albus (Cel5) and a cellulose binding domain (CBD) of Clostridium stercorarium. It accumulates in both the chloroplast and cytoplasm, but severe growth defects occur when expressed in the cytoplasm. Therefore, other subcellular compartments such as endoplasmic reticulum (ER) and vacuole must be evaluated and compared to determine the best co partment for production and activity of cellulases. Since elastin-like polypeptide (ELP) has also been shown to increase recombinant protein accumulation in plants, this study evaluated the effects of incorporating an ELP tag and a retrieval signal peptide on the expression levels of Cel5-CBM6.

  14. Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering.

    Science.gov (United States)

    Nakanishi, Akihito; Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2012-10-23

    To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

  15. Postglacial Indian Ocean

    Digital Repository Service at National Institute of Oceanography (India)

    Naidu, P.D.

    radiation that causes intense heating over carbon and carbonate as paleoproducrivity proxles: Examples northern India and the Tibetan Plateau. This pattern of heating causes ascending airflow and the develop- ment of an intense low pressure celI...

  16. Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinera and strawberry

    DEFF Research Database (Denmark)

    Mamarabadi, Mojtaba; Jensen, Birgit; Jensen, Søren Dan Funck

    2008-01-01

    Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucan......Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two...... endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for ß-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry......, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated...

  17. Powdery mildew of Chrysanthemum × morifolium: phylogeny and taxonomy in the context of Golovinomyces species on Asteraceae hosts.

    Science.gov (United States)

    Bradshaw, Michael; Braun, Uwe; Götz, Monika; Meeboon, Jamjan; Takamatsu, Susumu

    2017-01-01

    The taxonomic history of the common powdery mildew of Chrysanthemum × morifolium (chrysanthemum, florist's daisy), originally described in Germany as Oidium chrysanthemi, is discussed. The position of O. chrysanthemi was investigated on the basis of morphological traits and molecular phylogenetic analyses. Based on the results of this study, this species, which is closely related to Golovinomyces artemisae, was reassessed and reallocated to Golovinomyces. The phylogenetic analysis and taxonomic reassessment of the chrysanthemum powdery mildew is supplemented by a morphological description, a summary of its worldwide distribution data, and a brief discussion of the introduction of this fungus to North America. G. chrysanthemi differs from true G. artemisiae in that it has much longer conidiophores, is not constricted at the base, and has much larger and most importantly longer conidia. The close affinity of Golovinomyces to Artemisia and Chrysanthemum species signifies a coevolutionary event between the powdery mildews concerned and their host species in the subtribe Artemisiinae (Asteraceae tribe Anthemideae). This conclusion is fully supported by the current phylogeny and taxonomy of the host plant genera and the coevolution that occurred with the host and pathogen. The following powdery mildew species, which are associated with hosts belonging to the tribe Anthemideae of the Asteraceae, are epitypified: Alphitomorpha depressa β artemisiae (≡ Alphitomorpha artemisiae), Erysiphe artemisiae, and Oidium chrysanthemi. Erysiphe macrocarpa is neotypified. Their sequences were retrieved from the epitype collections and have been added to the phylogenetic tree. Golovinomyces orontii, an additional powdery mildew species on Chrysanthemum ×morifolium, is reported. This species is rarely found as a spontaneous infection and was obtained from inoculation experiments.

  18. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  19. Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi.

    Science.gov (United States)

    Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui

    2017-07-24

    Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel

  20. Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for 'D. solani', new biovar 3 variant on potato in Europe.

    Science.gov (United States)

    Van Vaerenbergh, Johan; Baeyen, Steve; De Vos, Paul; Maes, Martine

    2012-01-01

    Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

  1. Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for 'D. solani', new biovar 3 variant on potato in Europe.

    Directory of Open Access Journals (Sweden)

    Johan Van Vaerenbergh

    Full Text Available Worldwide, Dickeya (formerly Erwinia chrysanthemi is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

  2. Guide to the NITRD Program FY 2004-FY 2005. Supplement to the President’s Budget for FY 2005

    Science.gov (United States)

    2004-12-01

    and Professor University of California, Berkeley J. Carter Beese , Jr. President Riggs Capital Partners Pedro Celis, Ph.D. Software Architect Microsoft...University of North Carolina at Chapel Hill William J. Hannigan President AT&T Jonathan C. Javitt, M.D., M.P.H. Senior Fellow Potomac Institute for Policy

  3. Effects of cell wall degrading enzymes on carbohydrate fractions and metabolites in stomach and ileum of pigs fed wheat bran based diets

    NARCIS (Netherlands)

    Meulen, van der J.; Inborr, J.; Bakker, J.G.M.

    2001-01-01

    Pigs were fed diets containing 40 heat bran incubated with a water:acetic acid mixture (control, C) and a cellulase (Cel-i) or xylanase (Xyl-i) preparation or with addition of the cellulase (Cel-a) or xylanase (Xyl-a) preparation immediately before feeding. Stomach and ileal samples were analysed

  4. Nová francouzská revoluce – k událostem ve vyspělém post-industriálním státě

    Czech Academy of Sciences Publication Activity Database

    Benáček, Vladimír

    -, č. 5 (2006), s. 9-12 ISSN 1214-1720 R&D Projects: GA ČR GA403/05/2769 Institutional research plan: CEZ:AV0Z70280505 Keywords : labour contracts Subject RIV: AO - Sociology, Demography http://www.socioweb.cz/upl/editorial/download/129_socioweb%205-06%20cely%201.pdf

  5. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  6. Aromatic amino acids in the cellulose binding domain of Penicillium crustosum endoglucanase EGL1 differentially contribute to the cellulose affinity of the enzyme.

    Directory of Open Access Journals (Sweden)

    Jiang-Ke Yang

    Full Text Available The cellulose binding domain (CBD of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y451-Y452 and Y477-Y478-Y479, among which three amino acids Y451, Y477, and Y478 structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y451-Y452 appears to contribute more to the cellulose binding than Y477-Y478-Y479. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y451, Y477, and Y478 which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y451-Y452 of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids.

  7. Pectinolytic bacteria and their secreted pectate lyases: agents for the maceration and solubilization of phytomass for fuels production

    Energy Technology Data Exchange (ETDEWEB)

    Preston, J.F. III; Rice, J.D.; Chow, M.C. (Florida Univ., Gainesville, FL (United States). Dept. of Microbiology and Cell Science)

    1993-01-01

    The objectives of this research have been to identify the pectinolytic enzymes secreted by bacteria and apply these towards the enhanced maceration and solubilization of plant material, focusing on the pectate lyases secreted by the phytopathogenic strains of Erwinia chrysanthemi, the ruminant resident Lachnospira multiparus, and the wood digestor isolate, Clostridium populeti. An HPLC approach has been developed that permits the kinetic analysis of each enzyme with respect to the formation of individual products during the pectate depolymerization process. This approach has demonstrated that each of these organisms secretes a nonrandom trimer-generating pectate lyase with a combination of endolytic and exolytic depolymerizing mechanisms. Two different strains of E. chrysanthemi secrete a battery of pectate lyases that include random endolytic as well as nonrandom dimer - and nonrandom trimer-generating endolytic/exolytic mechanisms. (author)

  8. Identification of Two Candidate Tumor Suppressor Genes on Chromosome 17p13.3: Assessment of Their Roles in Breast and Ovarian Carcinogenesis

    Science.gov (United States)

    2001-07-01

    mismatched substrates are also recognized by attack both DNA and RNA (8). S 1 nuclease from Aspergillus CELI and cut on one of the two DNA strands for...researcher. Later we will be able to add proteomics data for more integrated analysis since the system is flexible. The maximum efficiency is gained by making

  9. Species-specific detection of Dickeya sp. (Pectobacterium ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... (Pectobacterium chrysanthemi) in infected banana tissues .... tetraacetic (EDTA) disodium salt pH 8.0, 100 mm NaCl and 1% .... lane 7, Erwinia carotovora subsp. carotovora; lane 8, Xanthomonas campestris pv.musacearum;.

  10. Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering.

    Science.gov (United States)

    Zhang, Yun; Liu, Xiaomei; Zhang, Jinju; Zhang, Chun

    2016-09-01

    To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins. AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed "CELiD" DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with "CELiD" DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %. The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.

  11. Exogenous cellulases of thermophilic micromycetes. Pt. 1. Selection of producers

    Energy Technology Data Exchange (ETDEWEB)

    Kvesitadze, G; Kvachadze, L; Aleksidze, T; Chartishvili, D K

    1986-01-01

    More than 600 micromycetes - representatives of different genera have been investigated for their ability to produce exogenous cellulases. Most of the investigated cultures were found to produce these enzymes, 24 cultures being thermophilic, and 18 thermotolerant. Cellulase or its derivatives proved to be the most favourable carbon source for cellulase secretion. None of the thermophilic cultures studied manifested the ability of exogenous exoglucanase biosynthesis. Using UV-rays as mutagen, a mutant strain A. terreus T-49 has been obtained being characterized by an increased endo-glucanase and cellobiase activity, as compared to the initial strains. The cellulase preparations of thermophilic micromycetes contain one cellulasic component: endo-glucanase, or two: endo-glucanase and cellobiase.

  12. Is Erwinia te beheersen? Een literatuurstudie over rotproblemen in diverse gewassen om met deze kennis Erwinia in bolgewassen beter te kunnen aanpakken

    NARCIS (Netherlands)

    Doorn, van J.; Wolf, J.

    2005-01-01

    Het meeste onderzoek aan Erwinia-bacteriën is uitgevoerd bij aardappel. In dit gewas worden drie ziekteverwekkende Erwinia-soorten gevonden: Erwinia carotovora subsp. atroseptica (Eca), Erwinia chrysanthemi (Echr) en Erwinia carotovora subsp. carotovora (Ecc). Eca speelt in gematigde

  13. Draft Genome Sequences of 17 Isolates of the Plant Pathogenic Bacterium Dickeya

    OpenAIRE

    Pritchard, Leighton; Humphris, Sonia; Saddler, Gerry S.; Elphinstone, John G.; Pirhonen, Minna; Toth, Ian K.

    2013-01-01

    Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.

  14. Draft genome sequences of 17 isolates of the plant pathogenic bacterium dickeya.

    Science.gov (United States)

    Pritchard, Leighton; Humphris, Sonia; Saddler, Gerry S; Elphinstone, John G; Pirhonen, Minna; Toth, Ian K

    2013-11-21

    Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.

  15. State of the Science Meeting: Burn Care: Goals for Treatment and Research

    Science.gov (United States)

    2006-11-01

    13. Celis, MM, Suman, OE, Huang, TT, Yen, P, Herndon, DN. Effect of a Supervised Exercise and Physiotherapy Program on Surgical Interventions in...the following: surgeons, nurses, therapists, nutritionists, pharmacists, residents, medical students , scientists, healthcare administration, industry...specialists, and students interact to pose research questions based on clinical problems, to look for answers in the laboratories and then in the clinical

  16. Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

    Science.gov (United States)

    Paul, Sujay; Zhang, Angel; Ludeña, Yvette; Villena, Gretty K; Yu, Fengan; Sherman, David H; Gutiérrez-Correa, Marcel

    2017-06-10

    Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Nieuwe Erwinia-varianten vragen meer kennis en specifieke toetsen

    NARCIS (Netherlands)

    Wolf, van der J.M.

    2015-01-01

    Lange tijd kenden we in Nederland twee soorten Erwinia bacteriën namelijk Erwinia carotovora subsp. atroseptica en Erwinia chrysanthemi. Deze zijn/waren verantwoordelijk voor de aardappelziekten zwartbenigheid en stengelnatrot. De naamgeving van deze ziekteverwekkende bacteriën is echter op de schop

  18. Black women’s ‘two-ness’ in african-american literature: can black and white worlds join together? = A dualidade de mulheres negras na literatura afro-americana: os mundos negro e branco podem se unir?

    Directory of Open Access Journals (Sweden)

    José Endoença Martins

    2010-01-01

    Full Text Available The article discusses how black women keep contacts with both black and white worlds in novels written by African-American female writers. In Toni Morrison’s (1970 The Bluest Eye, Pecola Breedlove keeps contact with the white world through her assimilationist behavior; in Alice Walker’s (1982 The Color Purple, Celie freezes herself in the black world by playing the role of the nationalist Negro; finally, in Lorraine Hansberry’s (1987 A Raisin in the Sun, Mama Younger joins black and white worlds together when she develops a catalyst agenda, as she moves to a white neighborhood.O artigo discute como mulheres negras mantêm contato com os mundos negro e branco em romances de escritoras afroamericanas. Em O Olho Mais Azul, de Toni Morrison (1970, Pecola Breedlove se alia ao mundo branco pelo comportamento assimilacionista; em A Cor Púrpura, de Alice Walker (1982, Celie se isola no mundo negro ao assumir o papel do Negro nacionalista; por fim, em Uma Cereja ao Sol, de Lorraine Hansberry (1987, Mama Younger aproxima o mundo negro e branco quando se torna catalista, indo morar num bairro branco.

  19. Erwinia besmet de plant ook via de wortels

    NARCIS (Netherlands)

    Wolf, van der J.M.; Czajkowski, R.L.; Velvis, H.; Doorn, van J.

    2008-01-01

    Door een viertal Nederlandse wetenschappers is onlangs aanvullend onderzoek verricht naar besmettingen van Erwinia chrysanthemi in plantenwortels. Daaruit is gebleken dat de bacterie veel makkelijker de plant binnenkomt en zich in de plant verspreidt dan ooit gedacht. Binnen één dag kan de bacterie

  20. Efectos de la entrevista motivacional en el tratamiento de la diabetes mellitus tipo 2 /

    OpenAIRE

    García Flores, Raquel

    2012-01-01

     tesis que para obtener el grado de Doctorado en Psicología, presenta Raquel Garcia Flores ; asesor Juan José Sánchez Sosa, Samuel Jurado Cárdenas, Ana Luisa González Celis Rangel, Gilda Gómez Pérez Mitré, Javier Nieto Gutiérrez235 páginas : ilustracionesDoctorado en Psicología UNAM, Facultad de Psicología, 2012

  1. CELLULASES FROM THE BASIDIO - MYCETES CULTURAL LIQUID

    Directory of Open Access Journals (Sweden)

    К. G. Dreval

    2013-04-01

    Full Text Available Adsorption of cellulases on substrate taking place during the cultivation process was determined. Adsorbed enzymes can be eluted by buffer solution with high ionic strength, but for determine their activity they should be transferred into the aqueous solution. On the basis of the results a method for obtaining of cellulases preparations from cultural liquids of basidiomycetes was developed. This method is the elution of cellulases from the cultivation substrate of basidiomycetes. It was found that using of the last allows to obtain enzymatic preparations with a high degree of purification in 3 stages (salting out of proteins — dialysis — gelchromatography. Cellulase preparations received original products of basidiomycetes strains К-1, А-Дон-02, Д-1 Irpex lacteus and AnSc-1 Daedaleopsis confragosa f. confragosa were obtained. They contained different proteins, enzymes with specific peaks out of column and their activity. However, common to them was a distinct maximum of outing from the column by endoglucanases or cellobiases, which may indicate that the studied cellulolytic complexes of basidiomycetes do not contain multiple forms of cellulases with different molecular mass. This method allowed to obtain preparations with different degree of purification in comparing with the original culture filtrate 7,3 for endoglucanase and 33,3 for cellobiase of strain А-Дон-02 I. lacteus; 13,1 for endoglucanase and 25,5 for cellobiase of strain Д-1 I. lacteus; 29,9 for endoglucanase and 90,1 for cellobiase of strain К-1 I. lacteus; 2,1 for endoglucanase and 30,6 for cellobiase of strain AnSc-1 D. confragosa f. confragosa.

  2. Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

    2014-02-01

    A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

  3. 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.).

  4. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.; Ku, Maurice S.

    2011-12-10

    Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in

  5. Dickeya species: an emerging problem for potato production in Europe

    NARCIS (Netherlands)

    Toth, I.K.; Wolf, van der J.M.; Saddler, G.; Lojkowska, E.; Hélias, V.; Pirhonen, M.; Tsror, L.; Elphinstone, J.G.

    2011-01-01

    Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world-wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5

  6. Well, what about intraspecific variation? Taxonomic and phylogenetic characters in the genus Synoeca de Saussure (Hymenoptera, Vespidae).

    Science.gov (United States)

    Carpenter, James M; Andena, Sergio R; Noll, Fernando B; Wenzel, John W

    2013-01-01

    Cely and Sarmiento (2011) took issue with the cladistic analysis of relationships among species of the genus Synoeca by Andena et al. (2009a), and presented a reanalysis. They claimed that intraspecific variation in the genus is meaningful, and proper consideration yields a conclusion different from that of Andena et al. Both their critique and reanalysis are vitiated by numerous errors, as is shown in the present paper.

  7. Cyber Security: A Crisis of Prioritization

    Science.gov (United States)

    2005-02-01

    Society (CITRIS) and Professor University of California, Berkeley J. Carter Beese , Jr. President Riggs Capital Partners Pedro Celis, Ph.D. Software...Science University of North Carolina at Chapel Hill William J. Hannigan President AT&T Jonathan C. Javitt, M.D., M.P.H. Senior Fellow Potomac Institute...CHAIR F. Thomson Leighton MEMBERS J. Carter Beese , Jr. Patricia Thomas Evans Luis E. Fiallo Harold Mortazavian David A. Patterson Alice G

  8. 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.

  9. Optimization, purification and characterization of recombinant L ...

    African Journals Online (AJOL)

    We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with ...

  10. Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

    NARCIS (Netherlands)

    Wolf, van der J.M.; Haas, de B.H.; Hoof, van R.A.; Haan, de E.G.; Bovenkamp, van den G.W.

    2014-01-01

    TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic

  11. PRODUCTION AND CHARACTERIZATION OF CELLULOLYTIC ENZYMES BY ASPERGILLUS NIGER AND RHIZOPUS SP . BY SOLID STATE FERMENTATION OF PRICKLY PEAR

    Directory of Open Access Journals (Sweden)

    TAMIRES CARVALHO DOS SANTOS

    2016-01-01

    Full Text Available Prickly palm cactus husk was used as a solid - state fermentation support substrate for the production of cellulolytic enzymes using Aspergillus niger and Rhizopus sp. A Box - Behnken design was used to evaluate the effects of water activity, fermentation time and temperature on endoglucanase and total cellulase production. Response Surface Methodology showed that optimum conditions for endoglucanase production were achieved at after 70.35 h of fermentation at 29.56°C and a water activity of 0.875 for Aspergillus niger and after 68.12 h at 30.41°C for Rhizopus sp. Optimum conditions for total cellulase production were achieved after 74.27 h of fermentation at 31.22°C for Aspergillus niger and after 72.48 h and 27.86°C for Rhizopus sp . Water activity had a significant effect on Aspergillus niger endoglucanase production only. In industrial applications, enzymatic characterization is important for optimizing variables such as temperature and pH. In this study we showed that endoglucanase and total cellulase had a high level of thermostability and pH stability in all the enzymatic extracts. Enzymatic deactivation kinetic experiments indicated that the enzymes remained active after the freezing of the crude extract. Based on the results, bioconversion of cactus is an excellent alternative for the production of thermostable enzymes.

  12. Computational Science: Ensuring America’s Competitiveness

    Science.gov (United States)

    2005-06-01

    Interest of Society (CITRIS) and Professor University of California, Berkeley J. Carter Beese , Jr. President Riggs Capital Partners Pedro Celis, Ph.D...and Library Science University of North Carolina at Chapel Hill William J. Hannigan President AT&T Jonathan C. Javitt, M.D., M.P.H. Senior Fellow...Vannier, Ph.D., Professor of Radiology, University of Chicago • Jonathan C. Silverstein, M.D., M.S., FACS, Assistant Professor of Surgery, University of

  13. Water relations of Pseudomonas solanacearum

    African Journals Online (AJOL)

    GREGORY

    2010-08-30

    Aug 30, 2010 ... pathogens of plants, especially, Erwinia chrysanthemi. (Mildenhall et al., 1988; Prior et al., 1994; Gouesbet et al., 1995; Gouesbet et al., 1996). This helps to determine how they survive in dry soil for long periods as saprophytes. Experiments undertaken here on P. solanacearum give a glimpse of what may ...

  14. The influence of carbon source and calcium on the production of ...

    African Journals Online (AJOL)

    E. chrysanthemi targets the host's middle lamella, which was mainly made of pectic substances containing calcium, for colonization. In this study, it is reported that a pectic substance, such as sodium polypectate (NaPP) may be preferable, not only for the production of hydrolytic enzymes, but for the production of protease as ...

  15. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    Directory of Open Access Journals (Sweden)

    Chou Hong

    2011-12-01

    Full Text Available Abstract Background Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. Results In this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1 gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice

  16. Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence-assisted carbohydrate electrophoresis.

    Science.gov (United States)

    Zhang, Qing; Zhang, Xiaomei; Wang, Peipei; Li, Dandan; Chen, Guanjun; Gao, Peiji; Wang, Lushan

    2015-03-01

    Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Production and characterization of compounds based on MgB4O7 for application in dosimetry

    International Nuclear Information System (INIS)

    Souza, Luiza Freire de

    2016-01-01

    Many materials with luminescent properties are used for ionizing radiation dosimetry through the thermoluminescence (TL) and optically stimulated luminescence (OSL) techniques. Detectors based on lithium fluoride (LiF), calcium sulphate (CaSO 4 ) and aluminum oxide (Al 2 O 3 ), doped or codoped with various elements, are the TL or OSL commercial dosimeters most widely used currently. However, several researches are focused to the development of new TL /OSL materials in intention to enhance the dosimetric properties, in view that no TL/OSL dosimeter has all the ideal characteristics for monitoring the radiation. In this context, magnesium tetraborate (MgB 4 O 7 ), which has been presented in the literature as a material for dosimetry TL, was investigated in this work. As there are no reports on the structural characterization of this material or regarding to its applicability on OSL dosimetry, the proposal of the present work was to develop compounds based on MgB 4 O 7 , with different doping, by solid state synthesis. It was made the structural, optical, TL and OSL characterization of the compound to verify it feasibility for application on radiation dosimetry. Initially, it was determined the calcination temperature and time for MgB 4 O 7 formation, with the use of thermal analyses and x ray diffraction. The ideal calcination was found at 900 °C for 7 hours. It were produced , in powder form, the compounds: MgB 4 O 7 , MgB 4 O 7 :Dy, MgB 4 O 7 :Dy,Li, MgB 4 O 7 :Ce, MgB 4 O 7 :Ce,Li, MgB 4 O 7 :Nd and MgB 4 O 7 :Nd,Li. For TL and OSL analyses it were produced pellets sintering at 950 °C for 2 hours. The radioluminescence (RL) analyses of MgB 4 O 7 :Dy and MgB 4 O 7 :Dy,Li shows wavelength emissions at 490, 590, 670 and 760 nm. For MgB 4 O 7 :Ce and MgB 4 O 7 :Ce,Li RL was observed wide emission band in the ultraviolet region. For the MgB 4 O 7 :Nd and MgB 4 O 7 :Nd,Li RL were not observed emission from the ultraviolet to infrared. With the results obtained from

  18. Antioxidant effects of 14 Chinese traditional medicinal herbs against human low-density lipoprotein oxidation

    Directory of Open Access Journals (Sweden)

    Hsin-Hung Lin

    2015-01-01

    Full Text Available The relationship between the antioxidant activities and inhibitory effect of 14 Chinese medicinal herbs against oxidized low-density lipoprotein (LDL formation was evaluated. Prolongation of the lag phase of LDL oxidation depended on the concentration of the herbs. The concentration of each herb that was able to prolong the lag time by about two-fold was calculated and expressed as doubling-time concentration. The lower the doubling-time concentration, the stronger the inhibitory effect exhibited toward LDL oxidation. Among them, Chrysanthemi Flos (Chrysanthemum morifolium ramat; 甘菊花 gān jú huā, Crataegi Fructus (Crataegus pinnatifida Bge. var. major N.E.Br.; 山楂 shān zhā, and Roselle (Hibiscus sabdariffa Linn.; 洛神 luò shén showed significant inhibitory effects. Correlation coefficients between doubling-time concentration and radical-scavenging activities were high; the total phenolic content was also high. In conclusion, phenolic compounds contributed not only to antioxidant activities, but also to the inhibitory effect against LDL oxidation. Chrysanthemi Flos, Crataegi Fructus, and H. sabdariffa, with lower doubling-time concentrations, could be potent phytochemical agents to reduce LDL oxidation and prevent the progression of atherosclerosis.

  19. Bacterial Diseases of Bananas and Enset: Current State of Knowledge and Integrated Approaches Toward Sustainable Management

    Directory of Open Access Journals (Sweden)

    Guy Blomme

    2017-07-01

    Full Text Available Bacterial diseases of bananas and enset have not received, until recently, an equal amount of attention compared to other major threats to banana production such as the fungal diseases black leaf streak (Mycosphaerella fijiensis and Fusarium wilt (Fusarium oxysporum f. sp. cubense. However, bacteria cause significant impacts on bananas globally and management practices are not always well known or adopted by farmers. Bacterial diseases in bananas and enset can be divided into three groups: (1 Ralstonia-associated diseases (Moko/Bugtok disease caused by Ralstonia solanacearum and banana blood disease caused by R. syzygii subsp. celebesensis; (2 Xanthomonas wilt of banana and enset, caused by Xanthomonas campestris pv. musacearum and (3 Erwinia-associated diseases (bacterial head rot or tip-over disease Erwinia carotovora ssp. carotovora and E. chrysanthemi, bacterial rhizome and pseudostem wet rot (Dickeya paradisiaca formerly E. chrysanthemi pv. paradisiaca. Other bacterial diseases of less widespread importance include: bacterial wilt of abaca, Javanese vascular wilt and bacterial fingertip rot (probably caused by Ralstonia spp., unconfirmed. This review describes global distribution, symptoms, pathogenic diversity, epidemiology and the state of the art for sustainable disease management of the major bacterial wilts currently affecting banana and enset.

  20. The industrial applicability of purified cellulase complex indigenously produced by Trichoderma viride through solid-state bio-processing of agro-industrial and municipal paper wastes

    Directory of Open Access Journals (Sweden)

    Muhammad Irshad

    2013-02-01

    Full Text Available An indigenous strain of Trichoderma viride produced high titers of cellulase complex in solid-state bio-processing of agro-industrial orange peel waste, which was used as the growth-supporting substrate. When the conditions of the SSF medium containing 15 g orange peel (50% w/w moisture inoculated with 5 mL of inoculum were optimal, the maximum productions of endoglucanase (655 ± 5.5 U/mL, exoglucanase (412 ± 4.3 U/mL, and β-glucosidase (515 ± 3.7 U/mL were recorded after 4 days of incubation at pH 5 and 35 °C. The enzyme with maximum activity (endoglucanase was purified by ammonium sulfate fractionation and Sephadex G-100 column gel filtration chromatographic technique. Endoglucanase was 5.5-fold purified with specific activity of 498 U/mg in comparison to the crude enzyme. The enzyme was shown to have a molecular weight of 58 kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE. The shelf life profile revealed that the enzyme could be stored at room temperature (30 °C for up to 45 days without losing much of its activity.

  1. Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi.

    Science.gov (United States)

    Chen, Qing; Rehman, S; Smant, G; Jones, John T

    2005-07-01

    RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.

  2. Enzymes and other agents that enhance cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Dickeya dadantii, a plant pathogenic bacterium producing Cyt-like entomotoxins, causes septicemia in the pea aphid Acyrthosiphon pisum

    OpenAIRE

    Costechareyre, Denis; Balmand, Severine; Condemine, Guy; Rahbé, Yves

    2012-01-01

    International audience; Dickeya dadantii (syn. Erwinia chrysanthemi) is a plant pathogenic bacteria that harbours a cluster of four horizontally-transferred, insect-specific toxin genes. It was recently shown to be capable of causing an acute infection in the pea aphid Acyrthosiphon pisum (Insecta: Hemiptera). The infection route of the pathogen, and the role and in vivo expression pattern of these toxins, remain unknown. Using bacterial numeration and immunolocalization, we investigated the ...

  5. Production and characterization of compounds based on MgB{sub 4}O{sub 7} for application in dosimetry; Producao e caracterizacao de compostos a base de MgB{sub 4}O{sub 7} para aplicacao em dosimetria

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Luiza Freire de

    2016-07-01

    Many materials with luminescent properties are used for ionizing radiation dosimetry through the thermoluminescence (TL) and optically stimulated luminescence (OSL) techniques. Detectors based on lithium fluoride (LiF), calcium sulphate (CaSO{sub 4}) and aluminum oxide (Al{sub 2}O{sub 3}), doped or codoped with various elements, are the TL or OSL commercial dosimeters most widely used currently. However, several researches are focused to the development of new TL /OSL materials in intention to enhance the dosimetric properties, in view that no TL/OSL dosimeter has all the ideal characteristics for monitoring the radiation. In this context, magnesium tetraborate (MgB{sub 4}O{sub 7}), which has been presented in the literature as a material for dosimetry TL, was investigated in this work. As there are no reports on the structural characterization of this material or regarding to its applicability on OSL dosimetry, the proposal of the present work was to develop compounds based on MgB{sub 4}O{sub 7}, with different doping, by solid state synthesis. It was made the structural, optical, TL and OSL characterization of the compound to verify it feasibility for application on radiation dosimetry. Initially, it was determined the calcination temperature and time for MgB{sub 4}O{sub 7} formation, with the use of thermal analyses and x ray diffraction. The ideal calcination was found at 900 °C for 7 hours. It were produced , in powder form, the compounds: MgB{sub 4}O{sub 7}, MgB{sub 4}O{sub 7}:Dy, MgB{sub 4}O{sub 7}:Dy,Li, MgB{sub 4}O{sub 7}:Ce, MgB{sub 4}O{sub 7}:Ce,Li, MgB{sub 4}O{sub 7}:Nd and MgB{sub 4}O{sub 7}:Nd,Li. For TL and OSL analyses it were produced pellets sintering at 950 °C for 2 hours. The radioluminescence (RL) analyses of MgB{sub 4}O{sub 7}:Dy and MgB{sub 4}O{sub 7}:Dy,Li shows wavelength emissions at 490, 590, 670 and 760 nm. For MgB{sub 4}O{sub 7}:Ce and MgB{sub 4}O{sub 7}:Ce,Li RL was observed wide emission band in the ultraviolet region. For the Mg

  6. Production of Cellulolytic and Hemicellulolytic Enzymes From Aureobasidium pulluans on Solid State Fermentation

    Science.gov (United States)

    Leite, Rodrigo Simões Ribeiro; Bocchini, Daniela Alonso; da Silva Martins, Eduardo; Silva, Dênis; Gomes, Eleni; da Silva, Roberto

    This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme remaining 100% active when incubated at 75°C for 1 h.

  7. Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

    Directory of Open Access Journals (Sweden)

    R. Navanietha Krishnaraj

    2017-04-01

    Full Text Available Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-glucosidase from a thermophilic bacterium,Clostridium thermocellumand their validation using Root Mean Square Deviation (RMSD and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and ß-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

  8. Use of nanostructure initiator mass spectrometry (NIMS to deduce selectivity of reaction in glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Kai eDeng

    2015-10-01

    Full Text Available Chemically synthesized nanostructure-initiator mass spectrometry (NIMS probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

  9. UPORABA SUPERKRITIČNEGA OGLIKOVEGA DIOKSIDA (SC CO 2 ) ZA IZOLACIJO ENCIMOV IN PROTEINOV IZ CELIC ČRNE KVASOVKE TRIMMATOSTROMA SALINUM

    OpenAIRE

    Bosilj, Monika

    2012-01-01

    Namen diplomske naloge je določiti vpliv superkritičnega ogljikovega dioksida (SC CO2) na preživetje črnih kvasovk Trimmatostroma salinum in izločanje proteinov in nukleinskih kislin iz celične suspenzije T. salinum. Suspenzijo s kulturo T. salinum smo v visokotlačnem šaržnem reaktorju izpostavili SC CO2 pri tlaku 100 bar in 35 °C. Rezultati so pokazali, da se je s podaljševanjem časa inkubacije v SC CO2 preživelost celic T. salinum zmanjševala. Merili smo tudi aktivnost encimov celulaze, α-a...

  10. Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Madsen, P S; Hokland, M; Ellegaard, J

    1988-01-01

    markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those...... of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition...

  11. ORF Sequence: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002945 gi|31792282 >gi|31792282|ref|NP_854775.1| PROBABLE CELLULASE CELA2A (ENDO-1,4-BETA-GLUCA...NASE) (ENDOGLUCANASE) (CARBOXYMETHYL CELLULASE) [Mycobacterium bovis AF2122/97] MNGAAPTNGAPLSYPSICEGVHWGHLVGGHQPAY

  12. ORF Sequence: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_000962 gi|57116825 >gi|57116825|ref|YP_177638.1| PROBABLE CELLULASE CELA2A (ENDO-1,4-BETA-GLUCA...NASE) (ENDOGLUCANASE) (CARBOXYMETHYL CELLULASE) [Mycobacterium tuberculosis H37Rv] MNGAAPTNGAPLSYPSICEGVHWGHLVGGHQPAY

  13. Isolation and characterization of β-glucosidase producing bacteria ...

    African Journals Online (AJOL)

    Administrator

    2011-10-26

    Oct 26, 2011 ... lase enzyme system, along with endoglucanase and cellobiohydrolase. ... biomass substrates, for synthesis of useful glucosides, in flavor industry for ... 2007) and in the bioconversion of phenolic anti-oxidants from defatted ...

  14. Thermostable crude endoglucanase produced by Aspergillus ...

    African Journals Online (AJOL)

    Cellulases are used in many industries worldwide and there is an ever increasing need to isolate, produce or develop thermostable cellulases. Manipulation of fermentation techniques in order to obtain desirable product(s) can be one line of action. In this study Aspergillus fumigatus was grown on chopped wheat straw in a ...

  15. EFEITO ANTIBIÓTICO DO PRÓPOLIS SOBRE BACTÉRIAS FITOPATOGÊNICAS

    OpenAIRE

    BIANCHINI, L.; BEDENDO, I.P.

    1998-01-01

    O efeito antibiótico de extrato aquoso de própolis, em várias concentrações, foi avaliado para cinco espécies de bactérias fitopatogênicas. Agrobacterium tumefaciens, Clavibacter michiganensis subsp. michiganensis e Xanthomonas axonopodis pv. phaseoli foram completamente inibidas em meio de cultura contendo 10% de extrato de própolis. Erwinia chrysanthemi foi parcialmente inibida, enquanto Pseudomonas syringae pv. tabaci se mostrou insensível ao extrato, desenvolvendo colônias idênticas àquel...

  16. Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

    Science.gov (United States)

    Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH)...

  17. ORF Alignment: NC_006274 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_006274 gi|52142820 >1kwfA 3 362 53 435 5e-85 ... ref|YP_084010.1| chitosanase; gly...cosyl hydrolases family 8; endoglucanase [Bacillus ... cereus ZK] gb|AAU17839.1| chitosanase; glycosy

  18. Browse Title Index

    African Journals Online (AJOL)

    Items 7901 - 7950 of 11090 ... ... of Zmda1-1 gene increases seed mass of corn, Abstract PDF ... gene in maize increases sethoxydim resistance and oil content, Abstract PDF ... and characterization of the Aspergillus niger endoglucanase, EglA, ...

  19. 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...

  20. Cellulolytic (cel) genes of Clostridium thermocellum F7 and the proteins encoded by them

    International Nuclear Information System (INIS)

    Piruzyan, E.S.; Mogutov, M.A.; Velikodvorskaya, G.A.; Pushkarskaya, T.A.

    1988-01-01

    This study is concerned with genes cell, ce12, and ce13 encoding the endoglucanase of the cellulolytic complex of the anaerobic thermophilic Clostridium thermocellum F7 bacteria, these genes having been closed by us earlier. The authors present the characteristics of proteins synthesized by the cel genes in the minicell system of the strain Escherichia coli K-12 X925. The molecular weights of the proteins encoded by genes cell, ce12, and ce13 are 30,000, 45,000, and 50,000 dalton, respectively. The study of the homology of the cloned section of the C. thermocellum DNA containing the endoglucanase genes, using Southern's blot-hybridization method, did not reveal their physical linkage in the genome. The authors detected a plasmid with a size of about 30 kb in the cells of the C. thermocellum F7 strain investigated

  1. Endogenous cellulases in stylet secretions of cyst nematodes

    NARCIS (Netherlands)

    Smant, G.

    1998-01-01

    This thesis describes the identification ofβ-1,4-endoglucanases (cellulases) in stylet secretions of the two cyst nematodes species, Globodera rostochiensis and Heterodera glycines . A novel method was developed to raise monoclonal antibodies that were

  2. Sequence Classification: 387772 [

    Lifescience Database Archive (English)

    Full Text Available E CELA2B (ENDO-1,4-BETA-GLUCANASE) (ENDOGLUCANASE) (CARBOXYMETHYL CELLULASE) || http://www.ncbi.nlm.nih.gov/protein/31792283 ... ...Non-TMB Non-TMH TMB TMB TMB Non-TMB >gi|31792283|ref|NP_854776.1| PROBABLE CELLULAS

  3. Sequence Classification: 397489 [

    Lifescience Database Archive (English)

    Full Text Available E CELA2B (ENDO-1,4-BETA-GLUCANASE) (ENDOGLUCANASE) (CARBOXYMETHYL CELLULASE) || http://www.ncbi.nlm.nih.gov/protein/15608230 ... ...Non-TMB Non-TMH TMB TMB TMB Non-TMB >gi|15608230|ref|NP_215606.1| PROBABLE CELLULAS

  4. Discovery and Characterization of Enzymes for Degradation of Xyloglucan and Extensin

    DEFF Research Database (Denmark)

    Feng, Tao; Mikkelsen, Jørn Dalgaard

    before the residual polymers are used in the bioethanol production. Therefore, mono-component, substrate-specific enzymes that could selectively degrade or modify plant cell wall components are required. In this PhD study, three enzymes, including two xyloglucan-specific endoglucanases and one...

  5. Sequence Classification: 396442 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB TMB Non-TMB Non-TMB >gi|57116687|ref|YP_177689.1| POSSIBLE CELLULASE CELA1 (ENDOGLUCA...NASE) (ENDO-1,4-BETA-GLUCANASE) (FI-CMCASE) (CARBOXYMETHYL CELLULASE) || http://www.ncbi.nlm.nih.gov/protein/57116687 ...

  6. Sequence Classification: 386728 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB TMB Non-TMB Non-TMB >gi|31791239|ref|NP_853732.1| POSSIBLE CELLULASE CELA1 (ENDOGLUCA...NASE) (ENDO-1,4-BETA-GLUCANASE) (FI-CMCASE) (CARBOXYMETHYL CELLULASE) || http://www.ncbi.nlm.nih.gov/protein/31791239 ...

  7. Determination of 16 insect growth regulators in edible Chinese traditional herbs by liquid chromatography electrospray tandem mass spectrometry.

    Science.gov (United States)

    Qian, Mingrong; Wu, Liqin; Zhang, Hu; Xu, Mingfei; Li, Rui; Wang, Xiangyun; Sun, Caixia

    2012-03-01

    A new sensitive multiresidue liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method for the determination of 16 insect growth regulator (IGR) residues-RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), halofenozide, methoxyfenozide, chromafenozide, fufenozide, tebufenozide, diflubenzuron, chlorbenzuron, triflumuron, hexaflumuron, novaluron, lufenuron, teflubenzuron, flucycloxuron, flufenoxuron, and chlorfluazuron-in herbs (Perilla frutescens, flos chrysanthemi, lily bulbs, and ginger) has been developed. After the herbs had been extracted with acetonitrile, a combined graphitized nonporous carbon/aminopropyl (ENVI-Carb/LC-NH(2)) cartridge and a Florisil cartridge were used to clean up the extracts. LC-MS/MS was performed in multiple reaction monitoring mode with two specific precursor ion-product ion transitions per IGR to confirm and quantitate the residues in herbs. Quantitation was performed on the basis of matrix-matched calibrations. The method showed excellent linearity (r(2) > 0.99) and precision (relative standard deviations of 13.6 or lower) for all the target insecticides. The limits of quantitation were 0.6-10 μg kg(-1) for the 16 insecticides in the four herbs. The average recoveries, measured at three concentrations (0.01, 0.1, 1 mg kg(-1)), were in the range 74.8-105.3%. The method was satisfactorily applied for the analysis of 60 herb samples (Perilla frutescens, flos chrysanthemi, lily bulbs, and ginger). Hexaflumuron was detected at concentrations of 0.029 and 0.051 mg kg(-1) in Perilla frutescens.

  8. Comparison between the cellulase systems of Trichoderma harzianum E58 and Trichoderma reesei C30

    Energy Technology Data Exchange (ETDEWEB)

    Saddler, J.N.; Hogan, C.M.; Louis-Seize, G.

    1985-06-01

    Nearly all of the filter paper, endoglucanase and ..beta..-glucosidase activities of T. harzianum E58 were located extracellularly, with low amounts of these activities detected in the cell extracts and relatively little associated with the cell wall. Most of the filter paper and endoglucanase activities of T. reesei C30 were detected extracellularly. The half lives of the different cellulase activities were assayed at various temperatures over a period of time. When the pH of the filtrate was adjusted to 4.8, the cellulase activities were considerably enhanced, with the average half-life at 50/sup 0/C extended to 25 hrs. When various lignocellulosic substrates were hydrolyzed by T. harzianum E58 cellulases approximately 90% of the reducing sugars were present as glucose while 50 - 60% of the reducing sugars were detected as glucose when T. reesei C30 cellulases were used.

  9. Rumen microbial genomics

    International Nuclear Information System (INIS)

    Morrison, M.; Nelson, K.E.

    2005-01-01

    Improving microbial degradation of plant cell wall polysaccharides remains one of the highest priority goals for all livestock enterprises, including the cattle herds and draught animals of developing countries. The North American Consortium for Genomics of Fibrolytic Ruminal Bacteria was created to promote the sequencing and comparative analysis of rumen microbial genomes, offering the potential to fully assess the genetic potential in a functional and comparative fashion. It has been found that the Fibrobacter succinogenes genome encodes many more endoglucanases and cellodextrinases than previously isolated, and several new processive endoglucanases have been identified by genome and proteomic analysis of Ruminococcus albus, in addition to a variety of strategies for its adhesion to fibre. The ramifications of acquiring genome sequence data for rumen microorganisms are profound, including the potential to elucidate and overcome the biochemical, ecological or physiological processes that are rate limiting for ruminal fibre degradation. (author)

  10. The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

    DEFF Research Database (Denmark)

    Hidayat, Budi J.; Weisskopf, Carmen; Felby, Claus

    2015-01-01

    or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method...

  11. Overexpression, purification and characterization of the Aspergillus ...

    African Journals Online (AJOL)

    Cellulases are industrially important hydrolytic enzymes applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, an endoglucanase from Aspergillus niger ATCC 10574, EglA, was expressed in the methylotrophic yeast Pichia pastoris and the properties of the recombinant protein were ...

  12. Induction and optimization of cellulases using various agro-wastes ...

    African Journals Online (AJOL)

    SAM

    2014-08-13

    Aug 13, 2014 ... This study presents optimization of various lignocellulosics and alkali pretreatment for maximum cellulase production by Trichoderma virdii sp. Maximum endoglucanase (642 IU/L) and exoglucanase. (187IU/L) activity was achieved with maize straw at 5% concentration. Oat hay was the most suitable.

  13. Cellulase Production from Spent Lignocellulose Hydrolysates by Recombinant Aspergillus niger▿

    Science.gov (United States)

    Alriksson, Björn; Rose, Shaunita H.; van Zyl, Willem H.; Sjöde, Anders; Nilvebrant, Nils-Olof; Jönsson, Leif J.

    2009-01-01

    A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard medium with a comparable monosaccharide content (e.g., 2,100 nkat/ml in spent bagasse hydrolysate compared to 480 nkat/ml in standard glucose-based medium). In addition, A. niger D15[egI] was also able to consume or convert other lignocellulose-derived compounds, such as acetic acid, furan aldehydes, and phenolic compounds, which are recognized as inhibitors of yeast during ethanolic fermentation. The results indicate that enzymes can be produced from the stillage stream as a high-value coproduct in second-generation bioethanol plants in a way that also facilitates recirculation of process water. PMID:19251882

  14. Comparative studies on production of cellulases from three strains of aspergillus niger

    International Nuclear Information System (INIS)

    Sohail, M.; Ahmad, A.; Khan, S.

    2014-01-01

    Three strains of Aspergillus niger were retrieved from culture collection of the Department of Microbiology, University of Karachi, Pakistan and were studied for their ability to produce cellulases. Cultivation at different temperatures and in presence of various carbon sources revealed that all the three strains produced more amounts of endoglucanase, glucosidase and filter-paperase activities at 35 degree C; carboxymethyl cellulose promotes the production of filter paperase and endoglucanase activities whereas salicin induced glucosidase activity. Experiments on growth and enzyme production kinetics showed that generation time and hence volumetric rate of biomass production is influenced by the carbon source used in the medium; simple carbon source, such as glucose favored the growth of all the strains. Cellulases from all the strains showed optimum activity at temperature >50 degree C and under acidic range of pH, while melting temperature was 64-65 degree C. These findings affirm that cellulases from A. niger are potential candidates as alternative to Trichoderma cellulases. (author)

  15. 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.

  16. NOVEL SOURCES OF FUNGAL CELLULASES OF THERMOPHILIC / THERMOTOLERANT FOR EFFICIENT DEINKING OF COMPOSITE PAPER WASTE

    Directory of Open Access Journals (Sweden)

    Rohit Soni

    2008-02-01

    Full Text Available Twenty thermophilic/thermotolerant fungal strains were isolated from compositing soils and screened for production of different enzymes (Endoglucanases, β-glucosidase, Fpase and xylanases to assess their deinking efficiency. Three isolates, Aspergillus sp. AMA, Aspergillus terreus AN1, and Myceliophthora fergusii T4I, identified on the basis of morphological and sequencing of amplified ITS1-5.8S-ITS2 rDNA region, showed significant deinking of composite waste paper (70% magazine and 30% Xerox copier/ laser print paper waste as well as improved properties (brightness, tensile strength, tear index of recycled paper sheets. The chosen strains Aspergillus sp. AMA, Aspergillus terreus AN1 and Myceliophthora fergusii T4I, showed 53, 52.7, and 40.32% deinking with increase in brightness by 4.32, 3.56, and 3.01 % ISO, respectively. These cultures were found to produce multiple endoglucanases and were characterized to lack a cellulose binding module (CBD, which may be responsible for their better deinking efficiency.

  17. Coordinative compounds of molybdenum and vanadium as possible stimulators of extracellular cellulases biosynthesis of micromycetes Penicillium expansum CNMN FD 05 C

    International Nuclear Information System (INIS)

    Chilochi, A.A.; Tyurina, Zh.P.; Klapko, S.F.; Lablyuk, S.V.; Pasha, L.I.; Bologa, O.A.; Koropchanu, Eh.B.; Rizha, A.P.

    2012-01-01

    The effect of coordinative compounds of molybdenum and vanadium on the biosynthesis of cellulosolytic enzymatic complex (endoglucanases, celobiohydrolases, β-glucosidases) of the fungal strain Penicillium expansum CNMN FD 05C was investigated. It was established that complexes of molybdenum, which contain amino acids in its composition, have a neutral effect on the activity of endoglucanases, inhibit celobiohydrolases (80-90%) and stimulate the activity of β-glucosidases. Among the most effective stimulators of β-glucosidases synthesis, the complex MoO 2 (ac.ac.)Gly may be mentioned, that increases the enzymatic activity by 47.8-67.0%. This complex can be used to obtain an enzymatic preparation with a high content of β-glucosidases. The metal complex (NH 4 ) 2 VO 3 Gly stimulates the activity of enzymes of the cellulosolytic complex of the fungi Penicillium expansum CNMN FD 05C, increasing the endogluconasic activity by 45%, cellobiohydrolasic by 32% and β-glucosidasic by 40%.

  18. Production and characterization of endoglucanase secreted by ...

    African Journals Online (AJOL)

    Cellulases are hydrolases of great importance to industries, especially due to their ability to produce ethanol via hydrolysis of cellulolytic materials. Actinomycetes are the producers of these enzymes, particularly the genus Streptomyces sp. The present study is the first report on the production and characterization of ...

  19. Factors affecting endoglucanase production by Trichoderma reesei ...

    African Journals Online (AJOL)

    Factors involved in the screening process were peptone concentration, urea ... ammonium sulfate concentration, calcium nitrate concentration, yeast extract ... pH, incubation time, initial moisture content, inoculum size and substrate amount.

  20. Kinetics of exoglucanase and endoglucanase produced by ...

    African Journals Online (AJOL)

    enoh

    2012-04-05

    Apr 5, 2012 ... Zn2+, Ca2+, Mn2+ and Co2+ enhanced the crude activity of EXG and EG ... processes for producing fuels and chemicals from plant ... increasing the yield of the fruit juices, oil extraction and in ... Trichoderma, Humicola and Aspergillus species were .... observation that stability of the fungal cellulases is.

  1. Kinetics of exoglucanase and endoglucanase produced by ...

    African Journals Online (AJOL)

    enoh

    2012-04-05

    Apr 5, 2012 ... cellulosic waste products can be used by fermentation for the production of useful ... Saleem et al., 2008). Aspergillus sp. is an impor- ... A 10 mL of liquid culture from the inoculum was transferred to 1000. mL Erlenmeyer ..... Cellulases from Penicillium funiculosum: production, properties and application to ...

  2. Production and characterization of endoglucanase secreted by ...

    African Journals Online (AJOL)

    Leonor

    2016-10-19

    Oct 19, 2016 ... 1Laboratory of Applied Chemistry and Technology, Chemical Engineering Course, ... 4Graduate Program in Biotechnology and Natural Resources, ... microorganism was identified at genus level by microculture method; and ...

  3. Factors affecting endoglucanase production by Trichoderma reesei ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... from the ANOVA analysis have a significant value of Pmodel>F= 0.0008 and R2 .... there are various environmental and nutritional factors ... reported to affect cellulase production from wheat straw ... many factors affecting simultaneously the fermentation ..... and control its stability (Kalra and Sandhu, 1986).

  4. Optimizing culture conditions for the production of endo-β-1,4 ...

    African Journals Online (AJOL)

    Among tested carbon sources (coconut fiber, coffee shell, corncob, dried tangerine skin, peanut shell, rice bran, saw dust, sugar-cane bagasse as organic wasters and glucose, lactose sucrose as pure carbon sources), corncob showed the highest endoglucanase production by A. awamori VTCC-F099 at the concentration ...

  5. Browse Title Index

    African Journals Online (AJOL)

    Vol 11, No 33 (2012), Chemical modification of β-endoglucanase from Trichoderma viridin by methanol and determination of the catalytic functional groups, Abstract PDF. Feng Cai, Yangang Xie, Xiaochun He, Tiejun Li. Vol 11, No 51 (2012), Chemical, physical, microbiological and quality attributes studies on River Nile ...

  6. Corn stover-enhanced cellulase production by Aspergillus niger ...

    African Journals Online (AJOL)

    The production of extracellular cellulases by Aspergilus niger NRRL 567 on corn stover was studied in liquid state fermentation. In this study, three cellulases, exoglucanase (EXG), endoglucanase (EG) and β-glucosidase (BGL) were produced by A. niger NRRL 567. The optimal pH, temperature and incubation time for ...

  7. Induction and optimization of cellulases using various agro-wastes ...

    African Journals Online (AJOL)

    This study presents optimization of various lignocellulosics and alkali pretreatment for maximum cellulase production by Trichoderma virdii sp. Maximum endoglucanase (642 IU/L) and exoglucanase (187IU/L) activity was achieved with maize straw at 5% concentration. Oat hay was the most suitable agro-waste for β ...

  8. Pectin-rich biomass as feedstock for fuel ethanol production.

    Science.gov (United States)

    Edwards, Meredith C; Doran-Peterson, Joy

    2012-08-01

    The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.

  9. Pectin-rich biomass as feedstock for fuel ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Meredith C.; Doran-Peterson, Joy [Georgia Univ., Athens, GA (United States). Dept. of Microbiology

    2012-08-15

    The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes. (orig.)

  10. Cloning, sequence and expression of the pel gene from an Amycolata sp.

    Science.gov (United States)

    Brühlmann, F; Keen, N T

    1997-11-20

    The pel gene from an Amycolata sp. encoding a pectate lyase (EC 4.2.2.2) was isolated by activity screening a genomic DNA library in Streptomyces lividans TK24. Subsequent subcloning and sequencing of a 2.3 kb BamHI BglII fragment revealed an open reading frame of 930 nt corresponding to a protein of 29,660 Da. The overall G + C content for the coding region was 65%, with a strong G + C preference in the third (wobble) codon position (93%). A putative ribosome-binding site 5'-GGGAG-3' preceded the translational start codon by 7 base pairs. The Amycolata pectate lyase contains a signal peptide of 26 amino acids, that is cleaved after the sequence Ala-Thr-Ala. The size of the deduced protein as well as its N-terminal amino-acid sequence match the wild-type pectate lyase from the Amycolata sp. Expression of the pel gene in S. lividans TK24 resulted in high pectate lyase activity in the culture supernatant, concomitant with the appearance of a dominant protein band on a sodium dodecyl polyacrylamide gel at 30 kDa. No pectate lyase activity was detected in E. coli BL21 with the pel gene under the strong T7 promotor. The deduced amino-acid sequence showed 40% identity with PelE from Erwinia chrysanthemi and the pectate lyase from Glomerella cingulata. The Amycolata pectate lyase clearly belongs to the pectate lyase superfamily, sharing all functional amino acids and likely has a similar structural topology as Pels from Erwinia chrysanthemi and Bacillus subtilis.

  11. Obtaining nanofibers from curauá and sugarcane bagasse fibers using enzymatic hydrolysis followed by sonication

    DEFF Research Database (Denmark)

    Campos, Adriana de; Correa, Ana Carolina; Cannella, David

    2013-01-01

    This paper is an initial study of the implementation of two new enzymes, an endoglucanase and a concoction of hemicellulases and pectinases to obtain cellulosic nanoparticles. In this study, curauá and sugarcane bagasse were dewaxed and bleached prior to enzymatic action for 72 h at 50 °C, and th...

  12. Computer says 2.5 litres--how best to incorporate intelligent software into clinical decision making in the intensive care unit?

    Science.gov (United States)

    Lane, Katie; Boyd, Owen

    2009-01-01

    What will be the role of the intensivist when computer-assisted decision support reaches maturity? Celi's group reports that Bayesian theory can predict a patient's fluid requirement on day 2 in 78% of cases, based on data collected on day 1 and the known associations between those data, based on observations in previous patients in their unit. There are both advantages and limitations to the Bayesian approach, and this test study identifies areas for improvement in future models. Although such models have the potential to improve diagnostic and therapeutic accuracy, they must be introduced judiciously and locally to maximize their effect on patient outcome. Efficacy is thus far undetermined, and these novel approaches to patient management raise new challenges, not least medicolegal ones.

  13. Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

    Directory of Open Access Journals (Sweden)

    Hui Wei

    Full Text Available Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory, 3 β-D-glucosidases (2 of them secretory and 243 other glycoside hydrolase (GH proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP strain by introducing a CBH (e.g. CBHI into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

  14. Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

    Science.gov (United States)

    Wei, Hui; Wang, Wei; Yarbrough, John M; Baker, John O; Laurens, Lieve; Van Wychen, Stefanie; Chen, Xiaowen; Taylor, Larry E; Xu, Qi; Himmel, Michael E; Zhang, Min

    2013-01-01

    Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

  15. 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.

  16. Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

    Science.gov (United States)

    Lo, Yung-Chung; Huang, Chi-Yu; Cheng, Chieh-Lun; Lin, Chiu-Yue; Chang, Jo-Shu

    2011-09-01

    A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Akihito; Bae, Jun Gu; Fukai, Kotaro; Tokumoto, Naoki; Kuroda, Kouichi; Ogawa, Jun; Shimizu, Sakayu; Ueda, Mitsuyoshi [Kyoto Univ. (Japan). Div. of Applied Life Sciences; Nakatani, Masato [Daiwa Kasei, Shiga (Japan)

    2012-05-15

    A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2{sup '}-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase. (orig.)

  18. Optimization of cellulase production by Penicillium sp.

    Science.gov (United States)

    Prasanna, H N; Ramanjaneyulu, G; Rajasekhar Reddy, B

    2016-12-01

    The production of cellulolytic enzymes (β-exoglucanase, β-endoglucanase and β-glucosidase) by Penicillium sp. on three different media in liquid shake culture conditions was compared. The organism exhibited relatively highest activity of endoglucanase among three enzymes measured at 7-day interval during the course of its growth on Czapek-Dox medium supplemented with 0.5 % (w/v) cellulose. Cellulose at 0.5 %, lactose at 0.5 %, sawdust at 0.5 %, yeast extract at 0.2 % as a nitrogen source, pH 5.0 and 30 °C temperature were found to be optimal for growth and cellulase production by Penicillium sp. Yields of Fpase, CMCase and β-glucosidase, attained on optimized medium with Penicillium sp. were 8.7, 25 and 9.52 U/ml, respectively with increment of 9.2, 5.9 and 43.8-folds over titers of the respective enzyme on unoptimised medium. Cellulase of the fungal culture with the ratio of β-glucosidase to Fpase greater than one will hold potential for biotechnological applications.

  19. Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

    2012-01-24

    The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  20. Toprak Solucanlarından Elde Edilen Vermikompostun Bazı Bitki Patojenleri Üzerindeki Antimikrobiyal Aktivitelerinin Araştırılması

    Directory of Open Access Journals (Sweden)

    Uğur Tutar

    2012-12-01

    Full Text Available Toprak solucanlarının, organik atıkları biyolojik olarak parçalayarak ayrıştırmaları ile oluşturdukları “vemikompost” un, bazı patojen bakteri ve funguslara karşı etkili oldukları yapılan çeşitli araştırmalarla saptanmıştır. Bu çalışmada, Eisenia fetida türü toprak solucanlarından elde edilen vermikompostun; etanol ve kloroform solventleri kullanılarak elde edilen ekstrelerinin, bitkilerde hastalıklara neden olan toprak kaynaklı patojen 9 adet bakteri ve 9 adet fungusa karşı etkinliklerinin belirlenmesi amacıyla “disk difüzyon” ve “MIC” testleri uygulanmıştır. Çalışma sonuçlarına göre, toprak solucanlarından elde edilen vermikompostun kloroform ile elde edilen ekstrelerinin Pseudomonas syringae, Xhantomonas carotae, Sclerotinia sclerotiorum, Fusarim oxysporum, Aspergillus humicola ve Aspergillus fumigatus’ a karşı etkileri güçlü olurken Erwinia chrysanthemi, Pseudomonas fluorescens, ve Penicillium brevicompactum’ a karşı etkilerinin daha zayıf olduğu görülmüştür. Vermikompostun etanol ile elde edilen ekstrelerinin ise Pseudomonas syringae, Xhantomonas campestris ve Aspergillus fumigatus’ a karşı etkilerinin güçlü olduğu, Erwinia herbicola, Erwinia chrysanthemi ve Sclerotinia sclerotiorum’ a  karşı ise daha zayıf bir etki gösterdiği saptanmıştır.

  1. EFEITO ANTIBIÓTICO DO PRÓPOLIS SOBRE BACTÉRIAS FITOPATOGÊNICAS

    Directory of Open Access Journals (Sweden)

    L. BIANCHINI

    1998-01-01

    Full Text Available O efeito antibiótico de extrato aquoso de própolis, em várias concentrações, foi avaliado para cinco espécies de bactérias fitopatogênicas. Agrobacterium tumefaciens, Clavibacter michiganensis subsp. michiganensis e Xanthomonas axonopodis pv. phaseoli foram completamente inibidas em meio de cultura contendo 10% de extrato de própolis. Erwinia chrysanthemi foi parcialmente inibida, enquanto Pseudomonas syringae pv. tabaci se mostrou insensível ao extrato, desenvolvendo colônias idênticas àquelas observadas em meios livres de própolis. Concentrações menores não foram suficientes para exercer um efeito antimicrobiano desejável sobre as bactérias pertencentes a todas as espécies testadas. Resultados idênticos foram obtidos quando própolis foi incorporado ao meio antes ou após a autoclavagem, demonstrando que a substância ativa presente no extrato não é termosensível. Foi demonstrado, portanto, o uso potencial do própolis como antibiótico para o controle de bactérias fitopatogênicas.The antibiotic effect of aqueous extract of propolis was evaluated in vitro against five species of bacteria. The growth of Agrobacterium tumefaciens, Clavibacter michiganensis subsp. michiganensis and Xanthomonas axonopodis pv. phaseoli was completely inhibited in a medium containing 10% of propolis. Erwinia chrysanthemi was partially inhibited and Pseudomonas syringae pv. tabaci was not affected. Lower concentration of the aqueous extract was not effective to promote a desirable antibiotic action against all bacterial species tested. The active ingredient present in the propolis was thermostable. The potential use of the propolis to control phytopathogenic bacteria was demonstrated.

  2. Estimativa da digestibilidade aparente de nutrientes em dietas para eqüinos, com o uso de óxido crômico e indicadores internos

    Directory of Open Access Journals (Sweden)

    Oliveira Chiara Albano de Araújo

    2003-01-01

    Full Text Available Foram realizados dois ensaios com o objetivo de avaliar a digestibilidade aparente de nutrientes em dietas para eqüinos, utilizando o método da coleta total de fezes e os indicadores óxido crômico, fibra em detergente ácido indigestível (FDAi, fibra em detergente neutro indigestível (FDNi, celulose indigestível (CELi, lignina (LIG e cinzas insolúveis em detergente ácido (CIDA. No primeiro ensaio, foram utilizados cinco potros mestiços, alimentados com cinco dietas contendo níveis diferenciados de proteína bruta, mantendo a relação concentrado e volumoso em 50:50. O óxido crômico foi utilizado como indicador externo. No segundo ensaio, foram utilizados quatro potros mestiços, alimentados com dietas compostas por feno de coastcross como volumoso e concentrado nas proporções de 40:60, 60:40, 80:20 e 100:00. Nos dois ensaios, os teores de fibra em detergente ácido indigestível, fibra em detergente neutro indigestível, celulose indigestível, lignina e cinzas insolúveis em detergente ácido foram obtidos após incubação in vitro. No primeiro ensaio, a FDAi mostrou-se adequada como indicador interno para estimar a digestibilidade; a CELi, em ambos os ensaios, foi adequada como indicador interno para a estimativa da digestibilidade aparente de nutrientes em dietas para eqüinos. O óxido crômico apresentou baixa recuperação fecal no primeiro ensaio e a LIG, baixa recuperação fecal nos dois ensaios, subestimando os coeficientes de digestibilidade dos nutrientes, sendo inadequada sua utilização para estimativa da digestibilidade. A CIDA, em função da metodologia de coleta fecal, também mostrou-se inadequada para as estimativas de digestibilidade em eqüinos.

  3. Clinical Note Creation, Binning, and Artificial Intelligence.

    Science.gov (United States)

    Deliberato, Rodrigo Octávio; Celi, Leo Anthony; Stone, David J

    2017-08-03

    The creation of medical notes in software applications poses an intrinsic problem in workflow as the technology inherently intervenes in the processes of collecting and assembling information, as well as the production of a data-driven note that meets both individual and healthcare system requirements. In addition, the note writing applications in currently available electronic health records (EHRs) do not function to support decision making to any substantial degree. We suggest that artificial intelligence (AI) could be utilized to facilitate the workflows of the data collection and assembly processes, as well as to support the development of personalized, yet data-driven assessments and plans. ©Rodrigo Octávio Deliberato, Leo Anthony Celi, David J Stone. Originally published in JMIR Medical Informatics (http://medinform.jmir.org), 03.08.2017.

  4. Premios para la Ciencia Médica: Suicidio celular, sida en niños y otras investigaciones.

    Directory of Open Access Journals (Sweden)

    Alfredo Jácome Roca

    2007-11-01

    Las menciones honoríficas en el área de básicas fueron dos y ambas correspondieron a profesores de la facultad de medicina de la Universidad Javeriana en Bogotá. La primera de ellas la recibió en grupo de ciencias fi siológicas, liderado por el doctor Gabriel Pascual, con la colaboración de los doctores Henry Adolfo Aceros y Darío Riascos Bernal, y fue presentada como tesis de grado por la bióloga Catalina Celis Borrero, investigadora en este campo. Se tituló “Evaluación del efecto del extracto de alcaloides de la piel de Dendrobates truncatus –DT– (Anura: Dendrobatidae sobre la unión neuromuscular en una preparación frénico-diafragma de rata”...

  5. Reframing Democracy -

    DEFF Research Database (Denmark)

    Siim, Birte

    2016-01-01

    economic crisis has inspired debatesabout equality and justice within and beyond the nation state and about theabilities of transnational civil society actors to influence the politicalagenda. Feminist scholarship has started to explore intersections of gender,class and diversity at national...... and citizenship include: GenderDiversities – Practicing Intersectionality in the European Union. Ethnicities. 2014 (14) 4 (with L.Rolandsen Agustin); “PoliticalIntersectionality and Democratic Politics in the European Public Sphere”, Politics & Gender, 2014 (10)01; “Conflictsand Negotiations about Framings...... of Gender and Ethnicity by Political Actors inthe European Public Sphere”; Journal ofInternational and Comparative Social Policy, 2014 (30) 1; Negotiations ofGender and Diversity in an Emergent European Public Sphere (ed. with M. Mokre), Palgrave/Macmillan 2013;“Citizenship” in K. Celis, V.Waylen (ed...

  6. Brcal: A Review of Structure and Putative Functions

    Directory of Open Access Journals (Sweden)

    James W.E. Paterson

    1998-01-01

    Full Text Available BRCA1 is a complex gene implicated in familial breast and ovarian cancer. Although it is almost certainly a tumour suppressor, it is also essential for the normal growth and development of embryonic cells. BRCA1 is probably involved in DNA damage and repair, in cell cycle regulation, and in differentiation of celis. It remains to be established whether all these functions are subserved by single mechanism or pathway. Since the cloning of BRCA1 in 1994, much has been learned about the function of the gene. However, a great deal more still has to be uncovered. The size of the protein coded by the BRCA1 gene and the variety of transcripts argues for a complexity of function and regulation that will provide intellectual and technical challenges for years to come.

  7. Compendium of Abstracts on Statistical Applications in Geotechnical Engineering.

    Science.gov (United States)

    1983-09-01

    motion and eogiteering prcedures for importnt i.t-la ions teo r ec fiv fault. 213. Lar A siat o o:iPft ea quk ampliftio specr frsuhern Celi frl site 213...iincck ’in tbcc n, ,acc.c -,c I- for.~~i .c.ccsc .o -o lf2 ~ ~ ~ ~ ~ ~ ~ ’ Ara ohrcc h-f c kc .,ccc v c ocl- ,c.c 184.c Iaelsosc I ccpecc-tc ot thecccall...Ii r. L’ Lf v. -C C Et KC . -C CIc c a, > LE r a o 7C -3C -C- - XC C . - C L- 0 L 0 1 f* *L U L c~ mC1 cc wz f ,c or -ft CL Q-t I C EC a a u C3’ a

  8. Investigation of stress tolerance of endoglucanases of the ...

    African Journals Online (AJOL)

    Ugochukwu Anieto

    2015-06-23

    Jun 23, 2015 ... The continual depletion of fossil fuel reserves, increase in the world's population ... Advantages of the cellulosome include (i) a direct and ... treatment; (ii) Ethanol treated cells indicated a decline in glutamic acid, a 2.8- fold ...

  9. Investigation of stress tolerance of endoglucanases of the ...

    African Journals Online (AJOL)

    Ugochukwu Anieto

    2015-06-23

    Jun 23, 2015 ... with technical merits, bioethanol continues to lead the pack in adoption and ... cultures of cellulose media three or more days old .... down to room temperature. ..... activity, e.g., the more ethanol, the faster the inactivation.

  10. Investigation of stress tolerance of endoglucanases of the ...

    African Journals Online (AJOL)

    Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself.

  11. Chemical modification of β-endoglucanase from Trichoderma viridin ...

    African Journals Online (AJOL)

    user

    2012-03-26

    Mar 26, 2012 ... Feng Cai#, Yangang Xie#, Xiaochun He and Tiejun Li* ..... m. pH 4.0. pH 5.0. pH 6.0. Figure 2. Effect of saturation degree of ammonium sulfate on the content of protein salted ..... JL, Feng JX (2007). Cloning ... Kim SJ, Lee CM, Kim MY, Yeo YS, Yoon SH, Kang HC, Koo. ... University Press, Beijing, China.

  12. Effectieve kolonisatie van aardappelplanten door Dickeya-soorten (Erwinia chrysanthemi) : themanummer fytobacteriologie

    NARCIS (Netherlands)

    Wolf, van der J.M.; Czajkowski, R.L.; Velvis, H.

    2009-01-01

    De bacterieziekten zwartbenigheid en stengelnatrot, veroorzaakt door Pectobacterium en Dickeya (Erwinia)- soorten, berokkenen grote schade aan de pootaardappelteelt. Bij PRI en HZPC wordt onderzoek verricht naar de verspreiding van deze pathogenen tijdens teelt- en (na)oogst. Het was al bekend dat

  13. The Effects of Bioprocess Parameters on Cellulase Production with Trichoderma viride CMIT35

    Directory of Open Access Journals (Sweden)

    Teodor Vintila

    2010-05-01

    Full Text Available Fungal cellulases are well-studied, and have various applications in industry, health or agriculture. Species of Trichoderma can produce substantial amounts of endoglucanase, exoglucanase (saccharifying cellulases, and some strains are able to produce important quantities of β-glucosidase. A number of fungi were isolated abroad and screened for cellulolytic potential. In this study, the kinetics of cellulase production from an indigenous strain of T. viride CMIT35 is reported. Product formation parameters of different types of cellulases indicate that the studied strain of T. viride is capable of producing important levels of cellulases when grown on Mandels medium with wheat bran as carbon source. Furthermore, it was observed that production of endoglucanase reaches its maximum during exponential phase of growth, while exoglucanase during the stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be more efficient than liquid state fermentation. High production of cellulase was noted at the following parameters for liquid cultures: 4% wheat bran, 5% inoculum, 180 r.p.m. agitation, pH 5; and 60% humidity in the case of solid state fermentation.

  14. High-solids enrichment of thermophilic microbial communities and their enzymes on bioenergy feedstocks

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A. P.; Allgaier, M.; Singer, S.W.; Hazen, T.C.; Simmons, B.A.; Hugenholtz, P.; VanderGheynst, J.S.

    2011-04-01

    Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

  15. A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues

    Directory of Open Access Journals (Sweden)

    Carlos Meneses

    2016-06-01

    Full Text Available Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol.

  16. A Metagenomic Advance for the Cloning and Characterization of a Cellulase from Red Rice Crop Residues.

    Science.gov (United States)

    Meneses, Carlos; Silva, Bruna; Medeiros, Betsy; Serrato, Rodrigo; Johnston-Monje, David

    2016-06-25

    Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol).

  17. Crystal structure and genetic modifications of FI-CMCase from Aspergillus aculeatus F-50.

    Science.gov (United States)

    Huang, Jian-Wen; Liu, Weidong; Lai, Hui-Lin; Cheng, Ya-Shan; Zheng, Yingying; Li, Qian; Sun, Hong; Kuo, Chih-Jung; Guo, Rey-Ting; Chen, Chun-Chi

    2016-09-16

    Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Growth and enzyme production by three Penicillium species on monosaccharides

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Krogh, Astrid Mørkeberg; Krogh, Kristian Bertel Rømer

    2004-01-01

    The growth and preference for utilisation of various sugar by the Penicillium species Penicillium pinophilum IBT 4186, Penicillium persicinum IBT 13226 and Penicillium brasilianum IBT 20888 was studied in batch cultivations using various monosaccharides as carbon source, either alone or in mixtur...... producing beta-glucosidase and endoglucanases. Xylose did not repress the enzyme production and it induced the production of endoxylanases and beta-xylosidases....

  19. Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

    OpenAIRE

    Wang, Min; Lu, Xuefeng

    2016-01-01

    Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in biomass to biofuels process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellob...

  20. Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.

    Science.gov (United States)

    Badhan, Ajay; Wang, Yuxi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim

    2014-04-26

    Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

  1. Exo-endo cellulase fusion protein

    Science.gov (United States)

    Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

    2012-01-17

    The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

  2. Ruminococcus flavefaciens 007C cellulosomes and cellulase consortium

    Directory of Open Access Journals (Sweden)

    Maša VODOVNIK

    2015-11-01

    Full Text Available Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its efficiency in degradation of (hemicellulosic substrates is associated with the production of remarkably intricate extracellular multienzyme complexes, named cellulosomes. In the present work we investigated the cellulolytic system of 007C. The bioinformatic analysis of the draft genome sequence revealed identical organization of sca gene cluster as has previously been found in four other strains of R. flavefaciens. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The cellulases of R. flavefaciens 007C belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 and GH9, whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only found coupled to GH9 catalytic domains. Zymogram analysis showed that larger endoglucanases were mostly constitutively expressed, wheras smaller enzymes were only detected in later phases of Avicel-grown cultures.

  3. Community dynamics and glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Gladden, J.M.; Allgaier, M.; Miller, C.S.; Hazen, T.C.; VanderGheynst, J.S.; Hugenholtz, P.; Simmons, B.A.; Singer, S.W.

    2011-05-01

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  4. Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.

    Science.gov (United States)

    Gladden, John M; Allgaier, Martin; Miller, Christopher S; Hazen, Terry C; VanderGheynst, Jean S; Hugenholtz, Philip; Simmons, Blake A; Singer, Steven W

    2011-08-15

    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

  5. Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential for lignocellulose decomposition

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Chaowei; Harrold, Duff R.; Claypool, Joshua T.; Simmons, Blake A.; Singer, Steven W.; Simmons, Christopher W.; VanderGheynst, Jean S.

    2017-01-01

    Microorganisms involved in biomass deconstruction are an important resource for organic waste recycling and enzymes for lignocellulose bioconversion. The goals of this paper were to examine the impact of nitrogen amendment on microbial community restructuring, secretion of xylanases and endoglucanases, and potential for biomass deconstruction. Communities were cultivated aerobically at 55 °C on green waste (GW) amended with varying levels of NH4Cl. Bacterial and fungal communities were determined using 16S rRNA and ITS region gene sequencing and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to predict relative abundance of genes involved in lignocellulose hydrolysis. Nitrogen amendment significantly increased secretion of xylanases and endoglucanases, and microbial activity; enzyme activities and cumulative respiration were greatest when nitrogen level in GW was between 4.13–4.56 wt% (g/g), but decreased with higher nitrogen levels. The microbial community shifted to one with increasing potential to decompose complex polymers as nitrogen increased with peak potential occurring between 3.79–4.45 wt% (g/g) nitrogen amendment. Finally, the results will aid in informing the management of nitrogen level to foster microbial communities capable of secreting enzymes that hydrolyze recalcitrant polymers in lignocellulose and yield rapid decomposition of green waste.

  6. Novas ocorrências de Erwinia carotovora subsp. carotovora e de E. chrysanthemi

    Directory of Open Access Journals (Sweden)

    Irene M. G. Almeida

    1997-05-01

    Full Text Available Em continuidade a trabalhos de caracterização de bactérias pectinolíticas do gênero Eruia ocorrendo no Brasil, são relacionadas novas ocorrências dessas fitobactérias em plantios comerciais, que ocasionam podridão mole em cinco espécies de plantas ornamentais. Testes bioquímicas, fisiológicos, culturais e de patogenicidade permitiram comprovar a ocorrência de Erwinia carotovora subsp. carotovora em plantas de afelandra, amarílis e copo-de-leite, e de Erwiniachr santhemiemcordilineekalanchoe.

  7. Pretreatment and enzymatic hydrolysis of wheat straw (Triticum aestivum L.) – The impact of lignin relocation and plant tissues on enzymatic accessibility

    DEFF Research Database (Denmark)

    Hansen, Mads Anders Tengstedt; Kristensen, Jan Bach; Felby, Claus

    2011-01-01

    , after 144 h of enzymatic hydrolysis the cortex had vanished, exposing the heavier lignified vascular tissue. Accumulation of lignin droplets and exposure of residual lignin could be part of the explanation for the decreasing hydrolysis rate. Flattening of macrofibrils after pretreatment together...... with more indentations on the surfaces was also observed, possibly caused by a proposed synergistic effect of cellobiohydrolases and endoglucanases. Keywords: Lignocellulose; Plant tissues; Lignin accumulation; Atomic Force Microscopy; Scanning Electron Microscopy...

  8. Catalysts of plant cell wall loosening [version 1; referees: 2 approved

    OpenAIRE

    Daniel J. Cosgrove

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyl...

  9. Production and localization of cellulases and. beta. -glucosidase from the thermophilic fungus Thielavia terrestris

    Energy Technology Data Exchange (ETDEWEB)

    Breuil, C; Wojtczak, G; Saddler, J N

    1986-01-01

    The enzyme production and localization of Thielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular ..beta..-glucosidase activity were detected in the former strain. The intracellular ..beta..-glucosidase of both strains were more thermostable than the extra-cellular enzyme; the half life of T. terrestris (C464) endoglucanase activity at 60 degrees C was greater than 96 hours. 12 references.

  10. Optically stimulated luminescence of MgB{sub 4}O{sub 7}:Ce,Li for gamma and neutron dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Yukihara, E.G.; Doull, B.A.; Gustafson, T. [Physics Department, Oklahoma State University, Stillwater, OK 74078 (United States); Oliveira, L.C. [Physics Department, Oklahoma State University, Stillwater, OK 74078 (United States); Departamento de Física, FFCLRP-Universidade de São Paulo, 14040-901 Ribeirão Preto-SP (Brazil); Kurt, K. [Physics Department, Oklahoma State University, Stillwater, OK 74078 (United States); Mersin University Science and Letter Faculty Physics Department 33343 Mersin (Turkey); Milliken, E.D. [Physics Department, Oklahoma State University, Stillwater, OK 74078 (United States); R& D Pigments, Ferro Corporation, 251 W. Wylie Ave, Washington, PA 15301 (United States)

    2017-03-15

    The objective of this work was to develop a new optically stimulated luminescence (OSL) material for dosimetry applications that is tissue equivalent and has high sensitivity to ionizing radiation, fast luminescence lifetime, and intrinsic neutron sensitivity. To achieve this combination of properties, we started with a host material with low effective atomic number, MgB{sub 4}O{sub 7} (Z{sub eff}=8.2){sub ,} with an appropriate dopant characterized by short luminescence lifetime (Ce{sup 3+}). The samples were synthesized using Solution Combustion Synthesis with excess boric acid to achieve the correct crystallographic phase and Li co-doping to enhance its sensitivity. We investigated the thermoluminescence (TL) and OSL properties as a function of annealing temperature, radiation dose, dopant concentration, and time elapsed after irradiation (i.e., signal fading). We also applied a step-annealing procedure to investigate the depth of the trapping centers associated with the OSL signal. The samples obtained are characterized by a dominant TL peak at ~210 °C with intensity comparable to LiF:Mg,Ti. The OSL intensity is ~50% of that from Al{sub 2}O{sub 3}:C when using Hoya U-340 filters and shows no saturation up to almost 1 kGy. The OSL signal seems to originate from trapping center with stability > 150 °C, which means that the OSL fading is expected to be small. After the first day, in which fading associated with shallow traps is observed, fading of the total OSL signal was <4% within 6 days. The possibility of enhancing the neutron sensitivity was also demonstrated by synthesizing the material with enriched {sup 10}B. Although further development and characterization of the material may be needed, this work demonstrates that this host/dopant combination can be a viable alternative in OSL dosimetry, particularly for 2D dose mapping and neutron dosimetry applications.

  11. Production of crude enzyme from Aspergillus nidulans AKB-25 using black gram residue as the substrate and its industrial applications

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    2016-06-01

    Full Text Available The production of crop residues in India is estimated to be about 500–550 million tons annually. It is estimated that about 93 million tons of crop residues is burnt annually which is not only wastage of valuable biomass resources but pollution of the environment with the production of green house gases also. Among different low cost crop residues, black gram residue as the substrate produced maximal endoglucanase, FPase, and β-glucosidase activities from Aspergillus nidulans AKB-25 under solid-state fermentation. During optimisation of cultural parameters A. nidulans AKB-25 produced maximal endoglucanase (152.14 IU/gds, FPase (3.42 FPU/gds and xylanase (2441.03 IU/gds activities. The crude enzyme was found effective for the saccharification of pearl millet stover and bio-deinking of mixed office waste paper. The crude enzyme from A. nidulans AKB-25 produced maximum fermentable sugars of 546.91 mg/g from alkali-pretreated pearl millet stover by saccharification process at a dose of 15 FPU/g of substrate. Pulp brightness and deinking efficiency of mixed office waste paper improved by 4.6% and 25.01% respectively and mitigated dirt counts by 74.70% after bio-deinking. Physical strength properties like burst index, tensile index and double fold number were also improved during bio-deinking of mixed office waste paper.

  12. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries

    DEFF Research Database (Denmark)

    Parisutham, Vinuselvi; Chandran, Sathesh-Prabu; Mukhopadhyay, Aindrila

    2017-01-01

    Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic...... hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by β-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less...

  13. Isolation and characterization of a cellulolytic actinomycete Microbispora bispora

    Energy Technology Data Exchange (ETDEWEB)

    Waldron, Jr, C R; Becker-Vallone, C A; Eveleigh, D E

    1986-09-01

    Protocols for the isolation of cellulolytic actinomycetes are described, and their use illustrated in the selection of thermophilic bacteria from soil. One isolate, Microbispora bispora, was selected for further study. It grew readily at 55/sup 0/C, produced an extracellular cellulase in good yield (endoglucanase, 5.9 U/ml) that had a broad pH range (pH 5.5 - 7.2) and was thermally stable. Its aryl-..beta..-glucosidase was cell-associated and was relatively resistant to end-product inhibition.

  14. Enzymatic degradation of cellulose for thermophilic actinomycete: isolation, characterization and cellulolytic activity determination

    Directory of Open Access Journals (Sweden)

    Pablo Ramírez

    2013-06-01

    Full Text Available One hundred and forty five cellulolytic thermophilic actinomycete strains were isolated from 71 compost, soil, hay and dung samples. Streptomyces sp. (50,63%, Thermomonospora curvata (15,82%, T. chromogena (13,92%, and other species were identified. Endoglucanase, exoglucanase and β-glucosidase activities were evaluated from 10 cellulolytic actinomycete strains. Among these the Streptomyces sp. 7CMC10 strain showed the biggest activity levels corresponding to 20,14; 2,61 and 5,40 UI/mg of protein, respectively.

  15. Bacterial leaf rot of Aloe vera L., caused byErwinia chrysanthemi biovar 3

    NARCIS (Netherlands)

    Laat, de P.C.A.; Verhoeven, J.T.W.; Danse, J.D.

    1994-01-01

    A severe attack of the bacteriumErwinia chrysantemi biovar 3 on the succulentAloe vera on the Carribean island of Aruba is described. Biochemical and pathological characteristics of strains are presented, including results of successful inoculation experiments onAloe vera. This is the first report

  16. Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2017-01-01

    in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species...... were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance....

  17. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

    International Nuclear Information System (INIS)

    Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

    1987-01-01

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

  18. Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

    Science.gov (United States)

    Bando, Hiroki; Hisada, Hiromoto; Ishida, Hiroki; Hata, Yoji; Katakura, Yoshio; Kondo, Akihiko

    2011-11-01

    A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

  19. Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

    2003-01-01

    Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities....... Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused...

  20. Hyperthermostable cellulolytic and hemicellulolytic enzymes and their biotechnological applications

    Directory of Open Access Journals (Sweden)

    Tipparat Hongpattarakere

    2002-07-01

    Full Text Available Hyperthermal cellulases and hemicellulases have been intensively studied due to their highly potential applications at extreme temperatures, which mimic industrial processes involving cellulose and hemicellulose degradation. More than 50 species of hyperthermophiles have been isolated, many of which possess hyperthermal enzymes required for hydrolyzing cellulose and hemicelluloses. Endoglucanases, exoglucanases, cellobiohydrolases, xylanases, β-glucosidase and β-galactosidase, which are produced by the hyperthermophiles, are resistant to boiling temperature. The characteristics of these enzymes and the ability to maintain their functional integrity at high temperature as well as their biotechnological application are discussed.

  1. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

    2015-11-04

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  2. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2015-09-08

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  3. Multifarious activities of cellulose degrading bacteria from Koala (Phascolarctos cinereus) faeces.

    Science.gov (United States)

    Singh, Surender; Thavamani, Palanisami; Megharaj, Mallavarapu; Naidu, Ravi

    2015-01-01

    Cellulose degrading bacteria from koala faeces were isolated using caboxymethylcellulose-Congo red agar, screened in vitro for different hydrolytic enzyme activities and phylogenetically characterized using molecular tools. Bacillus sp. and Pseudomonas sp. were the most prominent bacteria from koala faeces. The isolates demonstrated good xylanase, amylase, lipase, protease, tannase and lignin peroxidase activities apart from endoglucanase activity. Furthermore many isolates grew in the presence of phenanthrene, indicating their probable application for bioremediation. Potential isolates can be exploited further for industrial enzyme production or in bioremediation of contaminated sites.

  4. Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells.

    Science.gov (United States)

    Ngai, Patrick H K; Ng, T B

    2003-11-14

    From the fruiting bodies of the edible mushroom Lentinus edodes, a novel protein designated lentin with potent antifungal activity was isolated. Lentin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of lentin manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

  5. Promoting Secondary Analysis of Electronic Medical Records in China: Summary of the PLAGH-MIT Critical Data Conference and Health Datathon.

    Science.gov (United States)

    Li, Peiyao; Xie, Chen; Pollard, Tom; Johnson, Alistair Edward William; Cao, Desen; Kang, Hongjun; Liang, Hong; Zhang, Yuezhou; Liu, Xiaoli; Fan, Yong; Zhang, Yuan; Xue, Wanguo; Xie, Lixin; Celi, Leo Anthony; Zhang, Zhengbo

    2017-11-14

    Electronic health records (EHRs) have been widely adopted among modern hospitals to collect and track clinical data. Secondary analysis of EHRs could complement the traditional randomized control trial (RCT) research model. However, most researchers in China lack either the technical expertise or the resources needed to utilize EHRs as a resource. In addition, a climate of cross-disciplinary collaboration to gain insights from EHRs, a crucial component of a learning healthcare system, is not prevalent. To address these issues, members from the Massachusetts Institute of Technology (MIT) and the People's Liberation Army General Hospital (PLAGH) organized the first clinical data conference and health datathon in China, which provided a platform for clinicians, statisticians, and data scientists to team up and address information gaps in the intensive care unit (ICU). ©Peiyao Li, Chen Xie, Tom Pollard, Alistair Edward William Johnson, Desen Cao, Hongjun Kang, Hong Liang, Yuezhou Zhang, Xiaoli Liu, Yong Fan, Yuan Zhang, Wanguo Xue, Lixin Xie, Leo Anthony Celi, Zhengbo Zhang. Originally published in JMIR Medical Informatics (http://medinform.jmir.org), 14.11.2017.

  6. Isolation and enzyme bioprospection of endophytic bacteria associated with plants of Brazilian mangrove ecosystem.

    Science.gov (United States)

    Castro, Renata A; Quecine, Maria Carolina; Lacava, Paulo T; Batista, Bruna D; Luvizotto, Danice M; Marcon, Joelma; Ferreira, Anderson; Melo, Itamar S; Azevedo, João L

    2014-01-01

    The mangrove ecosystem is a coastal tropical biome located in the transition zone between land and sea that is characterized by periodic flooding, which confers unique and specific environmental conditions on this biome. In these ecosystems, the vegetation is dominated by a particular group of plant species that provide a unique environment harboring diverse groups of microorganisms, including the endophytic microorganisms that are the focus of this study. Because of their intimate association with plants, endophytic microorganisms could be explored for biotechnologically significant products, such as enzymes, proteins, antibiotics and others. Here, we isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. After identifying the isolates, the bacterial communities were evaluated for enzyme production. Protease activity was observed in 75% of the isolates, while endoglucanase activity occurred in 62% of the isolates. Bacillus showed the highest activity rates for amylase and esterase and endoglucanase. To our knowledge, this is the first reported diversity analysis performed on endophytic bacteria obtained from the branches of mangrove trees and the first overview of the specific enzymes produced by different bacterial genera. This work contributes to our knowledge of the microorganisms and enzymes present in mangrove ecosystems.

  7. 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)

  8. El patrimonio cultural nos incluye a todos. Fundación Eliécer Silva Celis : una organización inteligente

    OpenAIRE

    Ardila Silva, Sandra Ximena; Correal Cabezas, Ximena Alexandra

    2011-01-01

    El proyecto consta de tres componentes fundamentales los cuales son: (1) Crear un modelo organizacional inteligente que se articule a las demandas culturales de la comunidad de Sogamoso y contribuya a apropiación del patrimonio cultural en el departamento de Boyacá. (2) Formular un plan estratégico para la FESC que promueva procesos culturales de comunicación, intercambio y apropiación del patrimonio cultural. (3) Diseñar una estrategia de sostenibilidad para la FESC basada en la importancia ...

  9. Cellulase production by Trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Deschamps, F.; Giuliano, C.; Asther, M.; Huet, M.C.; Roussos, S.

    1985-09-01

    Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8, 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 hours. The extension from static column cultivation to stirred tank reactor of 65 l capacity gave similar yields of cellulase.

  10. Cellulase activities in biomass conversion: measurement methods and comparison.

    Science.gov (United States)

    Dashtban, Mehdi; Maki, Miranda; Leung, Kam Tin; Mao, Canquan; Qin, Wensheng

    2010-12-01

    Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

  11. Synthesis of O- and C-glycosides derived from β-(1,3)-D-glucans.

    Science.gov (United States)

    Marca, Eduardo; Valero-Gonzalez, Jessika; Delso, Ignacio; Tejero, Tomás; Hurtado-Guerrero, Ramon; Merino, Pedro

    2013-12-15

    A series of β-(1,3)-d-glucans have been synthesized incorporating structural variations specifically on the reducing end of the oligomers. Both O- and C-glucosides derived from di- and trisaccharides have been obtained in good overall yields and with complete selectivity. Whereas the O-glycosides were obtained via a classical Koenigs-Knorr glycosylation, the corresponding C-glycosides were obtained through allylation of the anomeric carbon and further cross-metathesis reaction. Finally, the compounds were evaluated against two glycosidases and two endo-glucanases and no inhibitory activity was observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. New genes and new biological roles for expansins

    Science.gov (United States)

    Cosgrove, D. J.

    2000-01-01

    Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

  13. Molecular basis of the activity of the phytopathogen pectin methylesterase

    Science.gov (United States)

    Fries, Markus; Ihrig, Jessica; Brocklehurst, Keith; Shevchik, Vladimir E; Pickersgill, Richard W

    2007-01-01

    We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel β-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs. PMID:17717531

  14. Pineapple Fruit Collapse: Newly Emerging Disease of Pineapple Fruit in Lampung, Indonesia

    Directory of Open Access Journals (Sweden)

    Joko Prasetyo

    2014-03-01

    Full Text Available ABSTRACT Pineapple fruit collapse: newly emerging disease of pineapple fruit in Lampung, Indonesia Recently, a new disease on pineapple fruit has occurred in Lampung. Symptoms of the disease are complex. Fruits rotted and exuded copious liquid from the inter- fruitlet tissues accompanied by gas bubbles. Open spaces were formed inside the rotten fruit. Dissection of diseased fruit showed many cavities within its sceletal fibres and bad odour was exerted from the rotten tissues. A bacterial entity was isolated  from the diseased materials. In a pathogenicity test, the isolated bacteria caused the same symptom as mentioned. In the growing-on test the crown of the heavily infected fruit  showed  heart rot symptom.  Those  indicated that the disease was pineapple fruit collapse. Both symptoms were known related to the same causal agent, Erwinia chrysanthemi (pineapple strain Dickeya sp.. In our opinion, this is the first report of pineapple fruit collapse in Indonesia.

  15. Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

    Science.gov (United States)

    Chen, Qiang; Kinde, Monica N; Arjunan, Palaniappa; Wells, Marta M; Cohen, Aina E; Xu, Yan; Tang, Pei

    2015-09-08

    Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

  16. Cellobiohydrolase and endoglucanase respond differently to surfactants during the hydrolysis of cellulose

    DEFF Research Database (Denmark)

    Hsieh, Chia-wen C.; Cannella, David; Jørgensen, Henning

    2015-01-01

    Background: Non-ionic surfactants such as polyethylene glycol (PEG) can increase the glucose yield obtained from enzymatic saccharification of lignocellulosic substrates. Various explanations behind this effect include the ability of PEG to increase the stability of the cellulases, decrease non-p...

  17. 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.

  18. Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

    Science.gov (United States)

    Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

    2016-12-09

    Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Expanding the knowledge on lignocellulolytic and redox enzymes of worker and soldier castes from the lower termite Coptotermes gestroi

    Directory of Open Access Journals (Sweden)

    João Paulo Lourenço Franco Cairo

    2016-10-01

    Full Text Available Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes. Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9 and beta-glucosidases (GH1 were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7 and endo-glucanases (GH5 and GH45, as well as classical xylanases (GH10 and GH11, were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs, which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

  20. Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi.

    Science.gov (United States)

    Franco Cairo, João P L; Carazzolle, Marcelo F; Leonardo, Flávia C; Mofatto, Luciana S; Brenelli, Lívia B; Gonçalves, Thiago A; Uchima, Cristiane A; Domingues, Romênia R; Alvarez, Thabata M; Tramontina, Robson; Vidal, Ramon O; Costa, Fernando F; Costa-Leonardo, Ana M; Paes Leme, Adriana F; Pereira, Gonçalo A G; Squina, Fabio M

    2016-01-01

    Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi . Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi . Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

  1. The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Arai, Sachiko; Suzuki, Kentaro; Kitaoka, Motomitsu; Fushinobu, Shinya

    2016-02-15

    Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity. © 2016 Authors; published by Portland Press Limited.

  2. Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems. Comprehensive report, 1 February 1981-15 September 1983

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1983-01-01

    We have explored the molecular mechanism of the repair of DNA at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian (including human) cells. We have demonstrated that uv endonuclease of phage T4 not only possesses pyrimidine dimer (PD)-DNA glycosylase activity but also apyrimidinic (AP) endonuclease activity. The demonstration of both activities provided an explanation for the specific endonucleosytic cleavage of DNA at sites of pyrimidine dimers catalyzed by this small protein. A new apurinic/apyrimidinic (AP) endonuclease, specific for sites of of base loss in single stranded DNA has been isolated from E. celi and presumably recognizes these lesions in single stranded regions of duplex DNA. We have partially purified this enzyme and have carried out a preliminary characterization of the activity. We treated xeroderma pigmentosum and normal cells with sodium butyrate in the hope of restoring normal levels of excision repair to the former. Although this result was not obtained, we established that all cells treated with sodium butyrate show enhanced levels of repair synthesis, thus providing a means for increasing the sensitivity of this commonly used technique for measuring DNA repair in mammalian cells in culture

  3. PURIFICATION AND SOME PROPERTIES OF CELLULASE FROM ODONTOTERMES FORMOSANUS (ISOPTERA: TERMITIDAE)

    Institute of Scientific and Technical Information of China (English)

    Tian-ciYang; Jian-chuMo; Jia-anCheng

    2004-01-01

    The purification of the cellulase from Odontotermes forrnosanus workers was achieved by using anion-exchange column of UNOsphere Q, BioLogic DuoFlow chromatography system. The purified cellulase was identified as an endoglucanase and some of its properties were investigated. The EGase activity was 807.5-fold as high as the initial enzyme activity using CMC as substrate and 14.4-fold using salicin as substrate. The enzyme preparations were homogeneous as judged by SDS-PAGE electrophoresis, molecular weight of which was 80 kDa and confirmed by 2-DE zymogram analysis. The enzyme was isoelectric at pH 6.4, which was active on CMC substrate.

  4. Composition of cellulase complex of Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Golovchenko, N P; Chuvil' skaya, N A; Akimenko, V K

    1985-01-01

    It is thought that the anaerobic thermophilic cellulolytic bacterium C. thermocellum has the potential for direct industrial bioconversion of cellulose into ethanol. Therefore, much attention has been given to the study of the cellulolytic properties of the culture and to the characteristics of the cellulose complex, which is still not completely understood. Hence, the activity and location of various cellulolytic enzymes of C. thermocellum were determined. C. thermocellum has 6 known cellulolytic enzymes. Endoglucanase, cellobiohydrolase and exoglucosidase are extracellular enzymes (99-100 percent of the activity is located outside the cells) while cellulobiases, cellobiose phosphorylase and cellodextrine phosphorylase are inside the cells (80-90% of the activity). 25 references.

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

    Science.gov (United States)

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

    2014-04-01

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

  6. Molecular and biochemical analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii.

    Science.gov (United States)

    Ye, Libin; Su, Xiaoyun; Schmitz, George E; Moon, Young Hwan; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-10-01

    A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol. 78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases from C. bescii led to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization by C. bescii.

  7. Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

    Directory of Open Access Journals (Sweden)

    Xi Bai

    2016-11-01

    Full Text Available Background Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, Citrobacter farmeri A1 which was isolated from a wood-inhabiting termite Reticulitermes labralis could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml. The objective of the present study was to clone and express the C. farmeri EglC gene in Escherichia coli to improve production level and determine the enzymatic properties of the recombinant enzyme. Methods The EglC gene was cloned from C. farmeri A1 by thermal asymmetric interlaced PCR. EglC was transformed into vector pET22b and functionally expressed in E. coli. The recombination protein EglC22b was purified for properties detection. Results SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5–8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30–40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co2+ and Fe3+, but inhibited by Cd2+, Zn2+, Li+, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA. Conclusion These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.

  8. Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

    Science.gov (United States)

    Adney, William S.; Baker, John O.; Decker, Stephen R.; Chou, Yat-Chen; Himmel, Michael E.; Ding, Shi-You

    2012-10-09

    Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

  9. Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

    Science.gov (United States)

    Adney, William S.; Baker, John O.; Decker, Stephen R.; Chou, Yat-Chen; Himmel, Michael E.; Ding, Shi-You

    2008-11-11

    Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

  10. PENAPISAN KHAMIR SELULOLITIK CRYPTOCOCCUS SP. YANG DIISOLASI DARI TANAH KEBUN BIOLOGI WAMENA, JAYA WIJAYA, PROPINSI PAPUA

    Directory of Open Access Journals (Sweden)

    Atit Kanti

    2007-06-01

    Full Text Available Cryptococcus sp. was isolated from Kebun Biologi Wamena, Papua. The isolate was able to grow in media with carboxymethyl cellulose as a sole carbon source implying that isolate produced 1-3 ? endo-glucanase. To study the effect of glucose and osmotic pressure, 0.1 % glucose and 0.1 % NaCl were amended into the medium containing CMC. Glucose significantly affected cellulolytic activity and biomass synthesis. At the beginning of cell cultivation glucose augmentation appear to slightly inhibit enzyme activity. Sodium chloride also significantly affected cellulolytic activity. Profile of pH varied dependent on cultivation media. Maximum growth of biomass was achieved after glucose addition, indicating that glucose stimulated cell growth.

  11. Production of hydrolytic enzymes by Trichoderma isolates with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa

    Directory of Open Access Journals (Sweden)

    Marco Janice Lisboa De

    2003-01-01

    Full Text Available Two isolates of Trichoderma, which reduce the incidence of witches'broom disease caused in cocoa by Crinipellis perniciosa, were evaluated for their potential to produce hydrolases in liquid medium. Very low or no hydrolytic activity was produced in the absence of any substrate. The activities of chitinase, N-acetylglucosaminidase, beta-1,3-glucanase, total cellulase, endoglucanase, aryl- beta-glucosidase, beta-glucosidase, protease and amylase increased dramatically within 72-120 h of growth in the presence of specific substrates. Except for N-acetylglucosaminidase and beta-glucosidase Trichoderma harzianum isolate 1051 produced the largest amounts of hydrolases. The possible involvement of these enzymes in the antagonistic interaction between Trichoderma and C. perniciosa is discussed.

  12. Cloning and Characterization of an Endoglucanase Gene from sp. Korean Native Goat 40

    Directory of Open Access Journals (Sweden)

    Sung Chan Kim

    2016-01-01

    Full Text Available A gene from Actinomyces sp. Korean native goat (KNG 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine–Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C. The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively.

  13. Las enfermedades vasculares del clavel en Colombia y en el mundo

    Directory of Open Access Journals (Sweden)

    Arbeláez Torres Germán

    1993-06-01

    Full Text Available El clavel es atacado por un gran número de enfermedades
    causadas por hongos, bacterias, nemátodos, virus y micoplasmas, las cuales se presentan afectando diferentes partes de la planta. Dentro de esas enfermedades, las más importantes son las enfermedades vasculares por las altas
    perdidas que ocasionan, por la facilidad de propagación
    a través de los esquejes, por su alta persistencia en el suelo y por el alto costo de algunas de las medidas de control utilizadas. Las enfermedades vasculares del clavel son ocasionadas por hongos y bacterias y, hasta el momento,
    se han reconocido las siguientes: 1. Marchitamiento bacterial, causado por Pseudomonas caryophylli (Burkh Starr et Burkh. 2. Enanismo bacterial, causado por Erwinia chrysanthemi Burkh. pv. dianthicola. 3. Marchitamiento lento, causado por una raza del hongo Rhizoctonia solani Kuhn. 4. Marchitamiento vascular, causado por el hongo Phialophora cinerescens (wollenw van Beyma. 5. Marchitamiento vascular, causado por el hongo Fusarium oxysporum Schlecht f.sp. dianthi (Prill et Dei. Snyder et Hansen.

  14. Inconsistency of residents' communication performance in challenging consultations.

    Science.gov (United States)

    Wouda, Jan C; van de Wiel, Harry B M

    2013-12-01

    Communication performance inconsistency between consultations is usually regarded as a measurement error that jeopardizes the reliability of assessments. However, inconsistency is an important phenomenon, since it indicates that physicians' communication may be below standard in some consultations. Fifty residents performed two challenging consultations. Residents' communication competency was assessed with the CELI instrument. Residents' background in communication skills training (CST) was also established. We used multilevel analysis to explore communication performance inconsistency between the two consultations. We also established the relationships between inconsistency and average performance quality, the type of consultation, and CST background. Inconsistency accounted for 45.5% of variance in residents' communication performance. Inconsistency was dependent on the type of consultation. The effect of CST background training on performance quality was case specific. Inconsistency and average performance quality were related for those consultation combinations dissimilar in goals, structure, and required skills. CST background had no effect on inconsistency. Physician communication performance should be of high quality, but also consistent regardless of the type and complexity of the consultation. In order to improve performance quality and reduce performance inconsistency, communication education should offer ample opportunities to practice a wide variety of challenging consultations. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Vera Hjelt and the calling of theosophical universal work, 1894–1904

    Directory of Open Access Journals (Sweden)

    Marjo Kaartinen

    2018-04-01

    Full Text Available This article discusses the theosophy of Vera Hjelt (1857–1947, who was inspired by Annie Besant. Hjelt led an active life as a schoolteacher, factory owner, writer, occupational safety inspector and member of parliament. Hjelt experienced a theosophic-al awakening at the latest during the summer of 1894, after which her theosophical endeavours in Besant’s spirit and in imitation of her are revealed in Hjelt’s letters to her friend Cely Mechelin. These letters have not previously been used in scholarly study. The article argues that it is not possible to understand the underlying ethos behind Hjelt’s activities without considering her esotericism. In the worldwide unity of all creations, all bad deeds done to one were done to all. Thus it was essential to improve the working conditions of women in factories, for instance. When Hjelt experienced difficulties in her position as an inspector during the Voikka strike, and became an object of hatred amongst the workers against all her wishes, she was comforted by her theosophical thinking. This article for its part shows the many ways in which Western esotericism exerted an influence on late nineteenth- and early twentieth-century Finnish culture, politics and policy making.

  16. Chitosan (biochikol 020 PC) in the control of some ornamental foliage diseases.

    Science.gov (United States)

    Wojdyła, A T

    2004-01-01

    Chitosan, (Biochikol 020 PC) a potential elicitor of plant defence and also an active inhibitor of fungal growth was used in experiments. The compound was used at concentrations 0.01 to 0.2% as a plant spray for rose protection against Sphaerotheca pannosa var. rosae, Peronospora sparsa and Diplocarpon rosae, Oidium chrysanthemi and Puccinia horiana on chrysanthemum, against Melampsora epitea on willow. Myrothecium roridum on dieffenbachia and against Lophodermium spp on Scots pine. Effectiveness of this product, applied curatively 2-times at 7-day-intervals after appearance of rose powdery mildew symptoms on most of plant parts, was about 32%. When compound was applied just after the first disease symptoms appearance, after 2-week-protection its effectiveness was about 43-60%. After 4 weeks, effectiveness of chitosan (Biochikol 020 PC) increased and ranged from 67 to 77%. In the control of P. sparsa on rose shrubs, growing in plastic tunnel, chitosan at conc. 0.025% was applied 4 times at weekly intervals. Effectiveness of chitosan against this pathogen was over 72%, similar as standard fungicide. Increase of chitosan (Biochikol 020 PC) concentration resulted in decrease of its effectiveness. In case of D. rosae control, chitosan used 9 times at weekly intervals was applied after first disease symptoms. After 3-week-protection effectiveness of the compound varied from 18 to 60% according to used concentration. After 9 weeks of protection effectiveness of tested product ranged from 16 to 23% and was connected with used concentration. Increase of chitosan concentration resulted in better protection of rose shrubs. Effectiveness of chitosan (Biochikol 020 PC) used 4-times at conc. 0.01 to 0.05% as chrysanthemum spray in the control of Oidium chrysanthemi ranged from 69 to 79%, whereas against Puccinia horiana from 54-97%. Two-time-spraying of willow with rust symptoms (Melampsora epitea) with chitosan at conc. 01-0.04% caused decrease of disease severity from 26 to

  17. Molecular cloning of cellulase genes from indigenous bacterial isolates

    International Nuclear Information System (INIS)

    Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

    2006-01-01

    Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

  18. Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail.

    Science.gov (United States)

    Bussamra, Bianca Consorti; Freitas, Sindelia; Costa, Aline Carvalho da

    2015-01-01

    The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5L converts 49.11%±0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Assessment of recent outbreaks of Dickeya sp (syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel

    NARCIS (Netherlands)

    Tsror, L.; Erlich, O.; Lebiush, S.; Hazanovsky, M.; Zig, U.; Slawiak, M.; Grabe, G.; Wolf, van der J.M.; Haar, van de J.J.

    2009-01-01

    Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004-2006, with disease incidence of 2-30% ( 10% in

  20. A coarse-grained model for synergistic action of multiple enzymes on cellulose

    Directory of Open Access Journals (Sweden)

    Asztalos Andrea

    2012-08-01

    Full Text Available Abstract Background Degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing β-1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, β-glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose, several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. Results We present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. Conclusions Our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures

  1. Growth and enzymatic activity of Leucoagaricus gongylophorus, a mutualistic fungus isolated from the leaf-cutting ant Atta mexicana, on cellulose and lignocellulosic biomass.

    Science.gov (United States)

    Vigueras, G; Paredes-Hernández, D; Revah, S; Valenzuela, J; Olivares-Hernández, R; Le Borgne, S

    2017-08-01

    A mutualistic fungus of the leaf-cutting ant Atta mexicana was isolated and identified as Leucoagaricus gongylophorus. This isolate had a close phylogenetic relationship with L. gongylophorus fungi cultivated by other leaf-cutting ants as determined by ITS sequencing. A subcolony started with ~500 A. mexicana workers could process 2 g day -1 of plant material and generate a 135 cm 3 fungus garden in 160 days. The presence of gongylidia structures of ~35 μm was observed on the tip of the hyphae. The fungus could grow without ants on semi-solid cultures with α-cellulose and microcrystalline cellulose and in solid-state cultures with grass and sugarcane bagasse, as sole sources of carbon. The maximum CO 2 production rate on grass (V max  = 17·5 mg CO 2  L g -1  day -1 ) was three times higher than on sugarcane bagasse (V max  = 6·6 mg CO 2  L g -1 day -1 ). Recoveries of 32·9 mg glucose  g biomass -1 and 12·3 mg glucose  g biomass -1 were obtained from the fungal biomass and the fungus garden, respectively. Endoglucanase activity was detected on carboxymethylcellulose agar plates. This is the first study reporting the growth of L. gongylophorus from A. mexicana on cellulose and plant material. According to the best of our knowledge, this is the first report about the growth of Leucoagaricus gongylophorus, isolated from the colony of the ant Atta mexicana, on semisolid medium with cellulose and solid-state cultures with lignocellulosic materials. The maximum CO 2 production rate on grass was three times higher than on sugarcane bagasse. Endoglucanase activity was detected and it was possible to recover glucose from the fungal gongylidia. The cellulolytic activity could be used to process lignocellulosic residues and obtain sugar or valuable products, but more work is needed in this direction. © 2017 The Society for Applied Microbiology.

  2. Utilization of waste cellulose. III. Comparative study of the activity of the cellulases of trichoderma viride and Aspergillus niger towards different cellulosic substrates

    Energy Technology Data Exchange (ETDEWEB)

    David, C.; Thiry, P.

    1981-01-01

    The kinetics of the saccharification of filter paper-derived cellulose by cellulases of Aspergillus niger and Tricoderma viride were studied. The formation of glucose and of total reducing sugar was measured as a function of time for the hydrolysis of cellulose by the same quantity of filter paper units from T. viride and (or) A. niger. Long term efficiency was lower for A. niger but an important synergistic effect was observed for the mixture of the enzymes. This synergistic action was attributed to a better balance of endo- and exoglucananses and to the addition to T. viride of thermally stable endoglucanases from A. niger. The beta-glucosidases formed in large quantity by A. niger were thermally unstable and susceptible to product inhibition and did not play any role in the observed synergistic action.

  3. The expression of extracellular fungal cell wall hydrolytic enzymes in different Trichoderma harzianum isolates correlates with their ability to control Pyrenochaeta lycopersici

    Directory of Open Access Journals (Sweden)

    LUZ MARÍA PÉREZ

    2002-01-01

    Full Text Available Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16 were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of ß -1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici

  4. Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

    Science.gov (United States)

    Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

    2018-03-01

    Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

  5. Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2018-01-01

    of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane......The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein...... recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast....

  6. Studying the ability of Fusarium oxysporum and recombinant Saccharomyces cerevisiae to efficiently cooperate in decomposition and ethanolic fermentation of wheat straw

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Topakas, Evangelos; Moukouli, Maria

    2011-01-01

    Fusarium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae F12 were used to ferment carbohydrates of wet exploded pre-treated wheat straw (PWS) directly to ethanol. Both microorganisms were first grown aerobically to produce cell mass and thereafter fermented PWS to ethanol under...... anaerobic conditions. During fermentation, soluble and insoluble carbohydrates were hydrolysed by the lignocellulolytic system of F. oxysporum. Mixed substrate fermentation using PWS and corn cobs (CC) in the ratio 1:2 was used to obtain an enzyme mixture with high cellulolytic and hemicellulolytic...... activities. Under these conditions, activities as high as 34300, 9100, 326, 24, 169, 27 and 254 U dm−3 of xylanase, endoglucanase, β-glucosidase, arabinofuranosidase, avicelase, feruloyl esterase and acetyl esterase, respectively, were obtained. The replacement of the enzyme production phase of F. oxysporum...

  7. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  8. Optimization of fermentation conditions for cellulases production by Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from Indian hot spring

    Directory of Open Access Journals (Sweden)

    Somen Acharya

    2012-08-01

    Full Text Available The aim of this work was to study the effect of some nutritional and environmental factors on the production of cellulases, in particular endoglucanase (CMCase and exoglucanases (FPase from Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from an Indian hot spring. The characterization study indicated that the optimum pH and temperature value was 6.5 to 7.0 and 50-55°C, respectively. Maximum cellulases production by both the isolates was detected after 60 h incubation period using wheat and rice straw. The combination of inorganic and organic nitrogen source was suitable for cellulases production. Overall, FPase production was much higher than CMCase production by both of the strains. Between the two thermophiles, the cellulolytic activity was more in B.licheniformis MVS1 than Bacillus sp. MVS3 in varying environmental and nutritional conditions.

  9. Enhanced production and application of acidothermophilic Streptomyces cellulase.

    Science.gov (United States)

    Budihal, Saikumar R; Agsar, Dayanand; Patil, Sarvamangala R

    2016-01-01

    An efficient cellulolytic and acidothermophilic actinobacterium was isolated from soil, adhered to decomposing tree bark and was identified as Streptomyces DSK59. Screening of synthetic media and the media components identified that, a medium based on starch casein minerals containing carboxy methyl cellulose (CMC) and beef extract (BE) could support enhanced cellulase production by the organism. CMC, BE, NaCl, temperature and pH were accounted as significant for cellulase production and these were optimized using a response surface central composite design (CCD). Optimization of cellulase production resulted in an enhancement of endoglucanase activity to 27IUml(-1). Acidothermophillic Streptomyces cellulase was found to be efficient for hydrolysis of pretreated sorghum stover and liberated 0.413gg(-1) of total reducing sugars which was higher than previously reported sugar yields obtained using fungal enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Morphogenesis and Production of Enzymes by Penicillium echinulatum in Response to Different Carbon Sources

    Directory of Open Access Journals (Sweden)

    Willian Daniel Hahn Schneider

    2014-01-01

    Full Text Available The effect of different carbon sources on morphology and cellulase and xylanase production of Penicillium echinulatum was evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, and β-glucosidases. However, sucrose and glucose showed β-glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol.

  11. Two Genomic Regions Involved in Catechol Siderophore Production by Erwinia carotovora

    Science.gov (United States)

    Bull, Carolee T.; Ishimaru, Carol A.; Loper, Joyce E.

    1994-01-01

    Two regions involved in catechol biosynthesis (cbs) of Erwinia carotovora W3C105 were cloned by functional complementation of Escherichia coli mutants that were deficient in the biosynthesis of the catechol siderophore enterobactin (ent). A 4.3-kb region of genomic DNA of E. carotovora complemented the entB402 mutation of E. coli. A second genomic region of 12.8 kb complemented entD, entC147, entE405, and entA403 mutations of E. coli. Although functions encoded by catechol biosynthesis genes (cbsA, cbsB, cbsC, cbsD, and cbsE) of E. carotovora were interchangeable with those encoded by corresponding enterobactin biosynthesis genes (entA, entB, entC, entD, and entE), only cbsE hybridized to its functional counterpart (entE) in E. coli. The cbsEA region of E. carotovora W3C105 hybridized to genomic DNA of 21 diverse strains of E. carotovora but did not hybridize to that of a chrysobactin-producing strain of Erwinia chrysanthemi. Strains of E. carotovora fell into nine groups on the basis of sizes of restriction fragments that hybridized to the cbsEA region, indicating that catechol biosynthesis genes were highly polymorphic among strains of E. carotovora. PMID:16349193

  12. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    Science.gov (United States)

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

    2015-01-01

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

  13. Portable hyperspectral fluorescence imaging system for detection of biofilms on stainless steel surfaces

    Science.gov (United States)

    Jun, Won; Lee, Kangjin; Millner, Patricia; Sharma, Manan; Chao, Kuanglin; Kim, Moon S.

    2008-04-01

    A rapid nondestructive technology is needed to detect bacterial contamination on the surfaces of food processing equipment to reduce public health risks. A portable hyperspectral fluorescence imaging system was used to evaluate potential detection of microbial biofilm on stainless steel typically used in the manufacture of food processing equipment. Stainless steel coupons were immersed in bacterium cultures, such as E. coli, Pseudomonas pertucinogena, Erwinia chrysanthemi, and Listeria innocula. Following a 1-week exposure, biofilm formations were assessed using fluorescence imaging. In addition, the effects on biofilm formation from both tryptic soy broth (TSB) and M9 medium with casamino acids (M9C) were examined. TSB grown cells enhance biofilm production compared with M9C-grown cells. Hyperspectral fluorescence images of the biofilm samples, in response to ultraviolet-A (320 to 400 nm) excitation, were acquired from approximately 416 to 700 nm. Visual evaluation of individual images at emission peak wavelengths in the blue revealed the most contrast between biofilms and stainless steel coupons. Two-band ratios compared with the single-band images increased the contrast between the biofilm forming area and stainless steel coupon surfaces. The 444/588 nm ratio images exhibited the greatest contrast between the biofilm formations and stainless coupon surfaces.

  14. In vitro antimicrobial efficacy of Rhynchostegium vagans A. Jaeger (moss against commonly occurring pathogenic microbes of Indian sub-tropics

    Directory of Open Access Journals (Sweden)

    Kavita Negi*

    2016-01-01

    Full Text Available Objective: To study the antimicrobial effect of organic extracts with a standard dose of Rhynchostegium vagans (R. vagans on pathogenic bacteria and fungi. Methods: R. vagans was extracted in solvents (ethanol and acetone and the extracts were evaluated for antimicrobial activity by using disc diffusion assay. Minimum inhibitory concentration and minimum bactericidal/fungicidal concentration was observed by employing micro broth dilution method. Mode of inhibition of ethanolic extract against Aspergillus flavus var. columnaris (A. flavus var. columnaris was assessed by scanning electron microscopy. Results: It was found that the ethanolic extract of R. vagans was the most potent with lowest minimum inhibitory concentration (3.91 to 61.25 µg/mL and minimum bactericidal/fungicidal concentration (3.91 to 500 µg/mL, respectively. Significant morphological and ultrastructural alterations were seen in A. flavus var. columnaris. Among microorganisms, Gram negative bacteria (Escherichia coli, Erwinia chrysanthemi and Salmonella enterica and fungi (A. flavus var. columnaris and Aspergillus parasiticus var. globosus were found more sensitive. Ethanolic extract was found superior over the antibiotics (chloramphenicol and fluconazole. Conclusions: R. vagans exhibited effective antimicrobial activity against all the microorganisms. The moss can be used as a broad spectrum herbal antimicrobial agent in pharmaceutics.

  15. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    Science.gov (United States)

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  16. Role of ornithine decarboxylase in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Wensheng Deng; Xian Jiang; Yu Mei; Jingzhong Sun; Rong Ma; Xianxi Liu; Hui Sun; Hui Tian; Xueying Sun

    2008-01-01

    Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis that decarboxylates ornithine to putrescine, has become a promising target for cancer research. The aim of this study is to investigate the role of ODC in breast cancer. We detected expression of ODC in breast cancer tissues and four breast cancer cell lines, and transfected breast cancer cells with an adenoviral vector carrying antisense ODC (rAd-ODC/Ex3as) and examined their growth and migration.ODC was overexpressed in breast cancer tissues and cell lines compared with non-tumor tissues and normal breast epithelial celis,and there was a positive correlation between the level of ODC mRNA and the staging of tumors.The expression of ODC correlated with cyclin D1,a cell cycle protein,in synchronized breast cancer MDA-MB-231 cells.Gene transfection of rAd-ODC/Ex3as markedly down-regulated expression Of ODC and cyclin D1,resulting in suppression of proliferation and cell cycle arrest at G0-G1 phase,and the inhibifion of colony formation,an anchorage-independent growth pattern,and the migratory ability of MDA-MB-231 cells.rAd-ODC/Ex3as also markedly reduced the concentration of putrescine,but not spermidine or spermine,in MDA-MB-231 cells.The results suggested that the ODC gene might act as aprognostic factor for breast cancer and it could be a promising therapeutic target.

  17. Periodismo Ambiental: un análisis a los medios digitales venezolanos Noticias 24 y Reporte 360

    Directory of Open Access Journals (Sweden)

    Herly Alejandra Quiñónez Gómez

    2012-03-01

    Full Text Available Para analizar la información ambiental en medios digitales, se hizo un estudio descriptivo y de análisis de contenido, donde se examinaron Reporte 360 y Noticias 24, con las categorías de Krisppendorff (1990 para el análisis de contenido; Aceves (2003 información ambiental y Cely (2004 para el medio digital. Se elaboró una clasificación de géneros periodísticos (tradicionales y ciberperiodísticos, se analizó el hipertexto, actualización, procedencia de la información, fuentes, temas, comentarios y explicación de términos. Como resultados destacan: la procedencia de la información de ambos medios es mayoritariamente de Agencias de Noticias internacionales y nacional, domina la fuente gobierno, escaso uso del lenguaje multimedia, Reporte 360 muestra un lento ritmo de actualización frente a Noticias 24 y ésta hace mayor uso de video. El género periodístico más empleado es la noticia, con la misma estructura del medio impreso. En ambos medios la agenda temática es: Contaminación, Desastres Naturales, Desechos y Lluvias. Los comentarios en Noticias 24 fueron muy críticos para los temas de contaminación y desechos y, los usuarios de Reporte 360, las reenviaron a las redes sociales. Se concluye que los medios digitales estudiados basan su contenido en las agencias de noticias.

  18. Structural characterization of novel L-galactose-containing oligosaccharide subunits of jojoba seed xyloglucans.

    Science.gov (United States)

    Hantus, S; Pauly, M; Darvill, A G; Albersheim, P; York, W S

    1997-10-28

    Jojoba seed xyloglucan was shown to be a convenient source of biologically active xyloglucan oligosaccharides that contain both L- and D-galactosyl residues [E. Zablackis et al., Science, 272 (1996) 1808-1810]. Oligosaccharides were isolated by liquid chromatography of the mixture of oligosaccharides generated by treating jojoba seed xyloglucan with a beta-(1-->4)-endoglucanase. The purified oligosaccharides were reduced with NaBH4, converting them to oligoglycosyl alditol derivatives that were structurally characterized by a combination of mass spectrometry and 2-dimensional NMR spectroscopy. This analysis established that jojoba xyloglucan oligosaccharides contain the novel side-chain [alpha-L-Gal p-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-], which is structurally homologous to the fucose-containing side-chain [alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-] found in other biologically active xyloglucan oligosaccharides.

  19. Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

    Science.gov (United States)

    Roper, M Caroline; Greve, L Carl; Warren, Jeremy G; Labavitch, John M; Kirkpatrick, Bruce C

    2007-04-01

    Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

  20. Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials

    Directory of Open Access Journals (Sweden)

    Peter S. Lübeck

    2013-09-01

    Full Text Available Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases.

  1. Plant cell-wall hydrolyzing enzymes from indigenously isolated fungi grown on conventional and novel natural substrates

    International Nuclear Information System (INIS)

    Kumari, D.; Sohail, M.; Jahangeer, S.; Abideen, Z.; Khan, M.A.

    2017-01-01

    Fungi elaborate a variety of plant-hydrolyzing enzymes including cellulases, xylanases, pectinases and amylases. Although these enzymes have potential biotechnological applications, their production at industrial level is limited because of higher costs of the purified substrates. Hence, the present study was aimed to explore the novel, natural and cheaper substrates for enzyme production. Indigenously isolated fungal strains of Aspergillus sp. were grown on banana-peels, grapefruit-peels, pomegranate-peels, sugarcane bagasse, Eucalyptus camaldulensis-leaves and shoots of two halophytic plants including Halopyrum mucronatum and Desmostachya bipinnata under solid-state fermentation (SSF) and submerged fermentation (Smf) conditions. The crude enzyme preparation was screened for cellulase (endoglucanase, beta-glucosidase and filter-paperase), hemicellulase (xylanase), pectinase and amylase production. The results revealed that among all investigated enzymes, the xylanase titers were highest using D. bipinnata- shoots and H. mucronatum- shoots as substrates under solid state fermentation conditions, suggesting their exploitation at commercial scale. (author)

  2. Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose.

    Science.gov (United States)

    Nozaki, Kouichi; Seki, Takahiro; Matsui, Keiko; Mizuno, Masahiro; Kanda, Takahisa; Amano, Yoshihiko

    2007-10-01

    Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.

  3. A novel marine bacterium Isoptericola sp. JS-C42 with the ability to saccharifying the plant biomasses for the aid in cellulosic ethanol production

    Directory of Open Access Journals (Sweden)

    Velayudhan Satheeja Santhi

    2014-06-01

    Full Text Available The ever growing demands for food products such as starch and sugar produces; there is a need to find the sources for saccharification for cellulosic bioethanol production. This study provides the first evidence of the lignocellulolytic and saccharifying ability of a marine bacterium namely Isoptericola sp. JS-C42, a Gram positive actinobacterium with the cocci cells embedded on mycelia isolated from the Arabian Sea, India. It exhibited highest filter paper unit effect, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, xylanase and ligninase effect. The hydrolytic potential of the enzymes displayed the efficient saccharification capability of steam pretreated biomass. It was also found to degrade the paddy, sorghum, Acacia mangium and Ficus religiosa into simple reducing sugars by its efficient lignocellulose enzyme complex with limited consumption of sugars. Production of ethanol was also achieved with the Saccharomyces cerevisiae. Overall, it offers a great potential for the cellulosic ethanol production in an economically reliable and eco-friendly point-of-care.

  4. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries.

    Science.gov (United States)

    Parisutham, Vinuselvi; Chandran, Sathesh-Prabu; Mukhopadhyay, Aindrila; Lee, Sung Kuk; Keasling, Jay D

    2017-09-01

    Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by β-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less challenging is to engineer microbes with the abilities to hydrolyze and assimilate the cellulosic-hydrolysate cellodextrin. Microorganisms engineered to metabolize cellobiose rather than the monomeric glucose can provide several advantages for lignocellulose-based biorefineries. This review describes the recent advances and challenges in engineering efficient intracellular cellobiose metabolism in industrial hosts. This review also describes the limitations of and future prospectives in engineering intracellular cellobiose metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor.

    Science.gov (United States)

    Ma, Zhenchuan; Zhu, Lin; Song, Tianqiao; Wang, Yang; Zhang, Qi; Xia, Yeqiang; Qiu, Min; Lin, Yachun; Li, Haiyang; Kong, Liang; Fang, Yufeng; Ye, Wenwu; Wang, Yan; Dong, Suomeng; Zheng, Xiaobo; Tyler, Brett M; Wang, Yuanchao

    2017-02-17

    The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae -soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae , however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems. Copyright © 2017, American Association for the Advancement of Science.

  6. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  7. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

    2017-03-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate

    Energy Technology Data Exchange (ETDEWEB)

    Singer, S.W.; Reddy, A. P.; Gladden, J. M.; Guo, H.; Hazen, T.C.; Simmons, B. A.; VanderGheynst, J. S.

    2010-12-15

    This work aims to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populationsdominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. The results of the study suggest tolerance to ionic liquids is a general property of Aspergilli. Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.

  9. Production of cellulases by fungal cultures isolated from forest litter soil

    Directory of Open Access Journals (Sweden)

    A. Sri Lakshmi

    2012-06-01

    Full Text Available The aims of this study were the isolation and screening of fungal cultures from forest litter soil for cellulases production. In the present study, four fungal cultures were isolated and identified. Among these fungal cultures, three belonged to the genus Aspergillus and one belonged to the genus Pencillium. These fungal cultures were tested to find their ability to produce cellulases, that catalyze the degradation of cellulose, which is a linear polymer made of glucose subunits linked by beta-1, 4 glycosidic bonds. The fungal isolate 3 (Aspergillus sp. was noticed to show maximum zone of hydrolysis of carboxy-methyl cellulose and produce higher titers of cellulases including exoglucanase, endoglucanase and beta -D-glucosidase. The activities of the cellulases were determined by Filter paper assay (FPA, Carboxy-methly cellulase assay (CMCase and beta -D-glucosidase assay respectively. The total soluble sugar and extracellular protein contents of the fungal filtrates were also determined.

  10. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

    Science.gov (United States)

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

  11. A novel marine bacterium Isoptericola sp. JS-C42 with the ability to saccharifying the plant biomasses for the aid in cellulosic ethanol production.

    Science.gov (United States)

    Santhi, Velayudhan Satheeja; Gupta, Ashutosh; Saranya, Somasundaram; Jebakumar, Solomon Robinson David

    2014-06-01

    The ever growing demands for food products such as starch and sugar produces; there is a need to find the sources for saccharification for cellulosic bioethanol production. This study provides the first evidence of the lignocellulolytic and saccharifying ability of a marine bacterium namely Isoptericola sp. JS-C42, a Gram positive actinobacterium with the cocci cells embedded on mycelia isolated from the Arabian Sea, India. It exhibited highest filter paper unit effect, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, xylanase and ligninase effect. The hydrolytic potential of the enzymes displayed the efficient saccharification capability of steam pretreated biomass. It was also found to degrade the paddy, sorghum, Acacia mangium and Ficus religiosa into simple reducing sugars by its efficient lignocellulose enzyme complex with limited consumption of sugars. Production of ethanol was also achieved with the Saccharomyces cerevisiae . Overall, it offers a great potential for the cellulosic ethanol production in an economically reliable and eco-friendly point-of-care.

  12. BIOPROCESS DEVELOPMENTS FOR CELLULASE PRODUCTION BY Aspergillus oryzae CULTIVATED UNDER SOLID-STATE FERMENTATION

    Directory of Open Access Journals (Sweden)

    R. D. P. B. Pirota

    Full Text Available Abstract Bioprocess development studies concerning the production of cellulases are of crucial importance due to the significant impact of these enzymes on the economics of biomass conversion into fuels and chemicals. This work evaluates the effects of solid-state fermentation (SSF operational conditions on cellulase production by a novel strain of Aspergillus oryzae using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The use of SSF cultivation under controlled conditions substantially improved cellulase production. Highest production of FPase (0.40 IU g-1, endoglucanase (123.64 IU g-1, and β-glucosidase (18.32 IU g-1 was achieved at 28 °C, using an initial substrate moisture content of 70%, with an inlet air humidity of 80% and an airflow rate of 20 mL min-1. Further studies of kinetic profiles and respirometric analyses were performed. The results showed that these data could be very useful for bioprocess development of cellulase production and scale-up.

  13. Characterization of the enzymes present in the cellulase system of Thielavia terrestris 255B

    Energy Technology Data Exchange (ETDEWEB)

    Gilbert, Michel; Breuil, Colette; Saddler, J N [Forintek Canada Corp., Ottawa, ON (CA). Dept. of Biotechnology and Chemistry

    1992-01-01

    The authors initiated a study of the cellulases from the thermophilic fungus Thielavia terrestris 255B to see how they compared with enzymes derived from mesophilic fungi such as Trichoderma. To try to obtain maximum production of a complete cellulase system, the fungus was first grown on a variety of soluble and insoluble substrates. As well as assaying the culture filtrates for cellulase activity and protein concentration, the enzyme profiles were compared using non-denaturing electrophoretic techniques (IEF and native-PAGE). The separation by native-PAGE and IEF was followed by activity staining methods to detect endoglucanase and xylanase activities. Native-PAGE could not be used to determine accurately the M{sub r} of the cellulases because of possible differences in mass/charge ratios. Bands with apparent M{sub r} values above 200000 were reproducibly detected. This suggested that the various cellulase components may be organized into high molecular weight complexes. (author).

  14. Application of thermophilic enzymes and water jet system to cassava pulp.

    Science.gov (United States)

    Chaikaew, Siriporn; Maeno, Yuka; Visessanguan, Wonnop; Ogura, Kota; Sugino, Gaku; Lee, Seung-Hwan; Ishikawa, Kazuhiko

    2012-12-01

    Co-production of fermentable sugars and nanofibrillated cellulose from cassava pulp was achieved by the combination of thermophilic enzymes (endoglucanase, β-glucosidase, and α-amylase) and a new atomization system (Star Burst System; SBS), which employs opposing water jets. The SBS represents a key technology for providing cellulose nanofibers and improving the enzymatic saccharification of cassava pulp. Depending on the enzymes used, the production of glucose from cassava pulp treated with the SBS was 1.2- to 2.5-fold higher than that from pulp not treated with the SBS. Nanofibrillated cellulose with the gel-like property in suspension was produced (yield was over 90%) by α-amylase treatment, which completely released trapped starch granules from the fibrous cell wall structure of cassava pulp pretreated with the SBS. The SBS provides an environmentally low-impact pretreatment system for processing biomass material into value-added products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

    Science.gov (United States)

    Dollhofer, Veronika; Callaghan, Tony M; Griffith, Gareth W; Lebuhn, Michael; Bauer, Johann

    2017-07-01

    Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. A Paratransgenic Strategy for the Control of Chagas Disease

    Directory of Open Access Journals (Sweden)

    Ivy Hurwitz

    2012-01-01

    Full Text Available Chagas disease results from infection with the parasite Trypanosoma cruzi. This disease remains a significant cause of morbidity and mortality in central and south America. Chagas disease now exists and is detected worldwide because of human migration. Control of Chagas disease has relied mainly on vector eradication however, the development of insect resistance to pesticides, coupled with cost and adverse health effects of insecticide treatments, has prompted our group to investigate novel methods of transmission control. Our laboratory has been instrumental in the development of the paratransgenic strategy to control vectorial transmission of T. cruzi. In this paper, we discuss various components of the paratransgenic approach. Specifically, we describe classes of molecules that can serve as effectors, including antimicrobial peptides, endoglucanases, and highly specific single chain antibodies that target surface glycoprotein tags on the surface of T. cruzi. Furthermore, we address evolving concepts related to field dispersal of engineered bacteria as part of the paratransgenic control strategy and attendant risk assessment evaluation.

  17. Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

    Energy Technology Data Exchange (ETDEWEB)

    Saddler, J N

    1982-11-01

    Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase (1,4-(1,3;1,4)-beta-D-glucan 4- glucanohydrolase, EC 3.2.1.4) activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) activity was approximately six to nine times greater. (Refs. 26).

  18. First report of latent infection of Cyperus rotundus caused by a biovar 3 Dickeya sp. (Syn. Erwinia chrysanthemi) in Israel

    NARCIS (Netherlands)

    Tsror, L.; Lebiush, S.; Erlich, O.; Ben-Daniel, B.; Wolf, van der J.M.

    2010-01-01

    Recent outbreaks of potato blackleg in Israel, caused by Dickeya spp., on plants grown from seed tubers imported from Northern Europe, are of a great concern. The warm climatic conditions during the growing season favour disease expression, and may result in the establishment of the pathogen in the

  19. Linarin Inhibits the Acetylcholinesterase Activity In-vitro and Ex-vivo

    Science.gov (United States)

    Feng, Xinchi; Wang, Xin; Liu, Youping; Di, Xin

    2015-01-01

    Linarin is a flavone glycoside in the plants Flos chrysanthemi indici, Buddleja officinalis, Cirsium setosum, Mentha arvensis and Buddleja davidii, and has been reported to possess analgesic, antipyretic, anti-inflammatory and neuroprotective activities. In this paper, linarin was investigated for its AChE inhibitory potential both in-vitro and ex-vivo. Ellman’s colorimetric method was used for the determination of AChE inhibitory activity in mouse brain. In-vitro assays revealed that linarin inhibited AChE activity with an IC50 of 3.801 ± 1.149 μM. Ex-vivo study showed that the AChE activity was significantly reduced in both the cortex and hippocampus of mice treated intraperitoneally with various doses of linarin (35, 70 and 140 mg/Kg). The inhibition effects produced by high dose of linarin were the same as that obtained after huperzine A treatment (0.5 mg/Kg). Molecular docking study revealed that both 4’-methoxyl group and 7-O-sugar moiety of linarin played important roles in ligand-receptor binding and thus they are mainly responsible for AChE inhibitory activity. In view of its potent AChE inhibitory activity, linarin may be a promising therapeutic agent for the treatment of some diseases associated with AChE, such as glaucoma, myasthenia gravis, gastric motility and Alzheimer’s disease. PMID:26330885

  20. A high-temperature tolerant species in clade 9 of the genus Phytophthora: P. hydrogena sp. nov.

    Science.gov (United States)

    Yang, Xiao; Gallegly, Mannon E; Hong, Chuanxue

    2014-01-01

    A previously unknown Phytophthora species was isolated from irrigation water in Virginia, USA. This novel species produces abundant noncaducous and nonpapillate sporangia in soil water extract solution. It sometimes produces chlamydospores and hyphal swellings in aged cultures and in Petri's solution. This species has optimum vegetative growth at 30 C and grows well at 35 C. The lowest and highest temperatures for growth are 5 and 40 C. All isolates examined in this study are compatibility type A1 and produce mostly plerotic oospores when paired with an A2 mating-type tester of P. cinnamomi. Sequence analyses of the rDNA internal transcribed spacer (ITS) regions and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene placed this species in clade 9 of the genus Phytophthora. These characteristics support the description of this taxon as a new species for which we propose the name P. hydrogena sp. nov. Further phylogenetic and physiological investigations of clade 9 species revealed a high-temperature tolerant cluster including P. hydrogena, P. aquimorbida, P. hydropathica, P. irrigata, P. chrysanthemi, P. insolita, P. polonica and P. parsiana. These species all grow well at 35 C. The monophyly of the species in this heat-tolerant cluster except P. insolita and P. polonica is highly supported by the maximum-likelihood analyses of the ITS and cox 1 sequences.

  1. Regulators Involved in Dickeya solani Virulence, Genetic Conservation and Functional Variability.

    Science.gov (United States)

    Potrykus, Marta; Golanowska, Małgorzata; Hugouvieux-Cotte-Pattat, Nicole; Lojkowska, Ewa

    2015-01-01

    Bacteria from the genus Dickeya (formerly Erwinia chrysanthemi) are plant pathogens causing severe diseases in many economically important crops. A majority of the strains responsible for potato disease in Europe belong to a newly identified Dickeya solani species. Although some ecological and epidemiological studies have been carried out, little is known about the regulation of D. solani virulence. The characterization of four D. solani strains indicates significant differences in their virulence on potato although they are genetically similar based on genomic fingerprinting profiles. A phenotypic examination included an analysis of virulence on potato, growth rate in culture, motility, Fe 3+ chelation, and pectate lyase, cellulase, protease, biosurfactant and blue pigment production. Mutants of four D. solani strains were constructed by inactivating the genes coding either for one of the main negative regulators of D. dadantii virulence (kdgR, pecS and pecT) or for the synthesis and perception of signaling molecules (expI and expR). Analysis of these mutants indicated that PecS, PecT and KdgR play a similar role in both species, repressing to different degrees the synthesis of virulence factors. The thermoregulator PecT seems to be a major regulator of D. solani virulence. This work also reveals the role of quorum sensing mediated by ExpI and ExpR in D. solani virulence on potato.

  2. Surface-enhanced Raman scattering spectra revealing the inter-cultivar differences for Chinese ornamental Flos Chrysanthemum: a new promising method for plant taxonomy

    Directory of Open Access Journals (Sweden)

    Heng Zhang

    2017-10-01

    Full Text Available Abstract Background Flos Chrysanthemi, as a part of Chinese culture for a long history, is valuable for not only environmental decoration but also the medicine and food additive. Due to their voluminously various breeds and extensive distributions worldwide, it is burdensome to make recognition and classification among numerous cultivars with conventional methods which still rest on the level of morphologic observation and description. As a fingerprint spectrum for parsing molecular information, surface-enhanced Raman scattering (SERS could be a suitable candidate technique to characterize and distinguish the inter-cultivar differences at molecular level. Results SERS spectra were used to analyze the inter-cultivar differences among 26 cultivars of Chinese ornamental Flos Chrysanthemum. The characteristic peaks distribution patterns were abstracted from SERS spectra and varied from cultivars to cultivars. For the bands distributed in the pattern map, the similarities in general showed their commonality while in the finer scales, the deviations and especially the particular bands owned by few cultivars revealed their individualities. Since the Raman peaks could characterize specific chemical components, those diversity of patterns could indicate the inter-cultivar differences at the chemical level in fact. Conclusion In this paper, SERS technique is feasible for distinguishing the inter-cultivar differences among Flos Chrysanthemum. The Raman spectral library was built with SERS characteristic peak distribution patterns. A new method was proposed for Flos Chrysanthemum recognition and taxonomy.

  3. The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius

    DEFF Research Database (Denmark)

    Linde, Tore; Zoglowek, Marta; Lübeck, Mette

    2016-01-01

    The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have ...

  4. Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition

    NARCIS (Netherlands)

    Ilari, A.; Fiorillo, A.; Angelaccio, S.; Florio, R.; Chiaraluce, R.; Oost, van der J.; Consalvi, V.

    2009-01-01

    Bacterial and archaeal endo-beta-1,3-glucanases that belong to glycoside hydrolase family 16 share a beta-jelly-roll fold, but differ significantly in sequence and in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus

  5. Protein cross-linking, peroxidase and beta-1,3-endoglucanase involved in resistance of pea against Orobanche crenata.

    Science.gov (United States)

    Pérez-de-Luque, Alejandro; González-Verdejo, Clara I; Lozano, M Dolores; Dita, Miguel A; Cubero, José I; González-Melendi, Pablo; Risueño, María C; Rubiales, Diego

    2006-01-01

    Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a beta-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.

  6. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass.

    Science.gov (United States)

    Duedu, Kwabena O; French, Christopher E

    2016-11-01

    Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design.

    Directory of Open Access Journals (Sweden)

    Jinfeng Zhang

    Full Text Available To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg(-1·min(-1 of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg(-1·min(-1 of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes.

  8. A novel pH-stable, endoglucanase (JqCel5A isolated from a salt-lake microorganism, Jonesia quinghaiensis

    Directory of Open Access Journals (Sweden)

    Ling Lin

    2016-11-01

    Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.

  9. 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.

  10. Enzymatic transformation of nonfood biomass to starch

    Science.gov (United States)

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

    2013-01-01

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

  11. Beta-Glucosidases from a new Aspergillus species can substitute commercial beta-glucosidases for saccharification of lignocellulosic biomass

    Energy Technology Data Exchange (ETDEWEB)

    Sorensen, Annette; Lubeck, Peter Stephensen; Lubeck, Mette; Teller, Philip Johan; Kiaer Ahring, Birgitte

    2011-07-01

    Exploitation of lignocellulosic biomasses for the production of biofuels and biochemicals gives a promising alternative to the world's limited fossil energy resources. Cellulose is of great interest in terms of producing sugars for biofuels and biochemicals, since its hydrolysis product, glucose, can readily be fermented into ethanol or converted into high-value chemicals. The hydrolysis of cellulose involves the synergistic action of cellobiohydrolases, endoglucanases and B-glucosidases, and B-glucosidases is key in ensuring final glucose release and the decrease of the accumulation of cellobiose and shorter cellodextrins, known as product inhibitors of the cellobiohydrolases. The aim of the present work was to search for efficient B-glucosidase-producing fungi using a screening strategy based on wheat bran as fermentation substrate. The fungi selected originated from several different countries and fungal fermentation broth were compared with an onsite enzyme production in mind. The broth of the best strain was tested against commercial enzyme preparations based on enzyme kinetics and it proved to be a valid substitute.

  12. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

    Science.gov (United States)

    Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

    2017-09-08

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Accessibility of Enzymatically Delignified Bambusa bambos for Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study.

    Science.gov (United States)

    Kuila, Arindam; Mukhopadhyay, Mainak; Tuli, D K; Banerjee, Rintu

    2011-01-01

    In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively). Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

  14. Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

    Science.gov (United States)

    Apte, Advait A; Senger, Ryan S; Fong, Stephen S

    2014-01-01

    Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement. PMID:24830736

  15. A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

    Science.gov (United States)

    Zhao, Xi-Hua; Wang, Wei; Tong, Bin; Zhang, Su-Ping; Wei, Dong-Zhi

    2016-01-01

    Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

  16. Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase.

    Science.gov (United States)

    Apiwatanapiwat, Waraporn; Murata, Yoshinori; Kosugi, Akihiko; Yamada, Ryosuke; Kondo, Akihiko; Arai, Takamitsu; Rugthaworn, Prapassorn; Mori, Yutaka

    2011-04-01

    In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

  17. Conversion of woody biomass into fermentable sugars by cellulase from Agaricus arvensis.

    Science.gov (United States)

    Jeya, Marimuthu; Nguyen, Ngoc-Phuong-Thao; Moon, Hee-Jung; Kim, Sang-Hwan; Lee, Jung-Kul

    2010-11-01

    Agaricus arvensis, a newly isolated basidiomycetous fungus, was found to secrete efficient cellulases. The strain produced the highest endoglucanase (EG), cellobiohydrolase (CBH) and beta-glucosidase (BGL) activities of 0.3, 3.2 and 8U/mg-protein, respectively, with rice straw as the carbon source. Saccharification of the woody biomass with A. arvensis cellulase as the enzyme source released a high level of fermentable sugars. Enzymatic hydrolysis of the poplar biomass was optimized using the response surface methodology in order to study the influence of the variables (pH, temperature, cellulases concentration and substrate concentration). The enzyme and substrate concentrations were identified as the limiting factors for the saccharification of poplar wood biomass. A total reducing sugar level of 29g/L (293mg/g-substrate) was obtained at an enzyme concentration of 65FPU/g-substrate after optimization of the hydrolysis parameters. The model validation showed a good agreement between the experimental results and the predicted responses. A. arvensis could be a good candidate for the production of reducing sugars from a cellulosic biomass.

  18. Isolation and Screening of Potential Cellulolytic and Xylanolytic Bacteria from Soil Sample for Degradation of Lignocellulosic Biomass

    Directory of Open Access Journals (Sweden)

    Bhupal Govinda Shrestha

    2016-11-01

    them with the aptitude to produce stable enzymes, little emphasis has been given to cellulose/xylanase production from bacteria. Seven soil samples were collected from eastern hilly districts of Nepal viz. Taplejung, Panchthar and Sankhuwasabha districts, from soil surface and at depth of 10cm to 20cm, and were isolated separately. From the seven soil samples, four bacterial isolates were obtained. Isolates (PSS, P1D, TLC, SNK were then screened for cellulolytic/xylanolytic activity using Congo red assay on Carboxymethylcellulose (CMC/xylan agar plates. The enzyme activity obtained from isolates was dependent on substrate concentration. The activity of enzymes produced by isolates were also measured and compared on pretreated sugarcane bagasse. Among those samples, the greatest zone of inhibition in both CMC (1.3 cm and xylan (1.0 cm agar media was seen in isolate P1D. It also produced the highest activity of endoglucanase and xylanase i.e. activity 0.035 U/mL and 0.050 U/mL respectively at 0.010 mg mL-1 standard substrate concentration of CMC and xylan.

  19. Designing novel cellulase systems through agent-based modeling and global sensitivity analysis.

    Science.gov (United States)

    Apte, Advait A; Senger, Ryan S; Fong, Stephen S

    2014-01-01

    Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement.

  20. Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and {beta}-glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Apiwatanapiwat, Waraporn; Rugthaworn, Prapassorn [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Kasetsart Univ., Bangkok (Thailand). Nanotechnology and Biotechnology Div.; Murata, Yoshinori; Kosugi, Akihiko; Arai, Takamitsu; Mori, Yutaka [Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki (Japan). Post-Harvest Science and Technology Div.; Yamada, Ryosuke; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering

    2011-04-15

    In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying {alpha}-amylase ({alpha}-AM), glucoamylase, endoglucanase, cellobiohydrase, and {beta}-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley {beta}-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes. (orig.)

  1. Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

    Directory of Open Access Journals (Sweden)

    Caitlin Siobhan Byrt

    2012-11-01

    Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

  2. Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers.

    Science.gov (United States)

    Rai, Rohit; Kaur, Baljit; Singh, Surender; Di Falco, Macros; Tsang, Adrian; Chadha, B S

    2016-09-01

    Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Enzymatic hydrolysis of oil palm empty fruits bunch fiber using Celluclast® and Accellerase® BG for sugar production

    Science.gov (United States)

    Salleh, Noor Shafryna; Murad, Abdul Munir Abdul

    2016-11-01

    In this work, the ability of commercial Trichoderma reesei cellulases preparation, Celluclast® or in combination with Accellerase®BG β-glucosidase to hydrolyse pretreated oil palm empty fruit bunch (OPEFB) was evaluated. Celluclast® alone hydrolyzed OPEFB to produce 2.41±0.44 mg glucose per gram OPEFB. However, the production of glucose was significantly improved with supplementation of Accellerase®BG (8.12±0.93 mg/g). This result suggested that the endoglucanases and exoglucanases in Celluclast® and β-glucosidase in Accellerase®BG able to work synergistically to increase the production of glucose from OPEFB. In addition, the production of xylose was also improved by 30% when the enzyme mixture was used. The result suggested that the mixture of Celluclast® with Accellerase®BG work synergistically to improve the production of sugars by removing the inhibition by cellobiose for complete cellulose hydrolysis. The production of glucose and xylose from OPEFB wastes showed the potential of this biomass as the source of renewable energy and fine chemicals production in Malaysia.

  4. [A study of the mechanisms of probiotic effect of Bacillus subtilis 8130 strain].

    Science.gov (United States)

    Ushakova, N A; Kotenkova, E V; Kozlova, A A; Nifatov, A V

    2006-01-01

    The wild-type Bacillus subtilis strain 8130 secreted metabolites that stimulated two to three times the growth of the test cultures of lactic acid bacteria. It exhibited endoglucanase activity that depended on the composition of nutrient medium. The addition of the product of two-stage culturing of B. subtilis 8130 to the diet of pigs (0.2% of fodder weight) made it possible to increase the daily weight gain by 19% and decrease the consumption of mixed fodder by 10%. Digestion of protein, fat, and other organic compounds increased by 3-4% and cellulose by 12%. It was shown that B. subtilis 8130 is a probiotic with targeted action stimulating digestion (primarily the digestion of cellulose). The enrichment of a dry-beer pellet with the product of solid-phase fermentation by bacillus (1 x 10(8) cells per gram dry pellet) allowed the pellet to entered into the diet of a calf (6% of the weight of fodder with probiotic), causing additional weight gain by 12% and a 10% economy of fodder consumption.

  5. Accessibility of Enzymatically Delignified Bambusa bambos for Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study

    Directory of Open Access Journals (Sweden)

    Arindam Kuila

    2011-01-01

    Full Text Available In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively. Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR, X-ray diffraction (XRD, and scanning electron microscopy (SEM. The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

  6. 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.

  7. Formulation and In Vitro Evaluation of Minoxidil Topical Gel

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    Rabinarayan PARHI

    2014-08-01

    Full Text Available The objective of the present investigation is to develop topical gel of minoxidil using model polymers such as hydroxypropyl methylcellulose K4M (HPMC K4M and hydroxypropyl cellulose (HPC at different concentrations (1, 2 and 3% individually and in combination. The dmg and polymers compatibility study was carried out by FTIR teclmique. The gels were evaluated for dmg content, viscosity, pH, homogenity, grittiness and in vitro dmg release. The FTIR spectra of dmg alone and in physical mixture with polymers did not show any slıift in majör peaks, wlıich indicates no dmg-polymer interaction. Ali the data obtained from above physicochemical parameters were satisfactory. In vitro dmg release of gels was performed using Franz diffusion celi of 25 ınL capacity with cellulose acetate membrane in phosphate buffer pH 6.8 as receptor medium. According to the release study, the dmg release was decreasing with the increasing polymer concentration in each fonnulation. The correlation coefficient (r values demonstrate that the dmg release pattem followed Higuclıi model and the release exponent (n values were witlıin 0.45 to 0.85 for ali fonnulations except FG3, FG4 and FG7. The above results showed that swelling and diffusion (Non-Fickian diffusion were the dmg release mechanism. To know the marketing feasibility, the release data of ali the fonnulations were compared with the marketed formulation (Tugain gel and it was found tlıat formulation FG2 was lıaving lıighest similarity with similarity factor (f2 of 77.79

  8. [Update on L-asparaginase treatment in paediatrics].

    Science.gov (United States)

    Moscardó Guilleme, C; Fernández Delgado, R; Sevilla Navarro, J; Astigarraga Aguirre, I; Rives Solà, S; Sánchez de Toledo Codina, J; Fuster Soler, J L; Parra Ramirez, L; Molina Garicaño, J; González Martínez, B; Madero López, L

    2013-11-01

    L-asparaginase (L-ASP) is one of the cornerstones of the treatment of acute lymphoblastic leukemia and non-Hodgkin lymphoma. It is an enzyme of bacterial origin capable of transforming L-asparagine to aspartic acid. The extracellular depletion of L-asparagine inhibits protein synthesis in lymphoblasts, inducing their apoptosis. Numerous studies have demonstrated that treatment with L-ASP improves survival of patients, but there are clear differences in the characteristics of the three currently available formulations. This article reviews the dosage, activity and side effects of the two L-ASP derived from Escherichia coli (native and pegylated), and the one derived from Erwinia chrysanthemi (Erwinia ASP). Despite its indisputable indication over the past50 years, there are still many points of contention, and its use is still marked by the side effects of the inhibition of protein synthesis. The short half-life of native forms, and the most frequently used parenteral administration by intramuscular injections, affects the quality of life of the patients. Therefore, recent studies claim to evaluate alternatives, such as the formulation of longer half-life pegylated L-ASP, and the use of intravenous formulations. There are encouraging results to date with both preparations. Still, further studies are needed to establish which should be the formulation and frontline indicated route of administration, optimal dosing, and management of adverse effects. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

  9. Isolation and Identification of L-asparaginase producing Erwinia strains which isolated from Potato Farms

    Directory of Open Access Journals (Sweden)

    Arastoo Badoei-Dalfard

    2016-09-01

    Full Text Available Introduction: L-Asparaginase can be effectively used for the treatment of lymphoblastic leukemia. The rapid growth of cancer cells are needed for L-asparagine abundant storage. L-asparaginase catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. The purpose of this study was to isolate and identify the L-asparaginase producing Erwinia strains from the potato farms of Jiroft. Materials and methods: Pectolytic Erwinia species isolated from crumbling potato in M9 medium. The desired L-asparaginase producing bacteria were isolated based on the color changes. Biochemical-microbial and the plant pathogenicity tests of these strains were also investigated with potato and geranium. The L-asparaginase production and molecular detection of these Erwinia strains were also investigated. Results: In this study, L-asparaginase producing Erwinia was isolated on the CVP and M9 mediums. The inoculation of Erwinia strains on the potato and geranium plants showed that Er8 and Er11 species have the ability to cause plant pathogenicity. Results showed that the maximum pathogenicity of Er8 and Er11 was observed after 48 and 15 h of inoculation in potato and geranium plants, respectively. 16S rDNA sequencing and phylogenetic analyses exhibited that Er8 and Er11 strains were similar to Erwinia chrysanthemi with 98% homology. Discussion and conclusion: Because of several applications of the Erwinia L-asparaginase in various fields, isolated Erwinia and their L-asparaginase can be suitable for applied utilization.

  10. Proteins from Erwinia asparaginase Erwinase ® and E. coli asparaginase 2 MEDAC ® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear.

    Science.gov (United States)

    Bae, Narkhyun; Pollak, Arnold; Lubec, Gert

    2011-07-01

    L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase(®))) and L-asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac(®))), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC-MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

    Science.gov (United States)

    Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

    2016-07-11

    The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N

  12. The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose.

    Directory of Open Access Journals (Sweden)

    Bjørge Westereng

    Full Text Available Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61, some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33, this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.

  13. 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.

  14. Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

    Science.gov (United States)

    Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

    2012-10-15

    Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

  15. Sur un deplacement de valeurs: traire et tirer

    Directory of Open Access Journals (Sweden)

    André Burger

    1972-12-01

    Full Text Available L'étymologie de firer est inconnue. Celie que le regretté W. von Wart burg a ern pouvoir avancer est inacceptable: firer serait sorti de marty rier par la grâce d'une fausse coupure mar tirier. Il est plus qu'improbable que cette «étymologie populaire» eût pu se produire si la langue ne possédait pas au préalable un verbe tirier. Au surplus aucun fait n'appuie cette hypothèse. Le FEW, t. VI, 1, p. 396, donne bien pour firer le sens de «torturer sur un treteam>, au XIIIe siècle, et p. 403, «démembrer en faisant tirer les quatre membles par des chevaux» et «torturer (qn en l'étendant sur un tréteau” au XIVe siècle; ces sens sont évidemment trop tardifs pour permettre des conclusions sur l'origine de firer, d'autant plus qu'ils s'expliquent sans peine par la valeur normale du mot, attesté dès la Chanson de Roland, sans aucun rapport avec martirie, mot savant de clerc, qui, dans le même texte s'applique deux fois sur quatre exemples aux Sarrasins, vv. 501 et 1467, où il ne signifie pas «martyre» mais <

  16. Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

    Science.gov (United States)

    Andersson, R A; Eriksson, A R; Heikinheimo, R; Mäe, A; Pirhonen, M; Kõiv, V; Hyytiäinen, H; Tuikkala, A; Palva, E T

    2000-04-01

    The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

  17. Characterization of the causal organism of blackleg and soft rot of potato, and management of the disease with balanced fertilization

    International Nuclear Information System (INIS)

    Ali, H.F.; Bibi, A.; Ahmad, M.; Junaid, M.; Ali, A.; Alam, S.

    2014-01-01

    Based upon colony morphology, physio-biochemical tests and polymerase chain reaction (using species or subspecies-specific primers) studies, 20 isolates (out of a total of 42) were found to be Erwinia carotovora subspecies atroseptica (Eca), 19 were identified as Erwinia carotovora subspecies carotovora (Ecc), and 3 as Erwinia chrysanthemi (Ech). Results of the subspecies-differentiating biochemical tests indicated that majority of the candidate Ecc isolates did not produce acid from methyle glucoside (as expected) but their reaction to the production of reducing substances from sucrose was variable. Likewise, some of our Eca and Ecc strains (unexpectedly) were sensitive to erythromycin. Also, most of our Eca strains unexpectedly grew at 36 degree C. Our strains slightly deviate from the standard description in some of their minor characteristics but they still remain the valid members of the Eca, Ecc or Ech group as similar variations in minor characteristics have been found by other workers. The occurrence of intermediate forms of Eca and Ecc (sharing some of the characteristics of both the groups) indicates variability happening among these strains. This variability indicates the potential ability of the pathogen to break the resistance of the host. The results of the effect of balanced nutrition in controlling blackleg and soft rot of potatoes indicated that the fertilizer combination of N3P1K3 (262/252/262 kg.ha-1) which is slightly higher than the normally practiced dose (247/247/247 kg.ha-1) was the best in bringing the disease to a minimum and subsequently increasing the yield. (author)

  18. The pectin lyase-encoding gene (pnl) family from Glomerella cingulata: characterization of pnlA and its expression in yeast.

    Science.gov (United States)

    Templeton, M D; Sharrock, K R; Bowen, J K; Crowhurst, R N; Rikkerink, E H

    1994-05-03

    Oligodeoxyribonucleotide primers were designed from conserved amino acid (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The polymerase chain reaction (PCR) was used with these primers to amplify genomic DNA from the plant pathogenic fungus Glomerella cingulata. Three different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes from Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-encoding gene from a lambda genomic DNA library prepared from G. cingulata. Nucleotide (nt) sequence data revealed that this gene has seven exons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the five introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein was secreted into the culture medium. Significantly higher expression was achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed yeast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Expression, purification and crystallization of Helicobacter pyloril-asparaginase

    Energy Technology Data Exchange (ETDEWEB)

    Dhavala, Prathusha [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland); Krasotkina, Julya [Institute for Biomedical Sciences, Russian Academy of Medical Sciences, Moscow (Russian Federation); Dubreuil, Christine; Papageorgiou, Anastassios C., E-mail: tassos.papageorgiou@btk.fi [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland)

    2008-08-01

    l-Asparaginase from H. pylori was overexpressed in E. coli, purified and crystallized. The crystals belonged to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule in the asymmetric unit. A complete data set to 1.6 Å resolution was collected using synchrotron radiation. The l-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to l-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards l-asparagine are highly desirable as better alternatives in cancer therapy. l-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16–19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 Å resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule of l-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure–activity relationship in l-asparaginases.

  20. Behavior of hybrid corn crop as second rot incidence in West Region Paraná

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    Jean Sérgio Rosset

    2013-09-01

    Full Text Available The objective of this study was to evaluate the incidence and influence of stalk rot and ear in cultivation of hybrid corn second crop in west region Paraná. The experiment was conducted in randomized block design with six transgenic corn hybrids (DKB 330PRO, P4285HX, P3646HX, 30F53HX, P3340HX and P3161HX with four replications at spacing of 0.90 m between rows and 0.20 m between plants. The characteristics evaluated were: number of healthy and symptomatic plants, number of ears healthy and symptomatic and total number of spikes. After harvest, we assessed the length of ears healthy and symptomatic, bulk grain ears healthy and symptomatic, thousand grain weight of ears healthy and symptomatic, and grain mass per spike weighted, thousand grain weight and weighted productivity. The hybrid P3646HX showed 100% of plants with stem base rot (Colletotrichum graminicola and soft rot cob (Erwinia chrysanthemi pv. Zeae and 100% of ears with symptoms of soft rot, followed by hybrid 30F53HX, DKB 330PRO with 34.9 and 29.1% of ears with symptoms of soft rot respectively. The hybrid DKB330PRO showed healthy spikes and patients with superior size, resulting in less interference in the grain yield. The hybrid P3340 productivity was higher, with 7952 kg ha-1 , followed by hybrid 30F53HX and DKB330PRO. A positive correlation between agronomic characteristics and grain yield.

  1. A STRUCTURAL OVERVIEW OF GH61 PROTEINS – FUNGAL CELLULOSE DEGRADING POLYSACCHARIDE MONOOXYGENASES

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    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  2. Engineering of family-5 glycoside hydrolase (Cel5A from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

    Directory of Open Access Journals (Sweden)

    Amar A Telke

    Full Text Available Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

  3. A structural overview of GH61 proteins – fungal cellulose degrading polysaccharide monooxygenases

    Directory of Open Access Journals (Sweden)

    Leila Lo Leggio

    2012-09-01

    Full Text Available Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

  4. Using heavy-ion mutagenesis technology to select cellulose enzyme vitality of mutants of Aspergillium niger

    International Nuclear Information System (INIS)

    Tang Jiahui; Yang Fumin; Wang Shuyang

    2012-01-01

    In order to improve the cellulose ion beam at 20, 40, 60, 80, 100, 120Gy and 140 enzyme vitality of Aspergillus niger (=AS3.316), heavy Gy doses was used for inducing mutation. Higher cellulose enzyme vitality strains were screened through the primary screening and secondary screening. The result showed that 5 mutants T2-1, T3-1, T5-1, T6-3, T6-4 were selected, and T6-4 had the highest cellulose enzyme activity. The activity of filter paper cellulose enzyme, endo-glucanase, exo-glucanase and 13-glucosidase of T6-4 was 61.3, 116.2, 29.9 U/mL and 35.9 U/mL respectively. Compared with the original A. niger (=AS3.316), the cellulose enzyme activity was increased by 3.5, 3.78, 2.76 and 2.52 times in turn. The activity of cellulose enzyme of the rest mutants sorted from strong to the weak were T6-3T5-1T3-1T2-1. The dose at 120 Gy showed the best mutagenesis effect. Mutants had different degree of changes in the genetic stability, but overall, the performance showed relatively stable

  5. Comparative Digital Gene Expression Analysis of Tissue-Cultured Plantlets of Highly Resistant and Susceptible Banana Cultivarsin Response to Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Yuqing Niu

    2018-01-01

    Full Text Available Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA cultivars differing in Foc susceptibility were used to reveal their differential responses to this pathogen using digital gene expression (DGE. Data were evaluated by various bioinformatic tools (Venn diagrams, gene ontology (GO annotation and Kyoto encyclopedia of genes and genomes (KEGG pathway analyses and immunofluorescence labelling method to support the identification of gene candidates determining the resistance of banana against Foc. Interestingly, we have identified MaWRKY50 as an important gene involved in both constitutive and induced resistance. We also identified new genes involved in the resistance of banana to Foc, including several other transcription factors (TFs, pathogenesis-related (PR genes and some genes related to the plant cell wall biosynthesis or degradation (e.g., pectinesterases, β-glucosidases, xyloglucan endotransglucosylase/hydrolase and endoglucanase. The resistant banana cultivar shows activation of PR-3 and PR-4 genes as well as formation of different constitutive cell barriers to restrict spreading of the pathogen. These data suggest new mechanisms of banana resistance to Foc.

  6. Genome assembly of Chryseobacterium sp. strain IHBB 10212 from glacier top-surface soil in the Indian trans-Himalayas with potential for hydrolytic enzymes

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    Mohinder Pal

    2017-09-01

    Full Text Available The cold-active esterases are gaining importance due to their catalytic activities finding applications in chemical industry, food processes and detergent industry as additives, and organic synthesis of unstable compounds as catalysts. In the present study, the complete genome sequence of 4,843,645 bp with an average 34.08% G + C content and 4260 protein-coding genes are reported for the low temperature-active esterase-producing novel strain of Chrysobacterium isolated from the top-surface soil of a glacier in the cold deserts of the Indian trans-Himalayas. The genome contained two plasmids of 16,553 and 11,450 bp with 40.54 and 40.37% G + C contents, respectively. Several genes encoding the hydrolysis of ester linkages of triglycerides into fatty acids and glycerol were predicted in the genome. The annotation also predicted the genes encoding proteases, lipases, amylases, β-glucosidases, endoglucanases and xylanases involved in biotechnological processes. The complete genome sequence of Chryseobacterium sp. strain IHBB 10212 and two plasmids have been deposited vide accession numbers CP015199, CP015200 and CP015201 at DDBJ/EMBL/GenBank.

  7. ENZIME ACTIVITY OF HIGHER BASIDIOMYCETES MUSHROOM GRIFOLA FRONDOSA

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    А. Бухало

    2011-02-01

    Full Text Available The purpose of this work was a revelation and evaluation of spectrum and activity of hydrolytic enzymes of higher basidiomycetes  Grifola frondosa in a surface and submerged culture. 8 strains of  Gf. frondosa,  mushrooms from culture collection of mushrooms at the M.G. Kholodny Institute of  Вotany National  Academy of Sciences of the Ukraine were object of investigation. Researches were conducted by standard microbiological, biochemical and biotechnological methods. All strains  on agar mediums were shown the  following enzymes: amylase, caseinase, polygalacturonase, pectattranselyminase, glucosidase, urease,  xylanase, lipase  and endoglucanase. The demonstration of oxidizing enzymes of laccase and tyrosinase  depended on a culture and did not depend on composition of medium. The estimation of presence and level of activity of hydrolytic enzymes at submerged cultivation indicate primary influence of components of complex nourishing medium on enzyme activity of Gr. frondosa. Strains biochemical features show up in the case of  oxidizing enzymes on agar mediums and for endo-1,4-β-glucanase on liquid mediums with glucose and molasses.

  8. Halo(natronoarchaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates

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    Dimitry Y Sorokin

    2015-09-01

    Full Text Available Until recently, extremely halophilic euryarchaeota were considered mostly as aerobic heterotrophs utilizing simple organic compounds as growth substrates. Almost nothing is known on the ability of these prokaryotes to utilize complex polysaccharides as cellulose, xylan and chitin. Although few haloarchaeal cellulases and chitinases were recently characterized, the analysis of currently available haloarchaeal genomes deciphered numerous genes encoding glycosidases (GHs of various families including endoglucanases and chitinases. However, all these haloarchaea were isolated and cultivated on simple substrates and their ability to grow on polysaccharides in situ or in vitro is unknown. This study examines several halo(natronoarchaeal strains from geographically distant hypersaline lakes for the ability to grow on insoluble polymers as a sole growth substrate in salt-saturated mineral media. Some of them belonged to known taxa, while other represented novel phylogenetic lineages within the class Halobacteria. All isolates produced extracellular extremely salt tolerant cellulases or chitinases, either cell-free or cell-bound. Obtained results demonstrate a presence of diverse population of haloarchaeal cellulo/chitinotrophs in hypersaline habitats indicating that euryarchaea participate in aerobic mineralization of recalcitrant organic polymers in salt-saturated environments.

  9. Reaction mechanism of dicofol removal by cellulase.

    Science.gov (United States)

    Wang, Ziyuan; Yang, Ting; Zhai, Zihan; Zhang, Boya; Zhang, Jianbo

    2015-10-01

    It remains unclear whether dicofol should be defined as a persistent organic pollutant. Its environmental persistence has gained attention. This study focused on its degradation by cellulase. Cellulase was separated using a gel chromatogram, and its degradation activity towards dicofol involved its endoglucanase activity. By analyzing the kinetic parameters of cellulase reacting with mixed substrates, it was shown that cellulase reacted on dicofol and carboxyl methyl cellulose through two different active centers. Thus, the degradation of dicofol was shown to be an oxidative process by cellulase. Next, by comparing the impacts of tert-butyl alcohol (a typical OH free-radical inhibitor) on the removal efficiencies of dicofol under both cellulase and Fenton reagent systems, it was shown that the removal of dicofol was initiated by OH free radicals produced by cellulase. Finally, 4,4'-dichloro-dibenzophenone and chloride were detected using gas chromatography mass spectrometry and ion chromatography analysis, which supported our hypothesis. The reaction mechanism was analyzed and involved an attack by OH free radicals at the orthocarbon of dicofol, resulting in the degradation product 4,4'-dichloro-dibenzophenone. Copyright © 2015. Published by Elsevier B.V.

  10. Genomic Characteristics of Bifidobacterium thermacidophilum Pig Isolates and Wild Boar Isolates Reveal the Unique Presence of a Putative Mobile Genetic Element with tetW for Pig Farm Isolates

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    Sayaka Tsuchida

    2017-08-01

    Full Text Available Genomic analysis was performed on seven strains of Bifidobacterium thermacidophilum, a Sus-associated Bifidobacterium. Three strains from the feces of domestic pigs (Sus scrofa domesticus and four strains from the rectal feces of free-range Japanese wild boars (S. s. scrofa were compared. The phylogenetic position of these isolates suggested by genomic analyses were not concordant with that suggested by 16S rRNA sequence. There was biased distribution of genes for virulence, phage, metabolism of aromatic compounds, iron acquisition, cell division, and DNA metabolism. In particular four wild boar isolates harbored fiber-degrading enzymes, such as endoglucanase, while two of the pig isolates obtained from those grown under an intensive feeding practice with routine use of antimicrobials, particularly tetracycline harbored a tetracycline resistance gene, which was further proved functional by disk diffusion test. The tetW gene is associated with a serine recombinase of an apparently non-bifidobacterial origin. The insertion site of the tetW cassette was precisely defined by analyzing the corresponding genomic regions in the other tetracycline-susceptible isolates. The cassette may have been transferred from some other bacteria in the pig gut.

  11. Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

    Science.gov (United States)

    Qian, Yuanchao; Zhong, Lixia; Hou, Yunhua; Qu, Yinbo; Zhong, Yaohua

    2016-01-01

    The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

  12. Characterization and strain improvement of a hypercellulytic variant, Trichoderma reesei SN1, by genetic engineering for optimized cellulase production in biomass conversion improvement

    Directory of Open Access Journals (Sweden)

    Qian Yuanchao

    2016-08-01

    Full Text Available The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG activity but lower β-glucosidase (BGL activity than those of QM9414 and RUT-C30. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

  13. Enzymology of lignocellulose bioconversion by Streptomyces viridosporus

    International Nuclear Information System (INIS)

    Ramachandra, M.

    1989-01-01

    Significant progress has been made in lignin biodegradation research since 1983, when lignin peroxidases were discovered in fungi. A similar breakthrough in bacterial lignin biodegradation research is anticipated. Several laboratories have successfully demonstrated the ability of bacteria to mineralize [ 14 C]-lignin lignocelluloses as well as 14 C-labelled synthetic lignins. Attempts are being made to identify the key enzymes involved. In this dissertation, two studies are presented which address the enzymology of lignin biodegradation by Streptomyces viridosporus. The first study compares selected extracellular enzyme of wild-type and genetically manipulated strains with enhanced abilities to produced a water soluble lignin degradation intermediate, designated acid-precipitable polymeric lignin (APPL). UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10 had higher and longer persisting peroxidase, esterase, and endoglucanase activity than did the wild type strain T7A. An extracellular lignocellulose-induced peroxidase with some similarities to fungal ligninases was described for the first time in Streptomyces. The second study describes purification and characterization of an extracellular lignin peroxidase produced by S. viridosporus T7A. This is the first report of a lignin peroxidase in any bacterium

  14. A Novel Carbohydrate-binding Module from Sugar Cane Soil Metagenome Featuring Unique Structural and Carbohydrate Affinity Properties*

    Science.gov (United States)

    Campos, Bruna Medeia; Alvarez, Thabata Maria; Zanphorlin, Letícia Maria; Ematsu, Gabriela Cristina; Barud, Hernane; Polikarpov, Igor; Ruller, Roberto; Gilbert, Harry J.; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2016-01-01

    Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked β1,3-β1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical β-sandwich fold comprising two β-sheets. The planar ligand binding site, observed in a parallel orientation with the β-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs. PMID:27621314

  15. Impact of cell wall-degrading enzymes on water-holding capacity and solubility of dietary fibre in rye and wheat bran.

    Science.gov (United States)

    Petersson, Karin; Nordlund, Emilia; Tornberg, Eva; Eliasson, Ann-Charlotte; Buchert, Johanna

    2013-03-15

    Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased. © 2012 Society of Chemical Industry.

  16. Ethanol from lignocellulosic biomasses

    International Nuclear Information System (INIS)

    Ricci, E.; Viola, E.; Zimbardi, F.; Braccio, G.; Cuna, D.

    2001-01-01

    In this report are presented results achieved on the process optimisation of bioethanol production from wheat straw, carried out within the ENEA's project of biomass exploitation for renewable energy. The process consists of three main steps: 1) biomass pretreatment by means of steam explosion; 2) enzymatic hydrolysis of the cellulose fraction; 3) fermentation of glucose. To perform the hydrolysis step, two commercial enzymatic mixtures have been employed, mainly composed by β-glucosidase (cellobiase), endo-glucanase and exo-glucanase. The ethanologenic yeast Saccharomyces cerevisiae has been used to ferment the glucose in he hydrolyzates. Hydrolysis yield of 97% has been obtained with steam exploded wheat straw treated at 220 0 C for 3 minutes and an enzyme to substrate ratio of 4%. It has been pointed out the necessity of washing with water the pretreated what straw, in order to remove the biomass degradation products, which have shown an inhibition effect on the yeast. At the best process conditions, a fermentation yield of 95% has been achieved. In the Simultaneous Saccharification and Fermentation process, a global conversion of 92% has been obtained, which corresponds to the production of about 170 grams of ethanol per kilogram of exploded straw [it

  17. Cellulose hydrolysis by Trichoderma reesei cellulases: studies on adsorption, sugar production and synergism of cellobiohydrolase I,II and endoglucanase II

    Energy Technology Data Exchange (ETDEWEB)

    Medve, J.

    1997-02-01

    Three major cellulases have been purified by ion-exchange chromatography in an FPLC system. Microcrystalline cellulose (Avicel) was hydrolyzed by the single enzymes and by equimolar mixtures of CBH I-CBH II and CBH I-EG II. Enzyme adsorption was followed indirectly by selectively quantifying the enzymes in the supernatant by ion-exchange chromatography in an FPLC system. The (synergistic) production of small, soluble sugars (glucose, cellobiose and cellotriose) by the enzymes was followed by HPLC. 76 refs

  18. Dickeya dadantii, a plant pathogenic bacterium producing Cyt-like entomotoxins, causes septicemia in the pea aphid Acyrthosiphon pisum.

    Science.gov (United States)

    Costechareyre, Denis; Balmand, Séverine; Condemine, Guy; Rahbé, Yvan

    2012-01-01

    Dickeya dadantii (syn. Erwinia chrysanthemi) is a plant pathogenic bacteria that harbours a cluster of four horizontally-transferred, insect-specific toxin genes. It was recently shown to be capable of causing an acute infection in the pea aphid Acyrthosiphon pisum (Insecta: Hemiptera). The infection route of the pathogen, and the role and in vivo expression pattern of these toxins, remain unknown. Using bacterial numeration and immunolocalization, we investigated the kinetics and the pattern of infection of this phytopathogenic bacterium within its insect host. We compared infection by the wild-type strain and by the Cyt toxin-deficient mutant. D. dadantii was found to form dense clusters in many luminal parts of the aphid intestinal tract, including the stomach, from which it invaded internal tissues as early as day 1 post-infection. Septicemia occurred soon after, with the fat body being the main infected tissue, together with numerous early infections of the embryonic chains showing embryonic gut and fat body as the target organs. Generalized septicemia led to insect death when the bacterial load reached about 10(8) cfu. Some individual aphids regularly escaped infection, indicating an effective partial immune response to this bacteria. Cyt-defective mutants killed insects more slowly but were capable of localisation in any type of tissue. Cyt toxin expression appeared to be restricted to the digestive tract where it probably assisted in crossing over the first cell barrier and, thus, accelerating bacterial diffusion into the aphid haemocel. Finally, the presence of bacteria on the surface of leaves hosting infected aphids indicated that the insects could be vectors of the bacteria.

  19. Dickeya dadantii, a plant pathogenic bacterium producing Cyt-like entomotoxins, causes septicemia in the pea aphid Acyrthosiphon pisum.

    Directory of Open Access Journals (Sweden)

    Denis Costechareyre

    Full Text Available Dickeya dadantii (syn. Erwinia chrysanthemi is a plant pathogenic bacteria that harbours a cluster of four horizontally-transferred, insect-specific toxin genes. It was recently shown to be capable of causing an acute infection in the pea aphid Acyrthosiphon pisum (Insecta: Hemiptera. The infection route of the pathogen, and the role and in vivo expression pattern of these toxins, remain unknown. Using bacterial numeration and immunolocalization, we investigated the kinetics and the pattern of infection of this phytopathogenic bacterium within its insect host. We compared infection by the wild-type strain and by the Cyt toxin-deficient mutant. D. dadantii was found to form dense clusters in many luminal parts of the aphid intestinal tract, including the stomach, from which it invaded internal tissues as early as day 1 post-infection. Septicemia occurred soon after, with the fat body being the main infected tissue, together with numerous early infections of the embryonic chains showing embryonic gut and fat body as the target organs. Generalized septicemia led to insect death when the bacterial load reached about 10(8 cfu. Some individual aphids regularly escaped infection, indicating an effective partial immune response to this bacteria. Cyt-defective mutants killed insects more slowly but were capable of localisation in any type of tissue. Cyt toxin expression appeared to be restricted to the digestive tract where it probably assisted in crossing over the first cell barrier and, thus, accelerating bacterial diffusion into the aphid haemocel. Finally, the presence of bacteria on the surface of leaves hosting infected aphids indicated that the insects could be vectors of the bacteria.

  20. Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

    Science.gov (United States)

    Samaratunga, Ashani; Kudina, Olena; Nahar, Nurun; Zakharchenko, Andrey; Minko, Sergiy; Voronov, Andriy; Pryor, Scott W

    2015-03-01

    Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

  1. Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides.

    Science.gov (United States)

    Devendran, Saravanan; Abdel-Hamid, Ahmed M; Evans, Anton F; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I; Cann, Isaac

    2016-10-17

    Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

  2. A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability, Broad Substrate Specificity and Potential Application in the Brewing Industry.

    Directory of Open Access Journals (Sweden)

    Yun Liu

    Full Text Available An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1-20, a catalytic domain of glycoside hydrolase family 7 (GH7, a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1. The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0-6.0 and excellent stability at pH3.0-8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg-1, moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.

  3. Solid state fermentation for production of microbial cellulases: Recent advances and improvement strategies.

    Science.gov (United States)

    Behera, Sudhanshu S; Ray, Ramesh C

    2016-05-01

    Lignocellulose is the most plentiful non-food biomass and one of the most inexhaustible renewable resources on the planet, which is an alternative sustainable energy source for the production of second generation biofuels. Lignocelluloses are composed of cellulose, hemicellulose and lignin, in which the sugar polymers account for a large portion of the biomass. Cellulases belong to the glycoside hydrolase family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. They are multi-enzymatic complex proteins and require the synergistic action of three key enzymes: endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) for the depolymerization of cellulose to glucose. Solid state fermentation, which holds growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged fermentation. The review summarizes the critical analysis of recent literature covering production of cellulase in solid state fermentation using advance technologies such as consolidated bioprocessing, metabolic engineering and strain improvement, and circumscribes the strategies to improve the enzyme yield. Copyright © 2016. Published by Elsevier B.V.

  4. 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.

  5. Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Yanase, Shuhei; Yamada, Ryosuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Hasunuma, Tomohisa; Tanaka, Tsutomu; Fukuda, Hideki [Kobe Univ. (Japan). Organization of Advanced Science and Technology

    2010-09-15

    To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 C and 37 C, while the activity of cellulolytic enzymes is highest at around 50 C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus {beta}-glucosidase on the cell surface, which successfully converts a cellulosic {beta}-glucan to ethanol directly at 48 C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of {beta}-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface. (orig.)

  6. 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.

  7. Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus

    Directory of Open Access Journals (Sweden)

    Lifeng Zhou

    2016-09-01

    Full Text Available Bursaphelenchus mucronatus (B. mucronatus isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus’ pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future.

  8. Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production.

    Science.gov (United States)

    Garcia-Kirchner, O; Muñoz-Aguilar, M; Pérez-Villalva, R; Huitrón-Vargas, C

    2002-01-01

    The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, beta-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly beta-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29 degrees C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.

  9. Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production

    Science.gov (United States)

    Muñoz, Camilo; Hidalgo, Catalina; Zapata, Manuel; Jeison, David; Riquelme, Carlos

    2014-01-01

    In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production. PMID:24795376

  10. Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture

    Directory of Open Access Journals (Sweden)

    Marli Camassola

    2014-01-01

    Full Text Available The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U·mL−1 was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher β-glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.

  11. Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose.

    Science.gov (United States)

    Yuki, Masahiro; Kuwahara, Hirokazu; Shintani, Masaki; Izawa, Kazuki; Sato, Tomoyuki; Starns, David; Hongoh, Yuichi; Ohkuma, Moriya

    2015-12-01

    Wood-feeding lower termites harbour symbiotic gut protists that support the termite nutritionally by degrading recalcitrant lignocellulose. These protists themselves host specific endo- and ectosymbiotic bacteria, functions of which remain largely unknown. Here, we present draft genomes of a dominant, uncultured ectosymbiont belonging to the order Bacteroidales, 'Candidatus Symbiothrix dinenymphae', which colonizes the cell surface of the cellulolytic gut protists Dinenympha spp. We analysed four single-cell genomes of Ca. S. dinenymphae, the highest genome completeness was estimated to be 81.6-82.3% with a predicted genome size of 4.28-4.31 Mb. The genome retains genes encoding large parts of the amino acid, cofactor and nucleotide biosynthetic pathways. In addition, the genome contains genes encoding various glycoside hydrolases such as endoglucanases and hemicellulases. The genome indicates that Ca. S. dinenymphae ferments lignocellulose-derived monosaccharides to acetate, a major carbon and energy source of the host termite. We suggest that the ectosymbiont digests lignocellulose and provides nutrients to the host termites, and hypothesize that the hydrolytic activity might also function as a pretreatment for the host protist to effectively decompose the crystalline cellulose components. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Interwell Connectivity Evaluation Using Injection and Production Fluctuation Data

    Science.gov (United States)

    Shang, Barry Zhongqi

    -off between processivity and jamming. Highly processive enzymes cleave a large fraction of a glucan chain during each processive run but are prone to jamming at obstacles. Less processive enzymes avoid jamming but cleave only a small fraction of a chain. Optimizing this trade-off maximizes the cellulose conversion rate. We also elucidate the molecular-scale kinetic origins for synergy among cellulases in enzyme mixtures. In contrast to the currently accepted theory, we show that the ability of an endoglucanase to increase the concentration of chain ends for exoglucanases is insufficient for synergy to occur. Rather, endoglucanases must enhance the rate of complexation between exoglucanases and the newly created chain ends. This enhancement occurs when the endoglucanase is able to partially decrystallize the cellulose surface. We show generally that the driving forces for complexation and jamming, which govern the kinetics of pure exoglucanases, also control the degree of synergy in endo-exo mixtures. In Part II, we focus our attention on a different multiscale problem. This challenge is the development of coarse-grained models from atomistic models to access larger length- and time-scales in a simulation. This problem is difficult because it requires a delicate balance between maintaining (1) physical simplicity in the coarse-grained model and (2) physical consistency with the atomistic model. To achieve these goals, we develop a scheme to coarse-grain an atomistic fluid model into a fluctuating hydrodynamics (FHD) model. The FHD model describes the solvent as a field of fluctuating mass, momentum, and energy densities. The dynamics of the fluid are governed by continuum balance equations and fluctuation-dissipation relations based on the constitutive transport laws. The incorporation of both macroscopic transport and microscopic fluctuation phenomena could provide richer physical insight into the behaviors of biophysical systems driven by hydrodynamic fluctuations, such as

  13. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  14. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

    Science.gov (United States)

    Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

    2016-09-01

    Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. PecS is an important player in the regulatory network governing the coordinated expression of virulence genes during the interaction between Dickeya dadantii 3937 and plants.

    Science.gov (United States)

    Mhedbi-Hajri, Nadia; Malfatti, Pierrette; Pédron, Jacques; Gaubert, Stéphane; Reverchon, Sylvie; Van Gijsegem, Frédérique

    2011-11-01

    Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  16. Interactions between crude drug extracts used in Japanese traditional Kampo medicines and organic anion-transporting polypeptide 2B1.

    Science.gov (United States)

    Iijima, Rie; Watanabe, Tomoki; Ishiuchi, Kan'ichiro; Matsumoto, Takashi; Watanabe, Junko; Makino, Toshiaki

    2018-03-25

    The use of herbal medicines has become popular worldwide, and the information on drug interactions between herbal medicines and chemical drugs is needed. We screened the inhibitory effects of crude drugs used in Kampo medicines used in Japan on organic anion-transporting polypeptide (OATP) 2B1 to predict potential interactions between Kampo medicines and chemical drugs used together. We chose 98 kinds of crude drugs frequently used as ingredients of Kampo formulations in Japan and prepared their boiling water extracts. We then screened their inhibitory effects on OATP2B1 by measuring the uptake of estrone 3-sulphate (E3S) by HEK293 cells stably expressing OATP2B1. At the concentration of 100µg/ml, the extracts prepared from 12 kinds of crude drugs, Scuteralliae Radix, Arecae Semen, Aurantii Fructus Immaturus, Perillae Herba, Panacis Japonici Rhizoma, Moutan Cortex, Polygalae Radix, Rhei Rhizoma, Cannabis Fructus, Chrysanthemi Flos, Eriobotryae Folium, and Querci Cortex, suppressed the function of OATP2B1 by less than 20%. The extract of bofutsushosan, a representative Kampo formulation, inhibited OATP2B1 function with sufficient levels to suppress absorption of OATP2B1 substrates in clinics. We further evaluated the inhibitory effects of several ingredients containing Rhei Rhizoma, Perillae Herba, and Moutan Cortex on OATP2B1. Because of crude drugs used in Kampo medicines might suppress absorption of OATP2B1 substrates, these results may contribute to the safe and effective use of Kampo medicine in clinics. A list of abbreviations: EC, (-)-epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, Epigallocatechin gallate; FBS, fetal bovine serum; grapefruit juice; HEK293, Human embryonic kidney; IC 50, The half inhibitory concentration; OATP, organic anion-transporting polypeptide; β-PGG, penta-O-galloyl-β-D-glucose; t.i.d, 3 times a day. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

    Science.gov (United States)

    Rajulapati, Vikky; Goyal, Arun

    2017-05-01

    Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca 2+ or Mg 2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca 2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.

  18. Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD

    Directory of Open Access Journals (Sweden)

    Moon Yuseok

    2009-01-01

    Full Text Available Abstract Background ATP binding cassette (ABC transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA, lipase ABC transporter domains (LARDs were designed for the secretion of fusion proteins. Results The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD to three types of proteins; green fluorescent protein (GFP, epidermal growth factor (EGF and cytoplasmic transduction peptide (CTP. These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant. Conclusion The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.

  19. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

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    Takuji Oyama

    Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  20. The Penicillium echinulatum secretome on sugar cane bagasse.

    Directory of Open Access Journals (Sweden)

    Daniela A Ribeiro

    Full Text Available Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases. Glycoside hydrolase (GH family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.

  1. Modeling the minimum enzymatic requirements for optimal cellulose conversion

    International Nuclear Information System (INIS)

    Den Haan, R; Van Zyl, W H; Van Zyl, J M; Harms, T M

    2013-01-01

    Hydrolysis of cellulose is achieved by the synergistic action of endoglucanases, exoglucanases and β-glucosidases. Most cellulolytic microorganisms produce a varied array of these enzymes and the relative roles of the components are not easily defined or quantified. In this study we have used partially purified cellulases produced heterologously in the yeast Saccharomyces cerevisiae to increase our understanding of the roles of some of these components. CBH1 (Cel7), CBH2 (Cel6) and EG2 (Cel5) were separately produced in recombinant yeast strains, allowing their isolation free of any contaminating cellulolytic activity. Binary and ternary mixtures of the enzymes at loadings ranging between 3 and 100 mg g −1 Avicel allowed us to illustrate the relative roles of the enzymes and their levels of synergy. A mathematical model was created to simulate the interactions of these enzymes on crystalline cellulose, under both isolated and synergistic conditions. Laboratory results from the various mixtures at a range of loadings of recombinant enzymes allowed refinement of the mathematical model. The model can further be used to predict the optimal synergistic mixes of the enzymes. This information can subsequently be applied to help to determine the minimum protein requirement for complete hydrolysis of cellulose. Such knowledge will be greatly informative for the design of better enzymatic cocktails or processing organisms for the conversion of cellulosic biomass to commodity products. (letter)

  2. Biomass sorghum as a novel substrate in solid-state fermentation for the production of hemicellulases and cellulases by Aspergillus niger and A. fumigatus.

    Science.gov (United States)

    Dias, L M; Dos Santos, B V; Albuquerque, C J B; Baeta, B E L; Pasquini, D; Baffi, M A

    2018-03-01

    We investigated the role of carbon and nitrogen sources in the production of cellulase and hemicellulase by Aspergillus strains. The strains Aspergillus niger SCBM1 and Aspergillus fumigatus SCBM6 were cultivated under solid-state fermentation (SSF), with biomass sorghum (BS) and wheat bran (WB) as lignocellulosic substrates, in different proportions, along with variable nitrogen sources. The best SSF condition for the induction of such enzymes was observed employing A. niger SCBM1 in BS supplemented with peptone; maximum production levels were achieved as follows: 72 h of fermentation for xylanase and exoglucanase (300·07 and 30·64 U g -1 respectively), 120 h for β-glucosidase and endoglucanase (54·90 and 41·47 U g -1 respectively) and 144 h for β-xylosidase (64·88 U g -1 ). This work demonstrated the viability of the use of BS for the production of hemi- and cellulolytic enzymes; the high concentration of celluloses in BS could be associated with the significant production of cellulases, mainly exoglucanase. This is the first study which presents the promising use of biomass sorghum (genetically modified sorghum to increase its biomass content) as an alternative carbon source for the production of enzymes by SSF. © 2017 The Society for Applied Microbiology.

  3. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Science.gov (United States)

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  4. Optimization of sodium hydroxide pretreatment and enzyme loading for efficient hydrolysis of rice straw to improve succinate production by metabolically engineered Escherichia coli KJ122 under simultaneous saccharification and fermentation.

    Science.gov (United States)

    Sawisit, Apichai; Jampatesh, Surawee; Jantama, Sirima Suvarnakuta; Jantama, Kaemwich

    2018-07-01

    Rice straw was pretreated with sodium hydroxide (NaOH) before subsequent use for succinate production by Escherichia coli KJ122 under simultaneous saccharification and fermentation (SSF). The NaOH pretreated rice straw was significantly enhanced lignin removal up to 95%. With the optimized enzyme loading of 4% cellulase complex + 0.5% xylanase (endo-glucanase 67 CMC-U/g, β-glucosidase 26 pNG-U/g and xylanase 18 CMC-U/g dry biomass), total sugar conversion reached 91.7 ± 0.8% (w/w). The physicochemical analysis of NaOH pretreated rice straw indicated dramatical changes in its structure, thereby favoring enzymatic saccharification. In batch SSF, succinate production of 69.8 ± 0.3 g/L with yield and productivity of 0.84 g/g pretreated rice straw and 0.76 ± 0.02 g/L/h, respectively, was obtained. Fed-batch SSF significantly improved succinate concentration and productivity to 103.1 ± 0.4 g/L and 1.37 ± 0.07 g/L/h with a comparable yield. The results demonstrated a feasibility of sequential saccharification and fermentation of rice straw as a promising process for succinate production in industrial scale. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Isolation of fungi from dung of wild herbivores for application in bioethanol production.

    Science.gov (United States)

    Makhuvele, Rhulani; Ncube, Ignatious; Jansen van Rensburg, Elbert Lukas; La Grange, Daniël Coenrad

    Producing biofuels such as ethanol from non-food plant material has the potential to meet transportation fuel requirements in many African countries without impacting directly on food security. The current shortcomings in biomass processing are inefficient fermentation of plant sugars, such as xylose, especially at high temperatures, lack of fermenting microbes that are able to resist inhibitors associated with pre-treated plant material and lack of effective lignocellulolytic enzymes for complete hydrolysis of plant polysaccharides. Due to the presence of residual partially degraded lignocellulose in the gut, the dung of herbivores can be considered as a natural source of pre-treated lignocellulose. A total of 101 fungi were isolated (36 yeast and 65 mould isolates). Six yeast isolates produced ethanol during growth on xylose while three were able to grow at 42°C. This is a desirable growth temperature as it is closer to that which is used during the cellulose hydrolysis process. From the yeast isolates, six isolates were able to tolerate 2g/L acetic acid and one tolerated 2g/L furfural in the growth media. These inhibitors are normally generated during the pre-treatment step. When grown on pre-treated thatch grass, Aspergillus species were dominant in secretion of endo-glucanase, xylanase and mannanase. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  6. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

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    Shazia Rehman

    2014-12-01

    Full Text Available Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE, in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase using a novel substrate, Banana Peels (BP for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  7. Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol.

    Science.gov (United States)

    Lee, Cho-Ryong; Sung, Bong Hyun; Lim, Kwang-Mook; Kim, Mi-Jin; Sohn, Min Jeong; Bae, Jung-Hoon; Sohn, Jung-Hoon

    2017-06-30

    To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6-2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

  8. Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia

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    W. P. Lokapirnasari

    2015-03-01

    Full Text Available Aim: This study aims to produce and assay cellulolytic enzyme activity (endo-(1,4-β-D-glucanase, exo-(1,4-β-Dglucanase, and β-glucosidase, at optimum temperature and optimum pH of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. Materials and Methods: To produce enzyme from a single colony of E. cloacae WPL 214, 98 × 1010 CFU/ml of isolates was put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C in accordance with growth time and optimum temperature of E. cloacae WPL 214. Further on, culture was centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulose enzyme activity was withdrawn to assay endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase, and β-glucosidase. Results: Cellulase enzyme of E. cloacae WPL 214 isolates had endoglucanase activity of 0.09 U/ml, exoglucanase of 0.13 U/ml, and cellobiase of 0.10 U/ml at optimum temperature 35°C and optimum pH 5. Conclusion: E. cloacae WPL 214 isolated from bovine rumen fluid waste produced cellulose enzyme with activity as cellulolytic enzyme of endo-(1,4-β-D-glucanase, exo-(1,4-β-D-glucanase and β-glucosidase.

  9. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    Science.gov (United States)

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

  10. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels.

    Science.gov (United States)

    Rehman, Shazia; Aslam, Hina; Ahmad, Aqeel; Khan, Shakeel Ahmed; Sohail, Muhammad

    2014-01-01

    Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  11. A Novel Multifunctional β-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove

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    Fábio Lino Soares

    2017-07-01

    Full Text Available The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a β-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a β-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-β-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-β-d-galactopyranoside, and 4-Nitrophenyl β-d-glucopyranoside, presenting β-N-acetylglucosaminidase, β-glucosidase, and β-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel β-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

  12. Ralstonia solanacearum biovar 1 associated with a new outbreak of potato brown rot in Portugal

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    Leonor Cruz

    2008-10-01

    Full Text Available In May 2007, potato plants exhibiting symptoms possibly of brown rot were collected in some potato fields in the Baixo Mondego region (Center, Portugal, as a part of a nationwide programme to monitor Ralstonia solanacearum. All laboratory procedures laid down in Commission Directive 2006/63/EC, including dilution plating on semi-selective medium SMSA, indirect imunofluorescence (IIF, polymerase chain reaction (PCR using specific primers and bioassays on tomato plants, were strictly followed and the causal agent of the disease was identified as Ralstonia solanacearum. The identity of the pure cultures of the isolated organism was confirmed by PCR, IIF and pathogenicity tests on several other plant species (eggplant, tobacco, pelargonium and eucalyptus. In biovar determination, the failure of the isolates to utilise/oxidise certain carbon sources indicated that the isolates were all biovar 1. This biovar has a broader host range than biovar 2 strains, and affects several crops of economic importance including ornamental plants and forest trees. Comparative analysis of 16S rRNA and endoglucanase (egl gene sequences of these isolates with sequences that have been deposited at the GenBank revealed a similarity higher than 99% for several Ralstonia solanacearum isolates from biovar 1, including isolate DAR 64836 (Accession number DQ011551. This is the first report of Ralstonia solanacearum biovar 1 in Portugal. All control measures specified in the Commission Directive are being implemented.

  13. Comparative characterization of proteins secreted by Neurospora sitophila in solid-state and submerged fermentation.

    Science.gov (United States)

    Li, Yanjun; Peng, Xiaowei; Chen, Hongzhang

    2013-10-01

    Although submerged fermentation (SmF) accounts for most of current enzyme industries, it has been reported that solid-state fermentation (SSF) can produce higher enzyme yields in laboratory scale. In order to understand the reasons contributing to high enzyme production in SSF, this study compared the cellulase activities and secretomes of Neurospora sitophila cultured in SSF and SmF using steam exploded wheat straw as carbon source and enzyme inducer. The total amounts of protein and biomass (glucosamine content) in SSF were respectively 30 and 2.8 times of those in SmF. The CMCase, FPA and β-glucoside activities in SSF were 53-181 times of those in SmF. Both in SSF and SmF, N. sitophila secreted the most critical cellulases and hemicellulases known for Trichoderma reesei, although a β-xylosidase was exclusively identified in SSF. Six endoglucanases were identified in N. sitophila secretion with the high CMCase activity. The non-enzyme proteins in SSF were involved in fungal mycelia growth and conidiation; while those in SmF were more related to glycometabolism and stress tolerance. This revealed that SSF more likely serves as a natural habitat for filamentous fungi to facilitate the enzyme secretion. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Extremely thermophilic microorganisms for biomass conversion: status and prospects.

    Science.gov (United States)

    Blumer-Schuette, Sara E; Kataeva, Irina; Westpheling, Janet; Adams, Michael Ww; Kelly, Robert M

    2008-06-01

    Many microorganisms that grow at elevated temperatures are able to utilize a variety of carbohydrates pertinent to the conversion of lignocellulosic biomass to bioenergy. The range of substrates utilized depends on growth temperature optimum and biotope. Hyperthermophilic marine archaea (T(opt)>or=80 degrees C) utilize alpha- and beta-linked glucans, such as starch, barley glucan, laminarin, and chitin, while hyperthermophilic marine bacteria (T(opt)>or=80 degrees C) utilize the same glucans as well as hemicellulose, such as xylans and mannans. However, none of these organisms are able to efficiently utilize crystalline cellulose. Among the thermophiles, this ability is limited to a few terrestrial bacteria with upper temperature limits for growth near 75 degrees C. Deconstruction of crystalline cellulose by these extreme thermophiles is achieved by 'free' primary cellulases, which are distinct from those typically associated with large multi-enzyme complexes known as cellulosomes. These primary cellulases also differ from the endoglucanases (referred to here as 'secondary cellulases') reported from marine hyperthermophiles that show only weak activity toward cellulose. Many extremely thermophilic enzymes implicated in the deconstruction of lignocellulose can be identified in genome sequences, and many more promising biocatalysts probably remain annotated as 'hypothetical proteins'. Characterization of these enzymes will require intensive effort but is likely to generate new opportunities for the use of renewable resources as biofuels.

  15. Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity.

    Science.gov (United States)

    Reynolds, Hannah T; Barton, Hazel A

    2014-01-01

    White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans' saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth.

  16. Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity.

    Directory of Open Access Journals (Sweden)

    Hannah T Reynolds

    Full Text Available White-nose Syndrome (WNS is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans, is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus, which may indicate a shift in selective pressure to the detriment of P. destructans' saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth.

  17. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

    Science.gov (United States)

    Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

    2010-08-06

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

  18. Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

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    Phillip B Pope

    Full Text Available Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus. Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1. Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5 but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

  19. Fermentation characteristics in hay from Cynodon and crop stubble treated with exogenous enzymes

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    Yânez André Gomes Santana

    Full Text Available ABSTRACT The effect of treatment with xylanase and β-glucanase was evaluated for gas production and the ruminal degradation of nutrients from the hay of Tifton 85 grass and the stubble of maize, sorghum, peanut, sunflower and sesame crops. Two commercial fibrolytic enzymes were used (Dyadic xylanase PLUS - Xylanase; BrewZyme LP-β-glucanase, added to the hay at doses of 7.5 units of endoglucanase and 0.46 units of xylanase per 500 mg/gDM, for the cellulase and xylanase products respectively. The chemical composition of the hay was determined for no enzyme application and 24 hours after enzyme treatment, and the in vitro gas production and in situ microbial degradation was estimated for dry matter, organic matter, neutral detergent fibre and truly-degradable organic matter after 24 hours of incubation in the rumen. Enzyme treatment of the hay from Tifton 85 grass and the stubble of maize, sorghum, sunflower, peanut and sesame crops with the exogenous fibrolytic enzymes β-glucanase and xylanase influences in vitro gas production, and the in situ degradation of dry matter, organic matter, neutral detergent fibre and truly-degradable organic matter in the rumen. This variation can be attributed to differences in the chemical composition of the hay from the grass and the crop stubble, and to the different ways the enzymes act upon the cell wall.

  20. Biochemical conversions of lignocellulosic biomass for sustainable fuel-ethanol production in the upper Midwest

    Science.gov (United States)

    Brodeur-Campbell, Michael J.

    species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

  1. Fermentation of Foc TR4-infected bananas and Trichoderma spp.

    Science.gov (United States)

    Yang, J; Li, B; Liu, S W; Biswas, M K; Liu, S; Wei, Y R; Zuo, C W; Deng, G M; Kuang, R B; Hu, C H; Yi, G J; Li, C Y

    2016-10-17

    Fusarium wilt (also known as Panama disease) is one of the most destructive banana diseases, and greatly hampers the global production of bananas. Consequently, it has been very detrimental to the Chinese banana industry. An infected plant is one of the major causes of the spread of Fusarium wilt to nearby regions. It is essential to develop an efficient and environmentally sustainable disease control method to restrict the spread of Fusarium wilt. We isolated Trichoderma spp from the rhizosphere soil, roots, and pseudostems of banana plants that showed Fusarium wilt symptoms in the infected areas. Their cellulase activities were measured by endoglucanase activity, β-glucosidase activity, and filter paper activity assays. Safety analyses of the Trichoderma isolates were conducted by inoculating them into banana plantlets. The antagonistic effects of the Trichoderma spp on the Fusarium pathogen Foc tropical Race 4 (Foc TR4) were tested by the dual culture technique. Four isolates that had high cellulase activity, no observable pathogenicity to banana plants, and high antagonistic capability were identified. The isolates were used to biodegrade diseased banana plants infected with GFP-tagged Foc TR4, and the compost was tested for biological control of the infectious agent; the results showed that the fermentation suppressed the incidence of wilt and killed the pathogen. This study indicates that Trichoderma isolates have the potential to eliminate the transmission of Foc TR4, and may be developed into an environmentally sustainable treatment for controlling Fusarium wilt in banana plants.

  2. Studies on the enzymology of cellulose degradation by the anaerobic bacterium Clostridium thermocellum and the anaerobic fungus Neocallimastix frontalis

    Energy Technology Data Exchange (ETDEWEB)

    Bhat, K.M.; Gow, L.A.; Wilson, C.A.; Wood, T.W. (Rowett Research Inst., Aberdeen (UK))

    1990-01-01

    The extracellular cellulases from the anaerobic bacterium Clostridium thermocellum and the anaerobic rumen fungus Neocallimastix frontalis are very active on crystalline cellulose. In both cases the activity resides in a high molecular weight complex. The complex from C. thermocellum (termed the cellulosome) was found to be readily dissociated at pH 5.0 and at room temperature by a mixture of SDS, EDTA and DTT. Virtually all the activity of the unfractionated cellulosome was recovered when the dissociated enzyme components were reassociated by dialysis. Thus, the route is now established for the first time for a meaningful study of the mechanism of cellulase action of this commercially important enzyme system. Nearly all of the activity to crystalline cellulose shown by the cellulase of N. frontalis was associated with a fraction which comprised only 2% of the extracellular protein, 3% of the endoglucanase and 3% of the {beta}-glucosidase. This fraction, which could be isolated by affinity chromatography on cellulose, was produced in greater quantity when the fungus was grown in co-culture with the methanogen, Methanobrevibacter smithii. The specific activity of the partially purified enzyme for degradation of crystalline cellulose was several-fold greater than that produced by the aerobic fungus T. reesei, which is being developed world-wide for its commercial potential for converting cellulose to fermentable soluble sugars. The cellulase of N. frontalis clearly has great commercial potential. 39 refs., 19 figs., 22 tabs.

  3. Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Cosgrove, Daniel J.

    2015-11-25

    The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.

  4. Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia.

    Science.gov (United States)

    Avellaneda-Torres, Lizeth Manuela; Pulido, Claudia Patricia Guevara; Rojas, Esperanza Torres

    2014-01-01

    A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

  5. Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia

    Directory of Open Access Journals (Sweden)

    Lizeth Manuela Avellaneda-Torres

    2014-12-01

    Full Text Available A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP, Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS of ribosomal DNA for fungi. Multivariate statistical analysis (MVA was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

  6. Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

    Science.gov (United States)

    Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

    2015-01-01

    Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

    Energy Technology Data Exchange (ETDEWEB)

    Khadempour, Lily [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Zoology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA; Burnum-Johnson, Kristin E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Baker, Erin S. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Nicora, Carrie D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Webb-Robertson, Bobbie-Jo M. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; White, Richard A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Huang, Eric L. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Currie, Cameron R. [Department of Bacteriology, University of Wisconsin-Madison, Madison WI 53706 USA; Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison WI 53706 USA

    2016-10-26

    Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles were significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.

  8. 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.

  9. Cellulase activity of a thermophilic Aspergillus fumigatus (fresenius) strain

    Energy Technology Data Exchange (ETDEWEB)

    Vandamme, E J; Logghe, J M; Geeraerts, A M

    1982-10-01

    A thermophilic fungus, isolated from horse manure on Whatman-cellulose CF-11 as sole carbon source was identified as Aspergillus fumigatus. It grew optimally at 45 degrees C and displayed highest cellulase activity at 55 degrees C and pH 5.0 towards a range of soluble and crude insolulble cellulosic substrates. Germination and outgrowth of the spore inoculum in carboxymethylcellulose (CMCellulose) medium was accompanied by high endoglucanase (E.C. 3.2.1.4) activity. The cellulose complex e.g. exo-beta-1,4-glucanase (E.C.3.2.1.-); endo-beta-1,4glucanase (E.C.3.2.1.4.) and beta-glucosidase (E.C.3.2.1.21.) displayed quite different properties depending on whether it was formed on low or highly substituted CMCellulose. Extracellular cellulase formation followed biomass accumulation. Upon prolonged incubation cell lysis occurred which resulted in a further increase in cellulase activity. Ball-milled crude cellulosics, such as newsprint, de-inked newsprint, glossy writing paper, toilet paper and beech sawdust were substantially solubilized with 110 hours of growth. The type of crude cellulosic material greatly influenced the pattern of enzyme production. The enzyme complex formed when A. fumigatus was grown on soluble CMC-4M6F, displayed a different activity spectrum towards crude cellulosics compared with that formed on ball-milled glossy writing paper. The extracellular cellulase of A. fumigatus looks promising for a rapid and substantial solubilisation and saccharification of crude cellulosics. (Refs. 32).

  10. Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.

    Science.gov (United States)

    Fitzpatrick, J; Kricka, W; James, T C; Bond, U

    2014-07-01

    To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol. cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases. We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes. The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ. © 2014 The Society for Applied Microbiology.

  11. Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases.

    Science.gov (United States)

    McGregor, Nicholas; Yin, Victor; Tung, Ching-Chieh; Van Petegem, Filip; Brumer, Harry

    2017-02-01

    The xyloglucan endotransglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodeling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endoglycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endoglucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endotranglycosylase (XET), and a xyloglucan endohydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  12. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum

    Science.gov (United States)

    The phytopathogen Ralstonia solanacearum is a species complex that contains a subset of strains that are quarantined or select agent pathogens. An unidentified R. solanacearum strain is considered a select agent in the US until proven otherwise, which can be done by phylogenetic analysis of a partia...

  13. Intake, digestibility, and nitrogen balance of rations containing different levels of murumuru meal in sheep diets

    Directory of Open Access Journals (Sweden)

    Bruno Peres de Menezes

    2016-02-01

    Full Text Available This study aimed to assess the potential use of increasing levels of murumuru cake Astrocaryum murumuru var. murumuru, M art. (MC in sheep diets as a replacement for Mombasa grass (Panicum maximum Jacq. Metabolic tests were performed with 20 castrated male sheep at Embrapa Amazônia Oriental, Belém, Pará, during 26 days. The experiment was arranged in a completely randomized design, with five diets and four replications. MC0: 100% grass; MC10: 10% MC and 90% grass; MC20: 20% MC and 80% grass; MC40: 40% MC and 60% grass; and MC60: 60% MC and 40% grass. The intake and the coefficient of apparent digestibility of dry matter (DMI and CDDM, organic matter (OMI and CDOM, crude protein (CPI and CDCP, neutral detergent fiber (NDFI and CDNDF, acid detergent fiber (ADFI and CDADF, ether extract (EEI and CDEE, cellulose (CELI and CDCEL, hemicellulose (HEMI and CDHEM, and nitrogen balance (NB of experimental diets were determined. The intake of mineral material (MMI and lignin (LIGI were also determined. The DMI, OMI, MMI, CPI, NDFI, and ADFI displayed a decreasing linear effect with the replacement of Mombasa grass by MC in the diet. The EEI and the LIGI presented quadratic effects according to the replacement levels of MC in the diet. The CDDM, CDOM, and CDHEM displayed increasing linear effects, between MC0 and MC60. The CDEE, CDNDF, CDADF, and CDCEL displayed a quadratic effect, with optimum replacement levels of 56.65%, 41%, 31.33%, and 27.46%, respectively. The nitrogen balance presented a negative linear effect in the range of 0% to 60% of murumuru cake. One can conclude that murumuru cake is an alternative to the dietary supplementation of ruminants in replacing the Mombasa grass because it provides an increase in the digestibility of nutrients for sheep. However, a limited replacement level must be implemented, considering that from 27.46%, 31.33%, 41%, and 56.65% replacement, a decline occurs in the CDCEL, CDADF, CDNDF, and CDEE, respectively

  14. Ventilation system in the RA reactor building - design specifications; Sistem ventilacije u objektu 'RA' - Tehnicki opis

    Energy Technology Data Exchange (ETDEWEB)

    Badrljica, R [Institute of Nuclear Sciences Boris Kidric, Vinca, Beograd (Serbia and Montenegro)

    1984-09-15

    Protective role of the ventilation system of nuclear facilities involve construction of ventilation barriers which prevent release of radioactive particulates or gases, elimination od radioactive particulates and gases from the air which is released from contaminated zones into the reactor environment. Ventilation barriers are created by dividing the building into a number of ventilation zones with different sub pressure compared to the atmospheric pressure. The RA reactor building is divided into four ventilation zones. First zone is the zone of highest risk. It includes reactor core with horizontal experimental channels, underground rooms of the primary coolant system (D{sub 2}O), helium system, hot cells and the space above the the reactor core. Second zone is the reactor hall and the room for irradiated fuel storage. The third zone includes corridors in the basement, ground floor and first floor where the probability of contamination is small. The fourth zone includes the annex where the contamination risk is low. There is no have natural air circulation in the reactor building. Ventilators for air input and outlet maintain the sub pressure in the building (pressure lower than the atmospheric pressure). This prevents release of radioactivity into the atmosphere. Zastitne uloge ventilacionog sistema kod nuklearnih postrojenja obuhvataju formiranje ventilacionih barijera koje onemugucavaju sirenje radioaktivnih cestica ili gasova putem cirkulacije vazduha; eliminaciju radioaktivnih cestica i gasova iz vazduha koji se evakuise iz kontaminiranih prostora u okolinu reaktorskog postrojenja. Formiranje zastitnih ventilacionih barijera ostvaruje se obicno podelom unutrasnjosti objekta na vise ventilacionih zona razlicitih podpritisaka u odnosu na spoljni atmosferski pritisak. Celi prostor zgrade reaktora RA podeljen je u cetiri ventilacione zone. Prva zona je zona najveceg rizika, u koju spadaju reaktorsko jezgro sa horizontalnim eksperimentalnim kanalima, tehnoloske

  15. The structure and significance of enterobacterial common antigen (ECA

    Directory of Open Access Journals (Sweden)

    Tomasz Kasper Goździewicz

    2015-09-01

    Full Text Available The enterobacterial common antigen (ECA is a carbohydrate-derived cell surface antigen present in all Gram-negative bacteria belonging to Enterobacteriaceae family. Biosynthetic pathways shared by ECA and LPS (endotoxin suggest close connections between these antigens. ECA occurs in three different forms: a phosphatidyl-linked linear polysaccharide anchored on the cell surface (ECAPG, a cyclic form built of 4-6 repeating units localized in the periplasm (ECACYC and as a linear polysaccharide covalently linked to LPS core oligosaccharide (ECALPS. Regardless of ECA form, poly- and oligosaccharides of ECA consist of the biological trisaccharide repeating units: →3-α-d-Fucp4NAc-(1→4-β-d-ManpNAcA-(1→4-α-d-GlcpNAc-(1→, where Fucp4NAc refers to 4-acetamido-2,4-dideoxygalactose, ManpNAcA to N-acetyl-mannosaminuronic acid and GlcpNAc to N-acetylglucosamine. ECAPG and ECALPS consisting of one unit with Fucp4NAc as a terminal sugar were also identified. The number of the studies shows its occurrence in all members of enteric bacteria with a few exceptions such as Erwinia chrysanthemi. The presence of ECA was also shown for such genera as Plesiomonas [4] and Yersinia [36], previously belonging to the Vibrionaceae and Pasteurellaceae families, respectively. It was one of the reasons to include these two taxa in the Enterobacteriaceae family. The function of ECA is not fully understood, but it was reported that its occurrence is important in resistance of bacterial cells to environmental conditions, such as bile salts in the human digestive tract. The immunogenicity of ECA seems very interesting in the fact that only sparse rough Gram-negative strains, such as Shigella sonnei phase II, Escherichia coli R1, R2, R4, K-12, and Yersinia enterocolitica O:3 are able to induce the production of specific anti-ECA antibodies. It is the effect of the ECALPS, and the evidence for the existence of such covalent linkage was provided by structural analysis of S

  16. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    Science.gov (United States)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  17. Molecular determinants of sporulation in Ashbya gossypii.

    Science.gov (United States)

    Wasserstrom, Lisa; Lengeler, Klaus B; Walther, Andrea; Wendland, Jürgen

    2013-09-01

    Regulation of development and entry into sporulation is critical for fungi to ensure survival of unfavorable environmental conditions. Here we present an analysis of gene sets regulating sporulation in the homothallic ascomycete Ashbya gossypii. Deletion of components of the conserved pheromone/starvation MAP kinase cascades, e.g., STE11 and STE7, results in increased sporulation. In kar3 mutants sporulation is severely reduced, while deletion of KAR4 as well as of homologs of central Saccharomyces cerevisiae regulators of sporulation, IME1, IME2, IME4, and NDT80, abolishes sporulation in A. gossypii. Comparison of RNAseq transcript profiles of sporulation-deficient mutants identified a set of 67 down-regulated genes, most of which were up-regulated in the oversporulating ste12 mutant. One of these differentially expressed genes is an endoglucanase encoded by ENG2. We found that Eng2p promotes hyphal fragmentation as part of the developmental program of sporulation, which generates single-celled sporangia. Sporulation-deficient strains are arrested in their development but form sporangia. Supply of new nutrients enabled sporangia to return to hyphal growth, indicating that these cells are not locked in meiosis. Double-strand break (DSB) formation by Spo11 is apparently not required for sporulation; however, the absence of DMC1, which repairs DSBs in S. cerevisiae, results in very poor sporulation in A. gossypii. We present a comprehensive analysis of the gene repertoire governing sporulation in A. gossypii and suggest an altered regulation of IME1 expression compared to S. cerevisiae.

  18. Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode.

    Science.gov (United States)

    Matthews, Benjamin F; Beard, Hunter; MacDonald, Margaret H; Kabir, Sara; Youssef, Reham M; Hosseini, Parsa; Brewer, Eric

    2013-05-01

    During pathogen attack, the host plant induces genes to ward off the pathogen while the pathogen often produces effector proteins to increase susceptibility of the host. Gene expression studies of syncytia formed in soybean root by soybean cyst nematode (Heterodera glycines) identified many genes altered in expression in resistant and susceptible roots. However, it is difficult to assess the role and impact of these genes on resistance using gene expression patterns alone. We selected 100 soybean genes from published microarray studies and individually overexpressed them in soybean roots to determine their impact on cyst nematode development. Nine genes reduced the number of mature females by more than 50 % when overexpressed, including genes encoding ascorbate peroxidase, β-1,4-endoglucanase, short chain dehydrogenase, lipase, DREPP membrane protein, calmodulin, and three proteins of unknown function. One gene encoding a serine hydroxymethyltransferase decreased the number of mature cyst nematode females by 45 % and is located at the Rhg4 locus. Four genes increased the number of mature cyst nematode females by more than 200 %, while thirteen others increased the number of mature cyst nematode females by more than 150 %. Our data support a role for auxin and ethylene in susceptibility of soybean to cyst nematodes. These studies highlight the contrasting gene sets induced by host and nematode during infection and provide new insights into the interactions between host and pathogen at the molecular level. Overexpression of some of these genes result in a greater decrease in the number of cysts formed than recognized soybean cyst nematode resistance loci.

  19. A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

    Science.gov (United States)

    Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

    1997-04-29

    Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

  20. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    Directory of Open Access Journals (Sweden)

    Chang Jui-Jen

    2012-07-01

    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  1. Adhesive properties of a symbolic bacterium from a wood-boreing marine shipworm

    International Nuclear Information System (INIS)

    Imam, S.H.; Greene, R.V.; Griffin, H.L.

    1990-01-01

    Adhesive properties of cellulolytic, nitrogen-fixing bacterium isolated from a marine shipworm are described. 35 S-labeled cells of the shipworm bacterium bound preferentially Whatman no.1 cellulose filter paper, compared with its binding to other cellulose substrata or substrata lacking cellulose. The ability of the bacteria to bind to Whatman no. 1 filter paper was significantly reduced by glutaraldehyde or heat treatment of cells. Pretreatment of cells with azide, valinomycin, gramicidin-D, bis-hexafluoroacetylacetone (1799), or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone inhibited adhesion activity. Cells pretreated with pronase or trypsin also exhibited reduced binding activity, but chymotrypsin and peptidase had no effect on adhesion activity. Cellodextrins and methyl cellulose 15 inhibited the adhesion of the shipworm bacteria to filter paper, whereas glucose, cellobiose, and soluble carboxymethyl cellulose had no significant effect. The divalent cation chelators EDTA and EGTA [ethylene hlycol-bis(β-aminoethyl ether)-N,N,N'N'-tetraacetic acid] had little or no effect on adhesive properties of shipworm bacteria. Also, preabsorbing the substratum with extracellular endoglucanase isolated from the ship worm bacterium or 1% bovine serum albumin had no apparent effect on bacterial binding. Low concentration (0.01%) of sodium dodecyl sulfate solubilized a fraction from whole cells, which appeared to be involved in cellular binding activity. After removal of sodium dodecyl, sulfate, several proteins in this fraction associated with intact cells. These cells exhibited up to 50% enhanced binding to filter paper in comparison to cells which had not been exposed to the sodium dodecyl sulfate-solubilized fraction

  2. Cellulolytic and xylanolytic enzymes from thermophilic Aspergillus terreus RWY.

    Science.gov (United States)

    Sharma, Reetika; Kocher, Gurvinder Singh; Bhogal, Ravinder Singh; Oberoi, Harinder Singh

    2014-12-01

    Thermophilic Aspergillus terreus RWY produced cellulases and xylanases in optimal concentrations at 45 °C in solid state fermentation process, though enzyme production was also observed at 50 and 55 °C. Filter paper cellulase (FP), endoglucanase (EG), β-glucosidase (BGL), cellobiohydrolase (CBH), xylanase, β-xylosidase, α-L-arabinofuranosidase and xylan esterase activities for A. terreus RWY at 45 °C in 72 h were 11.3 ± 0.65, 103 ± 6.4, 122.5 ± 8.7, 10.3 ± 0.66, 872 ± 22.5, 22.1 ± 0.75, 126.4 ± 8.4 and 907 ± 15.5 U (g-ds)(-1) , respectively. Enzyme was optimally active at temperatures and pH ranging between 50-60 °C and 4.0-6.0, respectively. The half life (T1/2 ) of 270 and 240 min at 70 and 75 °C, respectively for the enzyme indicates its stability at higher temperatures. The addition of MnCl2 , CoCl2 , and FeCl3 significantly enhanced cellulase activity. Enzyme demonstrated multiplicity by having seven, one and three isoform(s) for EG, CBH and BGL, respectively. Significant production of functionally active consortium of cellulolytic and xylanolytic enzymes from A. terreus RWY makes it a potential candidate in bioprocessing applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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.

  4. Differential gene expression in response to juvenile hormone analog treatment in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Archotermopsidae).

    Science.gov (United States)

    Cornette, Richard; Hayashi, Yoshinobu; Koshikawa, Shigeyuki; Miura, Toru

    2013-04-01

    Termite societies are characterized by a highly organized division of labor among conspicuous castes, groups of individuals with various morphological specializations. Termite caste differentiation is under control of juvenile hormone (JH), but the molecular mechanism underlying the response to JH and early events triggering caste differentiation are still poorly understood. In order to profile candidate gene expression during early soldier caste differentiation of the damp-wood termite, Hodotermopsis sjostedti, we treated pseudergates (workers) with a juvenile hormone analog (JHA) to induce soldier caste differentiation. We then used Suppressive Subtractive Hybridization to create two cDNA libraries enriched for transcripts that were either up- or downregulated at 24h after treatment. Finally, we used quantitative PCR to confirm temporal expression patterns. Hexamerins represent a large proportion of the genes upregulated following JHA treatment and have an expression pattern that shows roughly an inverse correlation to intrinsic JH titers. This data is consistent with the role of a JH "sink", which was demonstrated for hexamerins in another termite, Reticulitermes flavipes. A putative nuclear protein was also upregulated a few hours after JHA treatment, which suggests a role in the early response to JH and subsequent regulation of transcriptional events associated with soldier caste differentiation. Some digestive enzymes, such as endogenous beta-endoglucanase and chymotrypsin, as well as a protein associated to digestion were identified among genes downregulated after JHA treatment. This suggests that JH may directly influence the pseudergate-specific digestive system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants.

    Science.gov (United States)

    Peña, Maria J; Darvill, Alan G; Eberhard, Stefan; York, William S; O'Neill, Malcolm A

    2008-11-01

    Xyloglucan is a well-characterized hemicellulosic polysaccharide that is present in the cell walls of all seed-bearing plants. The cell walls of avascular and seedless vascular plants are also believed to contain xyloglucan. However, these xyloglucans have not been structurally characterized. This lack of information is an impediment to understanding changes in xyloglucan structure that occurred during land plant evolution. In this study, xyloglucans were isolated from the walls of avascular (liverworts, mosses, and hornworts) and seedless vascular plants (club and spike mosses and ferns and fern allies). Each xyloglucan was fragmented with a xyloglucan-specific endo-glucanase and the resulting oligosaccharides then structurally characterized using NMR spectroscopy, MALDI-TOF and electrospray mass spectrometry, and glycosyl-linkage and glycosyl residue composition analyses. Our data show that xyloglucan is present in the cell walls of all major divisions of land plants and that these xyloglucans have several common structural motifs. However, these polysaccharides are not identical because specific plant groups synthesize xyloglucans with unique structural motifs. For example, the moss Physcomitrella patens and the liverwort Marchantia polymorpha synthesize XXGGG- and XXGG-type xyloglucans, respectively, with sidechains that contain a beta-D-galactosyluronic acid and a branched xylosyl residue. By contrast, hornworts synthesize XXXG-type xyloglucans that are structurally homologous to the xyloglucans synthesized by many seed-bearing and seedless vascular plants. Our results increase our understanding of the evolution, diversity, and function of structural motifs in land-plant xyloglucans and provide support to the proposal that hornworts are sisters to the vascular plants.

  6. Benzoic acid fermentation from starch and cellulose via a plant-like β-oxidation pathway in Streptomyces maritimus

    Directory of Open Access Journals (Sweden)

    Noda Shuhei

    2012-04-01

    Full Text Available Abstract Background Benzoic acid is one of the most useful aromatic compounds. Despite its versatility and simple structure, benzoic acid production using microbes has not been reported previously. Streptomyces are aerobic, Gram-positive, mycelia-forming soil bacteria, and are known to produce various kinds of antibiotics composed of many aromatic residues. S. maritimus possess a complex amino acid modification pathway and can serve as a new platform microbe to produce aromatic building-block compounds. In this study, we carried out benzoate fermentation using S. maritimus. In order to enhance benzoate productivity using cellulose as the carbon source, we constructed endo-glucanase secreting S. maritimus. Results After 4 days of cultivation using glucose, cellobiose, or starch as a carbon source, the maximal level of benzoate reached 257, 337, and 460 mg/l, respectively. S. maritimus expressed β-glucosidase and high amylase-retaining activity compared to those of S. lividans and S. coelicolor. In addition, for effective benzoate production from cellulosic materials, we constructed endo-glucanase-secreting S. maritimus. This transformant efficiently degraded the phosphoric acid swollen cellulose (PASC and then produced 125 mg/l benzoate. Conclusions Wild-type S. maritimus produce benzoate via a plant-like β-oxidation pathway and can assimilate various carbon sources for benzoate production. In order to encourage cellulose degradation and improve benzoate productivity from cellulose, we constructed endo-glucanase-secreting S. maritimus. Using this transformant, we also demonstrated the direct fermentation of benzoate from cellulose. To achieve further benzoate productivity, the L-phenylalanine availability needs to be improved in future.

  7. Hydrolysis of cellulose and oil palm empty fruit bunches by using consortia of fungi isolated from the soil of Colombian high andean forest

    Directory of Open Access Journals (Sweden)

    Luz Aida Moya A

    2012-09-01

    Full Text Available Hydrolytic activity was evaluated in a mixture of supernatants produced by filamentous fungi grown individually on microcrystalline cellulose and empty fruit bunches. The strains that were used correspond to two types of isolates; the first was made from soil samples from a transect of a high andean forest of Colombia, in the Parque Natural Nacional de Los Nevados, where, based on previous studies, we selected the strains B7, B11, B11M and B19. The second isolate was obtained from a pool of oil palm empty fruit bunches (Eleaeis guinensis Jacq. in different states of decomposition on the Unipalma plantation located in the eastern plains; strains TA1 and TA2. To perform the hydrolysis of cellulose and empty fruit bunches, the previously obtained supernatants from each of the selected strains were cultivated for 300 hours in cellulose and characterized individually by endoglucanase, exoglucanase, and β-glucosidase activity. Individual supernatants were mixed at a 1:1 ratio to form consortia; and hydrolytic activity was evaluated in the substrates at two hours. The glucose concentration was determined by the 3,5-dinitrosalicylic acid (DNS method. The results show that hydrolysis of empty fruit bunch to glucose was favored by three pools of supernatants, with increases greater than 400% in comparison with the hydrolysis obtained by individual supernatants, demonstrating the potential to decompose palm empty fruit bunches; thereby contributing to the reduction of decay time of empty fruit bunches and the decrease of environmental and health problems

  8. Measurement and characterization of cellulase activity in sclerophyllous forest litter.

    Science.gov (United States)

    Criquet, Stéven

    2002-07-01

    Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.

  9. Microbial Consortium with High Cellulolytic Activity (MCHCA for enhanced biogas production.

    Directory of Open Access Journals (Sweden)

    Krzysztof ePoszytek

    2016-03-01

    Full Text Available The use of lignocellulosic biomass as a substrate in agricultural biogas plants is very popular and yields good results. However, the efficiency of anaerobic digestion, and thus biogas production, is not always satisfactory due to the slow or incomplete degradation (hydrolysis of plant matter. To enhance the solubilization of the lignocellulosic biomass various physical, chemical and biological pretreatment methods are used.The aim of this study was to select and characterize cellulose-degrading bacteria, and to construct a microbial consortium, dedicated for degradation of maize silage and enhancing biogas production from this substrate.Over one hundred strains of cellulose-degrading bacteria were isolated from: sewage sludge, hydrolyzer from an agricultural biogas plant, cattle slurry and manure. After physiological characterization of the isolates, sixteen strains (representatives of Bacillus, Providencia and Ochrobactrum genera were chosen for the construction of a Microbial Consortium with High Cellulolytic Activity, called MCHCA. The selected strains had a high endoglucanase activity (exceeding 0.21 IU/mL CMCase activity and a wide range of tolerance to various physical and chemical conditions. Lab-scale simulation of biogas production using the selected strains for degradation of maize silage was carried out in a two-bioreactor system, similar to those used in agricultural biogas plants.The obtained results showed that the constructed MCHCA consortium is capable of efficient hydrolysis of maize silage, and increases biogas production by even 38%, depending on the inoculum used for methane fermentation. The results in this work indicate that the mesophilic Microbial Consortium with High Cellulolytic Activity has a great potential for application on industrial scale in agricultural biogas plants.

  10. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  11. Genome, transcriptome, and secretome analysis of wood decay fungus postia placenta supports unique mechanisms of lignocellulose conversion

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Diego [Los Alamos National Laboratory; Challacombe, Jean F [Los Alamos National Laboratory; Misra, Monica [Los Alamos National Laboratory; Xie, Gary [Los Alamos National Laboratory; Brettin, Thomas [Los Alamos National Laboratory; Morgenstern, Ingo [CLARK UNIV; Hibbett, David [CLARK UNIV.; Schmoll, Monika [UNIV WIEN; Kubicek, Christian P [UNIV WIEN; Ferreira, Patricia [CIB, CSIC, MADRID; Ruiz - Duenase, Francisco J [CIB, CSIC, MADRID; Martinez, Angel T [CIB, CSIC, MADRID; Kersten, Phil [FOREST PRODUCTS LAB; Hammel, Kenneth E [FOREST PRODUCTS LAB; Vanden Wymelenberg, Amber [U. WISCONSIN; Gaskell, Jill [FOREST PRODUCTS LAB; Lindquist, Erika [DOE JGI; Sabati, Grzegorz [U. WISCONSIN; Bondurant, Sandra S [U. WISCONSIN; Larrondo, Luis F [U. CATHOLICA DE CHILE; Canessa, Paulo [U. CATHOLICA DE CHILE; Vicunna, Rafael [U. CATHOLICA DE CHILE; Yadavk, Jagiit [U. CINCINATTI; Doddapaneni, Harshavardhan [U. CINCINATTI; Subramaniank, Venkataramanan [U. CINCINATTI; Pisabarro, Antonio G [PUBLIC U. NAVARRE; Lavin, Jose L [PUBLIC U. NAVARRE; Oguiza, Jose A [PUBLIC U. NAVARRE; Master, Emma [U. TORONTO; Henrissat, Bernard [CNRS, MARSEILLE; Coutinho, Pedro M [CNRS, MARSEILLE; Harris, Paul [NOVOZYMES, INC.; Magnuson, Jon K [PNNL; Baker, Scott [PNNL; Bruno, Kenneth [PNNL; Kenealy, William [MASCOMA, INC.; Hoegger, Patrik J [GEORG-AUGUST-U.; Kues, Ursula [GEORG-AUGUST-U; Ramaiva, Preethi [NOVOZYMES, INC.; Lucas, Susan [DOE JGI; Salamov, Asaf [DOE JGI; Shapiro, Harris [DOE JGI; Tuh, Hank [DOE JGI; Chee, Christine L [UNM; Teter, Sarah [NOVOZYMES, INC.; Yaver, Debbie [NOVOZYMES, INC.; James, Tim [MCMASTER U.; Mokrejs, Martin [CHARLES U.; Pospisek, Martin [CHARLES U.; Grigoriev, Igor [DOE JGI; Rokhsar, Dan [DOE JGI; Berka, Randy [NOVOZYMES; Cullen, Dan [FOREST PRODUCTS LAB

    2008-01-01

    Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative {beta}-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC{center_dot}MSIMS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H202. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H202 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons to the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

  12. Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

    Science.gov (United States)

    Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin; Tran, Huu M.; Hsu, Nathan S. Y.; Dibble, Dean; Singh, Seema; Adams, Paul D.; Sapra, Rajat; Hadi, Masood Z.; Simmons, Blake A.; Sale, Kenneth L.

    2013-01-01

    Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding. PMID:24244549

  13. Improved activity of a thermophilic cellulase, Cel5A, from Thermotoga maritima on ionic liquid pretreated switchgrass.

    Directory of Open Access Journals (Sweden)

    Zhiwei Chen

    Full Text Available Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc] pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

  14. A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Seunghyun; Karim, Muhammad Nazmul [Texas Tech Univ., Lubbock, TX (United States). Dept. of Chemical Engineering

    2011-08-15

    In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum - endoglucanase (Cel5A), exoglucanase (Cel9E), and {beta}-glucosidase - on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 {+-} 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 {+-} 0.15%. Ethanol production was 0.30 {+-} 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 {+-} 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum-E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal-yeast systems. (orig.)

  15. Isolation of microbial native Stumps with cellulolytic activity of a compost process

    International Nuclear Information System (INIS)

    Jaramillo G, Marisol; Ruiz V Orlando Simon; Yepes P, Maria del Socorro; Montoya C, Olga Ines

    2003-01-01

    The isolation, selection adaptation and handling of native microorganisms coming from organic waste are an alternative to avoid the accumulation and the lack of the proper use of these undesirable materials. This organic waste is a source for obtaining microbial strains, which are potentially producers of Industrial enzymes and, at the same time, it works as substrate so that these organisms can transform it into compost or organic manure. In this work, 39 native strains of microorganisms with potential cellulolytic activity coming from the organic waste of the urban and rural sector, from the Compost Plant of Marinilla Antioquia) municipality, were isolated, evaluated and purified. The waste was previously selected and then submitted to an aerobic degradation or compost. The microbial strains were isolated in a selective medium with carboxymethyl cellulose (CMC), of the phases mesophile, termophile, cooling and maturation of the compost process. Eighty-two percent (82%)of the obtained colonies were identified, in principle as Bacillus, because of their morphology and their reaction to the Gram coloration. The fungi population was seen only during the cooling phase. Then, the potential cellulolytic activity was evaluated qualitatively in a solid medium with the Congo Red coloration, with which the Beta-endoglucanase activity was evaluated through the formation of clarified zones. Such staining was applied in two mediums with CMC with and without glucose It was observed that 33.3% of the isolated organisms produced the enzyme In both mediums; however, 25.6% of microorganisms did not show the production of this enzyme, and only 15.8% did not require the inducers to produce it

  16. Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

    Science.gov (United States)

    Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

    1996-05-01

    In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

  17. Soft rot erwiniae: from genes to genomes.

    Science.gov (United States)

    Toth, Ian K; Bell, Kenneth S; Holeva, Maria C; Birch, Paul R J

    2003-01-01

    SUMMARY The soft rot erwiniae, Erwinia carotovora ssp. atroseptica (Eca), E. carotovora ssp. carotovora (Ecc) and E. chrysanthemi (Ech) are major bacterial pathogens of potato and other crops world-wide. We currently understand much about how these bacteria attack plants and protect themselves against plant defences. However, the processes underlying the establishment of infection, differences in host range and their ability to survive when not causing disease, largely remain a mystery. This review will focus on our current knowledge of pathogenesis in these organisms and discuss how modern genomic approaches, including complete genome sequencing of Eca and Ech, may open the door to a new understanding of the potential subtlety and complexity of soft rot erwiniae and their interactions with plants. The soft rot erwiniae are members of the Enterobacteriaceae, along with other plant pathogens such as Erwinia amylovora and human pathogens such as Escherichia coli, Salmonella spp. and Yersinia spp. Although the genus name Erwinia is most often used to describe the group, an alternative genus name Pectobacterium was recently proposed for the soft rot species. Ech mainly affects crops and other plants in tropical and subtropical regions and has a wide host range that includes potato and the important model host African violet (Saintpaulia ionantha). Ecc affects crops and other plants in subtropical and temperate regions and has probably the widest host range, which also includes potato. Eca, on the other hand, has a host range limited almost exclusively to potato in temperate regions only. Disease symptoms: Soft rot erwiniae cause general tissue maceration, termed soft rot disease, through the production of plant cell wall degrading enzymes. Environmental factors such as temperature, low oxygen concentration and free water play an essential role in disease development. On potato, and possibly other plants, disease symptoms may differ, e.g. blackleg disease is associated

  18. Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

    Directory of Open Access Journals (Sweden)

    Nadal Anna

    2012-09-01

    Full Text Available Abstract Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER, analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP, had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM

  19. 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.

  20. Filamentous fungi and agro-industrial residues selection for enzyme production of biotechnological interestSeleção de fungos filamentosos e de resíduos agroindustriais para a produção de enzimas de interesse biotecnológico

    Directory of Open Access Journals (Sweden)

    Erivelton César Stroparo

    2012-12-01

    Full Text Available Many fungal enzymes have relevant applications in different industrial areas. The objective of this work was to select fungi producing hydrolytic enzymes, as well as establish agro-industrial wastes capable of inducing higher production levels. Xylanase, endoglucanase, amylase and poligalacturonase activities were determined by incubating the culture filtrates with their respective substrates. Subsequently, the reducing sugars determination was carried out using 3,5-dinitrosalicylic acid reagent.The protein determination was performed according the modified Bradford method. Among the fungal strains evaluated, Aspergillus niger J4 showed higher levels of xylanase production (8.73 ± 0.34 U/ml and this was greatest when brewer’s spent grain was used as substrate (9.80 ± 0.02 U/ml. Penicillium miczynskii produced the highest levels of endoglucanasic activity (0.13 ± 0.03 U/ml, which, in turn, was favored in the pineapple peel presence (0.18 ± 0.02 U/ml. In relation to amylase, A. niger J26 was selected as the best producer strain (6.10 ± 0.30 U/ml with wheat bran as the best substrate for their production (7.32 ± 0.14 U/ml. Penicillium verruculosum exhibited the highest level of poligalacturonase activity (8.65 ± 0.12 U/ml, especially when grown in orange peel presence (10.32 ± 0.10 U/ml. These wastes use in these enzymes production may not only reduce their production cost, but also substantially reduce the environmental impact caused by the deposition of these wastes on the environment.Muitas enzimas produzidas por fungos têm relevantes aplicações em diferentes áreas industriais. O objetivo deste trabalho foi selecionar fungos filamentosos produtores de enzimas hidrolíticas, bem como estabelecer os resíduos agroindustriais capazes de induzir maiores níveis de produção. As atividades xilanásica, endoglucanásica, amilásica e poligalacturonásica foram determinadas incubandose os filtrados de cultura com seus respectivos

  1. Radioactive Waste Control at the United Kingdom Atomic Energy Research Establishment, Harwell; Traitement des Dechets Radioactifs au Centre de Recherche sur l'Energie Atomique de Harwell; 041a 041e 041d 0422 0420 041e 041b 042c 0417 0410 0423 0414 ; Control de Desechos Radiactivos en el A.E.R.E., Harwell

    Energy Technology Data Exchange (ETDEWEB)

    Burns, R. H. [United Kingdom Atomic Energy Authority (United Kingdom)

    1960-07-01

    desechos solidos contaminados son almacenados o bien hundidos en el mar. Es importante reducir su volumen en la medida de lo posible; los metodos empleados para ello consisten en el embalado a presion, la fusion y la incineracion de los materiales combustibles. Pequenas cantidades de radiactividad pasan a la atmosfera traves de chimeneas. La purificacion de este 'aire que sale de las instalaciones se efectua generalmente mediante filtros de alta eficacia o con sistemas de filtracion por rociado. Las chimeneas son objeto de un control metodico, complementado con un amplio programa de muestreo por sectores. (author) [Russian] V dokumente izlagaetsja porjadok obrabotki radioaktivnyh othodov i kontrolja za ih udaleniem, prinjatyj v Harujelle. Zhidkie othody s nizkoj aktivnost'ju obrabatyvajutsja putem primenenija fosfatno-koaguljacionnogo metoda, a zatem otvodjatsja v Temzu. Othody so srednej aktivnost'ju otdeljajutsja i prohodjat himicheskuju obrabotku v dva jetapa, posle chego oni propuskajutsja cherez vermikulitovye kolonki. Jetot metod pozvoljaet jeffektivno udaljat' iz othodov radioaktivnyj cezij, kotoryj ne poddaetsja osazhdeniju. Zhidkie othody s vysokoj aktivnost'ju otvodjatsja v hranilishha. V nastojashhee vremja stroitsja novaja ustanovka, kotoraja pozvolit provodit' himicheskuju obrabotku takih othodov, budet imet' iono-obmenniki i daet vozmozhnost' osushhestvljat' vyparivanie othodov. Himicheskie materialy, ispol'zovannye dlja obrabotki othodov, posle obezvozhivanija i fil'trovanija otvodjatsja v more. Tverdye radioaktivnye othody libo hranjatsja, libo udaljajutsja v more. Ves'ma vazhno umen'shit' ob'em takih othodov. Dlja jetoj celi tverdye othody plavjatsja, razmel'chajutsja ili szhigajutsja. Nebol'shoe kolichestvo othodov otvoditsja v atmosferu cherez dymohodnye truby. Ochistka otvodimogo takim putem vozduha osushhestvljaetsja s pomoshh'ju vysokojeffektivnyh fil'trov. Reguljarno provoditsja dozimetricheskij kontrol' dymohodnyh trub i vzjatie prob vozduha dlja

  2. Effect of Different Lignocellulosic Diets on Bacterial Microbiota and Hydrolytic Enzyme Activities in the Gut of the Cotton Boll Weevil (Anthonomus grandis).

    Science.gov (United States)

    Ben Guerrero, Emiliano; Soria, Marcelo; Salvador, Ricardo; Ceja-Navarro, Javier A; Campos, Eleonora; Brodie, Eoin L; Talia, Paola

    2016-01-01

    Cotton boll weevils, Anthonomus grandis , are omnivorous coleopteran that can feed on diets with different compositions, including recalcitrant lignocellulosic materials. We characterized the changes in the prokaryotic community structure and the hydrolytic activities of A. grandis larvae fed on different lignocellulosic diets. A. grandis larvae were fed on three different artificial diets: cottonseed meal (CM), Napier grass (NG) and corn stover (CS). Total DNA was extracted from the gut samples for amplification and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Proteobacteria and Firmicutes dominated the gut microbiota followed by Actinobacteria, Spirochaetes and a small number of unclassified phyla in CM and NG microbiomes. In the CS feeding group, members of Spirochaetes were the most prevalent, followed by Proteobacteria and Firmicutes. Bray-Curtis distances showed that the samples from the CS community were clearly separated from those samples of the CM and NG diets. Gut extracts from all three diets exhibited endoglucanase, xylanase, β-glucosidase and pectinase activities. These activities were significantly affected by pH and temperature across different diets. We observed that the larvae reared on a CM showed significantly higher activities than larvae reared on NG and CS. We demonstrated that the intestinal bacterial community structure varies depending on diet composition. Diets with more variable and complex compositions, such as CS, showed higher bacterial diversity and richness than the two other diets. In spite of the detected changes in composition and diversity, we identified a core microbiome shared between the three different lignocellulosic diets. These results suggest that feeding with diets of different lignocellulosic composition could be a viable strategy to discover variants of hemicellulose and cellulose breakdown systems.

  3. Secretome analysis of the fungus Trichoderma harzianum grown on cellulose.

    Science.gov (United States)

    Do Vale, Luis H F; Gómez-Mendoza, Diana P; Kim, Min-Sik; Pandey, Akhilesh; Ricart, Carlos A O; Ximenes F Filho, Edivaldo; Sousa, Marcelo V

    2012-08-01

    Trichoderma harzianum is a mycoparasitic filamentous fungus that produces and secretes a wide range of extracellular hydrolytic enzymes used in cell wall degradation. Due to its potential in biomass conversion, T. harzianum draws great attention from biofuel and biocontrol industries and research. Here, we report an extensive secretome analysis of T. harzianum. The fungus was grown on cellulose medium, and its secretome was analyzed by a combination of enzymology, 2DE, MALDI-MS and -MS/MS (Autoflex II), and LC-MS/MS (LTQ-Orbitrap XL). A total of 56 proteins were identified using high-resolution MS. Interestingly, although cellulases were found, the major hydrolytic enzymes secreted in the cellulose medium were chitinases and endochitinases, which may reflect the biocontrol feature of T. harzianum. The glycoside hydrolase family, including chitinases (EC 3.2.1.14), endo-N-acetylglucosaminidases (EC 3.2.1.96), hexosaminidases (EC 3.2.1.52), galactosidases (EC 3.2.1.23), xylanases (EC 3.2.1.8), exo-1,3-glucanases (EC 3.2.1.58), endoglucanases (EC 3.2.1.4), xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), N-acetylhexosaminidases (EC 3.2.1.52), and other enzymes represented 51.36% of the total secretome. Few representatives were classified in the protease family (8.90%). Others (17.60%) are mostly intracellular proteins. A considerable part of the secretome was composed of hypothetical proteins (22.14%), probably because of the absence of an annotated T. harzianum genome. The T. harzianum secretome composition highlights the importance of this fungus as a rich source of hydrolytic enzymes for bioconversion and biocontrol applications. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

    Science.gov (United States)

    Yoav, Shahar; Barak, Yoav; Shamshoum, Melina; Borovok, Ilya; Lamed, Raphael; Dassa, Bareket; Hadar, Yitzhak; Morag, Ely; Bayer, Edward A

    2017-01-01

    Bioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium ( Ruminiclostridium ) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated. Glucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types. The results reported here highlight the finding that cellulosomes derived from cells grown on glucose

  5. Tissue-Specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus planipennis)

    Science.gov (United States)

    Mittapalli, Omprakash; Bai, Xiaodong; Bonello, Pierluigi; Herms, Daniel A.

    2010-01-01

    Background The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level. Methodology and Principal Findings Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level. Conclusions and Significance To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis. PMID:21060843

  6. Proteomics as a Tool to Identify New Targets Against Aspergillus and Scedosporium in the Context of Cystic Fibrosis.

    Science.gov (United States)

    Ramirez-Garcia, Andoni; Pellon, Aize; Buldain, Idoia; Antoran, Aitziber; Arbizu-Delgado, Aitana; Guruceaga, Xabier; Rementeria, Aitor; Hernando, Fernando L

    2018-02-01

    Cystic fibrosis (CF) is a genetic disorder that increases the risk of suffering microbial, including fungal, infections. In this paper, proteomics-based information was collated relating to secreted and cell wall proteins with potential medical applications from the most common filamentous fungi in CF, i.e., Aspergillus and Scedosporium/Lomentospora species. Among the Aspergillus fumigatus secreted allergens, β-1,3-endoglucanase, the alkaline protease 1 (Alp1/oryzin), Asp f 2, Asp f 13/15, chitinase, chitosanase, dipeptidyl-peptidase V (DppV), the metalloprotease Asp f 5, mitogillin/Asp f 1, and thioredoxin reductase receive a special mention. In addition, the antigens β-glucosidase 1, catalase, glucan endo-1,3-β-glucosidase EglC, β-1,3-glucanosyltransferases Gel1 and Gel2, and glutaminase A were also identified in secretomes of other Aspergillus species associated with CF: Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus. Regarding cell wall proteins, cytochrome P450 and eEF-3 were proposed as diagnostic targets, and alkaline protease 2 (Alp2), Asp f 3 (putative peroxiredoxin pmp20), probable glycosidases Asp f 9/Crf1 and Crf2, GPI-anchored protein Ecm33, β-1,3-glucanosyltransferase Gel4, conidial hydrophobin Hyp1/RodA, and secreted aspartyl protease Pep2 as protective vaccines in A. fumigatus. On the other hand, for Scedosporium/Lomentospora species, the heat shock protein Hsp70 stands out as a relevant secreted and cell wall antigen. Additionally, the secreted aspartyl proteinase and an ortholog of Asp f 13, as well as the cell wall endo-1,3-β-D-glucosidase and 1,3-β-glucanosyl transferase, were also found to be significant proteins. In conclusion, proteins mentioned in this review may be promising candidates for developing innovative diagnostic and therapeutic tools for fungal infections in CF patients.

  7. Bio-ethanol production from waste biomass of Pogonatherum crinitum phytoremediator: an eco-friendly strategy for renewable energy.

    Science.gov (United States)

    Waghmare, Pankajkumar R; Watharkar, Anuprita D; Jeon, Byong-Hun; Govindwar, Sanjay P

    2018-03-01

    In this study, we have described three steps to produce ethanol from Pogonatherum crinitum , which was derived after the treatment of textile wastewater. (a) Production of biomass: biomass samples collected from a hydroponic P. crinitum phytoreactor treating dye textile effluents and augmented with Ca-alginate immobilized growth-promoting bacterium, Bacillus pumilus strain PgJ (consortium phytoreactor), and waste sorghum husks were collected and dried. Compositional analysis of biomass (consortium phytoreactor) showed that the concentration of cellulose, hemicelluloses and lignin was 42, 30 and 17%, respectively, whereas the biomass samples without the growth-promoting bacterium (normal phytoreactor) was slightly lower, 40, 29 and 16%, respectively. (b) Hydrolysate (sugar) production: a crude sample of the fungus, Phanerochaete chrysosporium containing hydrolytic enzymes such as endoglucanase (53.25 U/ml), exoglucanase (8.38 U/ml), glucoamylase (115.04 U/ml), xylanase (83.88 U/ml), LiP (0.972 U/ml) and MnP (0.459 U/ml) was obtained, and added to consortium, normal and control phytoreactor derived biomass supplemented with Tween-20 (0.2% v/v). The hydrolysate of biomass from consortium phytoreactor produced maximum reducing sugar (0.93 g/l) than hydrolysates of normal phytoreactor biomass (0.82 g/l) and control phytoreactor biomass (0.79 g/l). FTIR and XRD analysis confirmed structural changes in treated biomass. (c) Ethanol production: the bioethanol produced from enzymatic hydrolysates of waste biomass of consortium and normal phytoreactor using Saccharomyces cerevisiae (KCTC 7296) was 42.2 and 39.4 g/l, respectively, while control phytoreactor biomass hydrolysate showed only 25.5 g/l. Thus, the amalgamation of phytoremediation and bioethanol production can be the truly environment-friendly way to eliminate the problem of textile dye along with bioenergy generation.

  8. Large-scale proteome comparative analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale.

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena

    2012-06-01

    Full Text Available Equisetum hyemale is a widespread vascular plant species, whose reproduction is mainly dependent on the growth and development of the rhizomes. Due to its key evolutionary position, the identification of factors that could be involved in the existence of the rhizomatous trait may contribute to a better understanding of the role of this underground organ for the successful propagation of this and other plant species. In the present work, we characterized the proteome of E. hyemale rhizomes using a GeLC-MS spectral-counting proteomics strategy. A total of 1,911 and 1,860 non-redundant proteins were identified in the rhizomes apical tip and elongation zone, respectively. Rhizome- characteristic proteins were determined by comparisons of the developing rhizome tissues to developing roots. A total of 87 proteins were found to be up-regulated in both E. hyemale rhizome tissues in relation to developing roots. Hierarchical clustering indicated a vast dynamic range in the expression of the 87 characteristic proteins and revealed, based on the expression profile, the existence of 9 major protein groups. Gene ontology analyses suggested an over-representation of the terms involved in macromolecular and protein biosynthetic processes, gene expression and nucleotide and protein binding functions. Spatial differences analysis between the rhizome apical tip and the elongation zone revealed that only eight proteins were up-regulated in the apical tip including RNA-binding proteins and an acyl carrier protein, as well as a KH-domain protein and a T-complex subunit; while only seven proteins were up-regulated in the elongation zone including phosphomannomutase, galactomannan galactosyltransferase, endoglucanase 10 and 25 and mannose-1-phosphate guanyltransferase subunits alpha and beta. This is the first large scale characterization of the proteome of a plant rhizome. Implications of the findings were discussed in relation to other underground organs and related

  9. In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn.

    Science.gov (United States)

    Ramos, Araceli M; Gally, Marcela; Szapiro, Gala; Itzcovich, Tatiana; Carabajal, Maira; Levin, Laura

    Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  10. Comparative genomic analysis of the microbiome [corrected] of herbivorous insects reveals eco-environmental adaptations: biotechnology applications.

    Directory of Open Access Journals (Sweden)

    Weibing Shi

    Full Text Available Metagenome analysis of the gut symbionts of three different insects was conducted as a means of comparing taxonomic and metabolic diversity of gut microbiomes to diet and life history of the insect hosts. A second goal was the discovery of novel biocatalysts for biorefinery applications. Grasshopper and cutworm gut symbionts were sequenced and compared with the previously identified metagenome of termite gut microbiota. These insect hosts represent three different insect orders and specialize on different food types. The comparative analysis revealed dramatic differences among the three insect species in the abundance and taxonomic composition of the symbiont populations present in the gut. The composition and abundance of symbionts was correlated with their previously identified capacity to degrade and utilize the different types of food consumed by their hosts. The metabolic reconstruction revealed that the gut metabolome of cutworms and grasshoppers was more enriched for genes involved in carbohydrate metabolism and transport than wood-feeding termite, whereas the termite gut metabolome was enriched for glycosyl hydrolase (GH enzymes relevant to lignocellulosic biomass degradation. Moreover, termite gut metabolome was more enriched with nitrogen fixation genes than those of grasshopper and cutworm gut, presumably due to the termite's adaptation to the high fiber and less nutritious food types. In order to evaluate and exploit the insect symbionts for biotechnology applications, we cloned and further characterized four biomass-degrading enzymes including one endoglucanase and one xylanase from both the grasshopper and cutworm gut symbionts. The results indicated that the grasshopper symbiont enzymes were generally more efficient in biomass degradation than the homologous enzymes from cutworm symbionts. Together, these results demonstrated a correlation between the composition and putative metabolic functionality of the gut microbiome and host

  11. High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

    Science.gov (United States)

    Akazawa, Shin-Ichi; Tokuyama, Haruka; Sato, Shunsuke; Watanabe, Toshinori; Shida, Yosuke; Ogasawara, Wataru

    2018-02-01

    Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

    2013-12-01

    Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

  13. Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa.

    Science.gov (United States)

    Backus, Elaine A; Andrews, Kim B; Shugart, Holly J; Carl Greve, L; Labavitch, John M; Alhaddad, Hasan

    2012-07-01

    A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away

  14. Genomics insights into different cellobiose hydrolysis activities in two Trichoderma hamatum strains.

    Science.gov (United States)

    Cheng, Peng; Liu, Bo; Su, Yi; Hu, Yao; Hong, Yahui; Yi, Xinxin; Chen, Lei; Su, Shengying; Chu, Jeffrey S C; Chen, Nansheng; Xiong, Xingyao

    2017-04-19

    Efficient biomass bioconversion is a promising solution to alternative energy resources and environmental issues associated with lignocellulosic wastes. The Trichoderma species of cellulolytic fungi have strong cellulose-degrading capability, and their cellulase systems have been extensively studied. Currently, a major limitation of Trichoderma strains is their low production of β-glucosidases. We isolated two Trichoderma hamatum strains YYH13 and YYH16 with drastically different cellulose degrading efficiencies. YYH13 has higher cellobiose-hydrolyzing efficiency. To understand mechanisms underlying such differences, we sequenced the genomes of YYH13 and YYH16, which are essentially identical (38.93 and 38.92 Mb, respectively) and are similar to that of the T. hamatum strain GD12. Using GeneMark-ES, we annotated 11,316 and 11,755 protein-coding genes in YYH13 and YYH16, respectively. Comparative analysis identified 13 functionally important genes in YYH13 under positive selection. Through examining orthologous relationships, we identified 172,655, and 320 genome-specific genes in YYH13, YYH16, and GD12, respectively. We found 15 protease families that show differences between YYH13 and YYH16. Enzymatic tests showed that exoglucanase, endoglucanase, and β-glucosidase activities were higher in YYH13 than YYH16. Additionally, YYH13 contains 10 families of carbohydrate-active enzymes, including GH1, GH3, GH18, GH35, and GH55 families of chitinases, glucosidases, galactosidases, and glucanases, which are subject to stronger positive selection pressure. Furthermore, we found that the β-glucosidase gene (YYH1311079) and pGEX-KG/YYH1311079 bacterial expression vector may provide valuable insight for designing β-glucosidase with higher cellobiose-hydrolyzing efficiencies. This study suggests that the YYH13 strain of T. hamatum has the potential to serve as a model organism for producing cellulase because of its strong ability to efficiently degrade cellulosic biomass

  15. Enhanced ethanol production from brewer's spent grain by a Fusarium oxysporum consolidated system

    Directory of Open Access Journals (Sweden)

    Christakopoulos Paul

    2009-02-01

    Full Text Available Abstract Background Brewer's spent grain (BG, a by-product of the brewing process, is attracting increasing scientific interest as a low-cost feedstock for many biotechnological applications. BG in the present study is evaluated as a substrate for lignocellulolytic enzyme production and for the production of ethanol by the mesophilic fungus Fusarium oxysporum under submerged conditions, implementing a consolidated bioconversion process. Fermentation experiments were performed with sugar mixtures simulating the carbohydrate content of BG in order to determine the utilization pattern that could be expected during the fermentation of the cellulose and hemicellulose hydrolysate of BG. The sugar mixture fermentation study focused on the effect of the initial total sugar concentration and on the effect of the aeration rate on fermenting performance of F. oxysporum. The alkali pretreatment of BG and different aeration levels during the ethanol production stage were studied for the optimization of the ethanol production by F. oxysporum. Results Enzyme yields as high as 550, 22.5, 6.5, 3225, 0.3, 1.25 and 3 U per g of carbon source of endoglucanase, cellobiohydrolase, β-D-glucosidase, xylanase, feruloyl esterase, β-D-xylosidase and α-L-arabinofuranosidase respectively, were obtained during the growth stage under optimized submerged conditions. An ethanol yield of 109 g ethanol per kg of dry BG was obtained with alkali-pretreated BG under microaerobic conditions (0.01 vvm, corresponding to 60% of the theoretical yield based on total glucose and xylose content of BG. Conclusion The enzymatic profile of the extracellular extract from F. oxysporum submerged cultures using BG and corn cob as the carbon source was proved efficient for a successful hydrolysis of BG. The fermentation study carried out using sugar mixtures simulating BG's carbohydrates content and consecutively alkali-pretreated and untreated BG, indicates that BG hydrolysis is the bottleneck

  16. Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

    International Nuclear Information System (INIS)

    Cripwell, Rosemary; Favaro, Lorenzo; Rose, Shaunita H.; Basaglia, Marina; Cagnin, Lorenzo; Casella, Sergio; Zyl, Willem van

    2015-01-01

    Highlights: • A cocktail of recombinant cellulases was proposed for wheat bran hydrolysis. • Optimal conditions for enzymatic hydrolysis of wheat bran were determined. • Recombinant amylolytic strains completely hydrolysed the starch in wheat bran. • Addition of cellulases to SSF with amylolytic strains enhanced ethanol yield. - Abstract: Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a β-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n[TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeast S. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran’s starch producing similar amounts of ethanol (5.3 ± 0.14 g/L and 5.0 ± 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisation. This study demonstrated that untreated wheat bran could be a promising ready-to-use substrate for ethanol production. The addition of crude recombinant cellulases improved ethanol yields in the SSF process and S. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] strains can efficiently convert wheat bran’s starch to ethanol.

  17. Simultaneous cell growth and ethanol production from cellulose by an engineered yeast consortium displaying a functional mini-cellulosome

    Directory of Open Access Journals (Sweden)

    Madan Bhawna

    2011-11-01

    Full Text Available Abstract Background The recalcitrant nature of cellulosic materials and the high cost of enzymes required for efficient hydrolysis are the major impeding steps to their practical usage for ethanol production. Ideally, a recombinant microorganism, possessing the capability to utilize cellulose for simultaneous growth and ethanol production, is of great interest. We have reported recently the use of a yeast consortium for the functional presentation of a mini-cellulosome structure onto the yeast surface by exploiting the specific interaction of different cohesin-dockerin pairs. In this study, we engineered a yeast consortium capable of displaying a functional mini-cellulosome for the simultaneous growth and ethanol production on phosphoric acid swollen cellulose (PASC. Results A yeast consortium composed of four different populations was engineered to display a functional mini-cellulosome containing an endoglucanase, an exoglucanase and a β-glucosidase. The resulting consortium was demonstrated to utilize PASC for growth and ethanol production. The final ethanol production of 1.25 g/L corresponded to 87% of the theoretical value and was 3-fold higher than a similar yeast consortium secreting only the three cellulases. Quantitative PCR was used to enumerate the dynamics of each individual yeast population for the two consortia. Results indicated that the slight difference in cell growth cannot explain the 3-fold increase in PASC hydrolysis and ethanol production. Instead, the substantial increase in ethanol production is consistent with the reported synergistic effect on cellulose hydrolysis using the displayed mini-cellulosome. Conclusions This report represents a significant step towards the goal of cellulosic ethanol production. This engineered yeast consortium displaying a functional mini-cellulosome demonstrated not only the ability to grow on the released sugars from PASC but also a 3-fold higher ethanol production than a similar yeast

  18. Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance.

    Directory of Open Access Journals (Sweden)

    Alexander G Bulakhov

    Full Text Available Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO displaying a synergism with cellulases.Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL, and eglIV, encoding LPMO (formerly endoglucanase IV from Trichoderma reesei (TrLPMO, were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3 varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples. The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10-43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable

  19. Structural and functional insights of β-glucosidases identified from the genome of Aspergillus fumigatus

    Science.gov (United States)

    Dodda, Subba Reddy; Aich, Aparajita; Sarkar, Nibedita; Jain, Piyush; Jain, Sneha; Mondal, Sudipa; Aikat, Kaustav; Mukhopadhyay, Sudit S.

    2018-03-01

    Thermostable glucose tolerant β-glucosidase from Aspergillus species has attracted worldwide interest for their potentiality in industrial applications and bioethanol production. A strain of Aspergillus fumigatus (AfNITDGPKA3) identified by our laboratory from straw retting ground showed higher cellulase activity, specifically the β-glucosidase activity, compared to other contemporary strains. Though A. fumigatus has been known for high cellulase activity, detailed identification and characterization of the cellulase genes from their genome is yet to be done. In this work we have been analyzed the cellulase genes from the genome sequence database of Aspergillus fumigatus (Af293). Genome analysis suggests two cellobiohydrolase, eleven endoglucanase and seventeen β-glucosidase genes present. β-Glucosidase genes belong to either Glycohydro1 (GH1 or Bgl1) or Glycohydro3 (GH3 or Bgl3) family. The sequence similarity suggests that Bgl1 and Bgl3 of A. fumagatus are phylogenetically close to those of A. fisheri and A. oryzae. The modelled structure of the Bgl1 predicts the (β/α)8 barrel type structure with deep and narrow active site, whereas, Bgl3 shows the (α/β)8 barrel and (α/β)6 sandwich structure with shallow and open active site. Docking results suggest that amino acids Glu544, Glu466, Trp408,Trp567,Tyr44,Tyr222,Tyr770,Asp844,Asp537,Asn212,Asn217 of Bgl3 and Asp224,Asn242,Glu440, Glu445, Tyr367, Tyr365,Thr994,Trp435,Trp446 of Bgl1 are involved in the hydrolysis. Binding affinity analyses suggest that Bgl3 and Bgl1 enzymes are more active on the substrates like 4-methylumbelliferyl glycoside (MUG) and p-nitrophenyl-β-D-1, 4-glucopyranoside (pNPG) than on cellobiose. Further docking with glucose suggests that Bgl1 is more glucose tolerant than Bgl3. Analysis of the Aspergillus fumigatus genome may help to identify a β-glucosidase enzyme with better property and the structural information may help to develop an engineered recombinant enzyme.

  20. Examination of the Abscission-Associated Transcriptomes for Soybean, Tomato, and Arabidopsis Highlights the Conserved Biosynthesis of an Extensible Extracellular Matrix and Boundary Layer.

    Science.gov (United States)

    Kim, Joonyup; Sundaresan, Srivignesh; Philosoph-Hadas, Sonia; Yang, Ronghui; Meir, Shimon; Tucker, Mark L

    2015-01-01

    Abscission zone (AZ) development and the progression of abscission (detachment of plant organs) have been roughly separated into four stages: first, AZ differentiation; second, competence to respond to abscission signals; third, activation of abscission; and fourth, formation of a protective layer and post-abscission trans-differentiation. Stage three, activation of abscission, is when changes in the cell wall and extracellular matrix occur to support successful organ separation. Most abscission research has focused on gene expression for enzymes that disassemble the cell wall within the AZ and changes in phytohormones and other signaling events that regulate their expression. Here, transcriptome data for soybean, tomato and Arabidopsis were examined and compared with a focus not only on genes associated with disassembly of the cell wall but also on gene expression linked to the biosynthesis of a new extracellular matrix. AZ-specific up-regulation of genes associated with cell wall disassembly including cellulases (beta-1,4-endoglucanases, CELs), polygalacturonases (PGs), and expansins (EXPs) were much as expected; however, curiously, changes in expression of xyloglucan endotransglucosylase/hydrolases (XTHs) were not AZ-specific in soybean. Unexpectedly, we identified an early increase in the expression of genes underlying the synthesis of a waxy-like cuticle. Based on the expression data, we propose that the early up-regulation of an abundance of small pathogenesis-related (PR) genes is more closely linked to structural changes in the extracellular matrix of separating cells than an enzymatic role in pathogen resistance. Furthermore, these observations led us to propose that, in addition to cell wall loosening enzymes, abscission requires (or is enhanced by) biosynthesis and secretion of small proteins (15-25 kDa) and waxes that form an extensible extracellular matrix and boundary layer on the surface of separating cells. The synthesis of the boundary layer