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Sample records for humicola insolens cutinase

  1. Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

    DEFF Research Database (Denmark)

    Kold, Daniel; Dauter, Zbigniew; Laustsen, Anne

    2014-01-01

    The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds...... a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown...... of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but...

  2. Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

    Science.gov (United States)

    Kold, David; Dauter, Zbigniew; Laustsen, Anne K; Brzozowski, Andrzej M; Turkenburg, Johan P; Nielsen, Anders D; Koldsø, Heidi; Petersen, Evamaria; Schiøtt, Birgit; De Maria, Leonardo; Wilson, Keith S; Svendsen, Allan; Wimmer, Reinhard

    2014-01-01

    The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 105 M−1). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad. PMID:24832484

  3. High level expression of a novel family 3 neutral β-xylosidase from Humicola insolens Y1 with high tolerance to D-xylose.

    Directory of Open Access Journals (Sweden)

    Wei Xia

    Full Text Available A novel β-xylosidase gene of glycosyl hydrolase (GH family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0-9.0 (incubated at 37°C for 1 h and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.

  4. Flash photolysis of cutinase

    DEFF Research Database (Denmark)

    Neves Petersen, Teresa; Klitgaard, Søren; Skovsen, Esben

    2009-01-01

    were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime...... kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation...

  5. Variant Humicola grisea CBH1.1

    Energy Technology Data Exchange (ETDEWEB)

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2017-05-09

    Disclosed are variants of Humicola grisea CeI7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  6. Extracellular protease from the antarctic yeast Candida humicola.

    OpenAIRE

    Ray, M K; Devi, K U; Kumar, G S; Shivaji, S

    1992-01-01

    The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulf...

  7. Expression and secretion of defined cutinase variants by Aspergillus awamori

    NARCIS (Netherlands)

    Gemeren, I.A. van; Beijersbergen, A.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1998-01-01

    Several cutinase variants derived by molecular modelling and site- directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae. The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori. However, the

  8. Application and comparison in biosynthesis and biodegradation by Fusarium solani and Aspergillus fumigatus cutinases.

    Science.gov (United States)

    Ping, Li-Feng; Chen, Xiao-Yang; Yuan, Xiao-Li; Zhang, Min; Chai, Yan-Jun; Shan, Sheng-Dao

    2017-11-01

    In this study, two synthesized cutinase genes from Fusarium solani and Aspergillus fumigatus were expressed in Pichia pastoris X33. The characteristics of these two cutinases were investigated and compared. The results indicated that F. solani and A. fumigatus cutinases hydrolyzed p-nitrophenyl substrates with different carbon chain lengths. A. fumigatus cutinase predominately hydrolyzed p-nitrophenyl butyrate, but F. solani cutinase preferred p-nitrophenyl decanoate. The abilities of polymer synthesis and bioplastic degradation were tested and compared between F. solani and A. fumigatus cutinases. The results showed that F. solani cutinase had degradation ability on poly(ε-caprolactone) (PCL) and synthesized polymer with a molecular weight (MW) of 2300 in organic solvent. However, A. fumigatus cutinase completely degraded PCL and synthesized molecules with a MW of 25,000, suggesting that A. fumigatus cutinase has more promising applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Cutinase of Fusarium solani F. sp. pisi: mechanism of induction and relatedness to other Fusarium species

    Energy Technology Data Exchange (ETDEWEB)

    Woloshuk, C.P.

    1986-01-01

    Three studies were made on the extracellular cutinase of the phytopathogenic fungus Fusarium solani f. sp. pisi. I. The production of cutinase was found to be induced in spores of F. solani f. sp. pisi, strain T-8, by cutin and cutin hydrolysate. Fractionation and analysis of the cutin hydrolysate indicated that dihydroxy-C/sub 16/ acid and trihydroxy-C/sub 18/ acid were the cutin monomers most active for inducing cutinase. Measurement of cutinase-specific RNA levels by dot-blot hybridization with a (/sup 32/P)-labeled cutinase cDNA showed that the cutinase gene transcripts could be detected within 15 min after addition of the inducers. The results indicated that the fungal spores have the capacity to recognize the unique monomer components of the plant cuticle and rapidly respond by the synthesis of cutinase. II. Analysis of the genomic DNA's of seven strains of F. solani f. sp. pisi indicated that both high and low cutinase-producing strains contain at least one copy of the cutinase structural gene and a homologous promoter region. The data suggest a different promoter sequence exists in these additional copies. III. Relatedness of five phytopathogenic Fusarium species to F. solani f. sp. pisi was determined by their cutinase antigenic properties and gene homologies of cutinase cDNA from F. solani f. sp. pisi. The results suggest that formae specialis of F. solani are phylogenetically identical and that F. solani is quite distinct from the other Fusarium species tested.

  10. Extracellular protease from the antarctic yeast Candida humicola.

    Science.gov (United States)

    Ray, M K; Devi, K U; Kumar, G S; Shivaji, S

    1992-06-01

    The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.

  11. A rapid screening method for cutinase producing microorganisms Seleção de microorganismos produtores de cutinase

    Directory of Open Access Journals (Sweden)

    Gabriela Alves Macedo

    2005-12-01

    Full Text Available Both cutinase and lipase belong to the esterase group of enzymes (EC 3.1.1.X, which are capable of catalyzing the hydrolysis of ester bonds. Cutinase catalyzes the hydrolysis of cutin, an insoluble biopolyester which is the structural component of plant cuticles. As cutinase is an efficient catalyst in watery or organic media, it is potentially appropriate for the detergent, food and cosmetic industries. The objective of this work was to isolate microorganisms from plants and perform a pre-selection of molds showing esterase producing ability. The selected strains were then submitted to fermentation in media containing cutin. The lipolytic and cutinolytic activities of the supernatant were determined in order to differentiate lipase producers from cutinase-producing strains. The mold strain selected as the best cutinase producer was identified as being Fusarium oxysporium.A cutinase pertence, como as lípases, ao grupo das esterases (EC 3.1.1.X, que são enzimas capazes de catalisar reações de hidrólise de ligações do tipo éster. A cutinase catalisa a hidrólise da cutina, um biopoliéster insolúvel que constitui o componente estrutural da cutícula das plantas. Esta enzima tem se mostrado um eficiente catalisador tanto em solução aquosa quanto em meios orgânicos, sendo potencialmente apropriada para usos em indústria de detergentes, alimentos e cosméticos. O objetivo deste trabalho foi isolar fungos de diversas fontes e realizar uma pré-seleção destes, quanto à habilidade em produzir esterase. As linhagens fúngicas pré-selecionadas como produtoras de esterase foram inoculadas em meio de cultivo contendo cutina extraída de maçã. As atividades cutinolítica e lipolítica foram medidas no sobrenadante das culturas a fim de diferenciar os microrganismos produtores de lipase dos produtores de cutinase. Uma linhagem isolada mostrou a maior atividade cutinolítica após 12 dias de fermentação em um meio contendo 1% de cutina e

  12. Modeling cutinase enzyme regulation in polyethylene terepthalate plastic biodegradation

    Energy Technology Data Exchange (ETDEWEB)

    Apri, M., E-mail: m.apri@math.itb.ac.id; Silmi, M. [Department of Mathematics, Institut Teknologi Bandung, Jalan Ganeca 10 Bandung, 40132 (Indonesia); Heryanto, T. E.; Moeis, M. R. [School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganeca 10 Bandung, 40132 (Indonesia)

    2016-04-06

    PET (Polyethylene terephthalate) is a plastic material that is commonly used in our daily life. The high production of PET and others plastics that can be up to three hundred million tons per year, is not matched by its degradation rate and hence leads to environmental pollution. To overcome this problem, we develop a biodegradation system. This system utilizes LC Cutinase enzyme produced by engineered escherichia coli bacteria to degrade PET. To make the system works efficaciously, it is important to understand the mechanism underlying its enzyme regulation. Therefore, we construct a mathematical model to describe the regulation of LC Cutinase production. The stability of the model is analyzed. We show that the designated biodegradation system can give an oscillatory behavior that is very important to control the amount of inclusion body (the miss-folded proteins that reduce the efficiency of the biodegradation system).

  13. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the

  14. Cytoplasmic inorganic polyphosphate participates in the heavy metal tolerance of Cryptococcus humicola.

    Science.gov (United States)

    Andreeva, Nadezhda; Ryazanova, Lubov; Dmitriev, Vladimir; Kulakovskaya, Tatiana; Kulaev, Igor

    2014-09-01

    The basidiomycetous yeast Cryptococcus humicola was shown to be tolerant to manganese, cobalt, nickel, zinc, lanthanum, and cadmium cations at a concentration of 2.5 mmol/L, which is toxic for many yeasts. The basidiomycetous yeast Cryptococcus terreus was sensitive to all these ions and did not grow at the above concentration. In the presence of heavy metal cations, С. humicola, as opposed to C. terreus, was characterized by the higher content of acid-soluble inorganic polyphosphates. In vivo 4',6'-diamino-2-phenylindole dihydrochloride staining revealed polyphosphate accumulation in the cell wall and cytoplasmic inclusions of С. humicola in the presence of heavy metals. In C. terreus, polyphosphates in the presence of heavy metals accumulate mainly in vacuoles, which results in morphological changes in these organelles and, probably, disturbance of their function. The role of polyphosphate accumulation and cellular localization as factors of heavy metal tolerance of Cryptococcus humicola is discussed.

  15. Molecular dynamics simulations in rational protein design : Stabilisation of fusarium solani pisi cutinase against anionic surfactants

    NARCIS (Netherlands)

    Creveld, Lucia Deborah

    2001-01-01

    Cutinases are enxymes with a molecular mass of 22-25 kDa that supposedly degrade ester bonds of the cutin polymer in the plant cell wall. They are serine esterases, also able to hydrolyse a wide variety of synthetic esters and triacylglycerols. As cutinase is an efficient catalyst both in solution

  16. Cutinase and pectinase in cotton bioscouring: an innovative and fast bioscouring process

    NARCIS (Netherlands)

    Agrawal, Pramod; Nierstrasz, Vincent; Bouwhuis, G.H.; Bouwhuis, G.H.; Warmoeskerken, Marinus

    2008-01-01

    This manuscript describes the potential of a cutinase from the fungus Fusarium solani pisi for cotton wax degradation in order to design an efficient low temperature scouring process. The main characteristics, relevant to cotton wax removal with F. solani pisi cutinase are given. The additive effect

  17. Mechanism by which contact with plant cuticle triggers cutinase gene expression in the spores of Fusarium solani f. sp. pisi

    Energy Technology Data Exchange (ETDEWEB)

    Woloshuk, C.P.; Kolattukudy, P.E.

    1986-03-01

    Spores of the phytopathogenic fungus Fusarium solani f. sp. pisi were shown to produce the extracellular enzyme, cutinase, only when cutin or cutin hydrolysate was added to the spore suspension. Dihydroxy-C/sub 16/ acid and trihydroxy-C/sub 18/ acid, which are unique cutin monomers, showed the greatest cutinase-inducing activity. Experiments with several compounds structurally related to these fatty acids suggested that both a omega-hydroxyl and a midchain hydroxyl are required for cutinase-inducing activity. Cutinase appeared in the medium 30-45 min after the addition of the inducers to the spore suspension, and the activity level increased for 6 hr. Addition of cycloheximide (5 ..mu..g/ml) completely inhibited cutinase production, suggesting that protein synthesis was involved in the increase of cutinase activity. Immunoblot analysis with rabbit antibodies prepared against cutinase showed that cutinase protein increased in parallel with the increase in enzyme activity. Measurement of cutinase-specific RNA levels by dot-blot hybridization with /sup 32/P-labeled cutinase cDNA showed that the cutinase gene transcripts could be detected within 15 min after addition of the inducers. Addition of exogenous cutinase greatly enhanced the level of cutinase gene transcripts induced by cutin. These results strongly suggest that the fungal spore senses that it is in contact with the plant by the production of small amounts of cutin monomers catalyzed by the low level of cutinase carried by the spore and that these monomers induce the synthesis of cutinase needed for penetration of the fungus into the plant.

  18. A new fungal endophyte, Scolecobasidium humicola, promotes tomato growth under organic nitrogen conditions.

    Science.gov (United States)

    Mahmoud, Rola S; Narisawa, Kazuhiko

    2013-01-01

    A new fungal endophyte, Scolecobasidium humicola, was identified as a common dark septate endophytic fungal (DSE) species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular, pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth with organic nitrogen sources.

  19. A new fungal endophyte, Scolecobasidium humicola, promotes tomato growth under organic nitrogen conditions.

    Directory of Open Access Journals (Sweden)

    Rola S Mahmoud

    Full Text Available A new fungal endophyte, Scolecobasidium humicola, was identified as a common dark septate endophytic fungal (DSE species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular, pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth with organic nitrogen sources.

  20. Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring▿ †

    Science.gov (United States)

    Zhang, Yao; Chen, Sheng; Xu, Meng; Cavoco-Paulo, Artur; Wu, Jing; Chen, Jian

    2010-01-01

    Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBMCel6A and cutinase-CBMCenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50°C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBMCel6A and by 28% for cutinase-CBMCenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase. PMID:20729325

  1. Flocculating performance of a bioflocculant produced by Arthrobacter humicola in sewage waste water treatment.

    Science.gov (United States)

    Agunbiade, Mayowa Oladele; Van Heerden, Esta; Pohl, Carolina H; Ashafa, Anofi Tom

    2017-06-12

    The discharge of poorly treated effluents into the environment has far reaching, consequential impacts on human and aquatic life forms. Thus, we evaluated the flocculating efficiency of our test bioflocculant and we report for the first time the ability of the biopolymeric flocculant produced by Arthrobacter humicola in the treatment of sewage wastewater. This strain was isolated from sediment soil sample at Sterkfontein dam in the Eastern Free State province of South Africa. Basic Local Alignment Search Tool (BLAST) analysis of the nucleotide sequence of the 16S rDNA revealed the bacteria to have 99% similarity to Arthrobacter humicola strain R1 and the sequence was deposited in the Gene bank as Arthrobacter humicola with accession number KC816574.1. Flocculating activity was enhanced with the aid of divalent cations, pH 12, at a dosage concentration of 0.8 mg/mL. The purified bioflocculant was heat stable and could retain more than 78% of its flocculating activity after heating at 100 °C for 25 min. Fourier Transform Infrared Spectroscopy analysis demonstrated the presence of hydroxyl and carboxyl moieties as the functional groups. The thermogravimetric analysis was used to monitor the pyrolysis profile of the purified bioflocculant and elemental composition revealed C: O: Na: P: K with 13.90: 41.96: 26.79: 16.61: 0.74 weight percentage respectively. The purified bioflocculant was able to remove chemical oxygen demand, biological oxygen demand, suspended solids, nitrate and turbidity from sewage waste water at efficiencies of 65.7%, 63.5%, 55.7%, 71.4% and 81.3% respectively. The results of this study indicate the possibility of using the bioflocculant produced by Arthrobacter humicola as a potential alternative to synthesized chemical flocculants in sewage waste water treatment and other industrial waste water.

  2. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle.

    Science.gov (United States)

    Degani, Ofir; Gepstein, Shimon; Dosoretz, Carlos G

    2002-01-01

    The present study characterized the ability of a bacterial cutinase to improve the wettability of raw cotton fabrics by specific hydrolysis of the cutin structure of the cuticle. The effect of cutinase was studied alone and in coreaction with pectin lyase. The changes in both the fabric and the reaction fluid were measured and compared to enzymatic hydrolysis with polygalacturonase, and to chemical hydrolysis with boiling NaOH. Water absorbancy, specific staining, fabric weight loss, and evaporative light-scattering reverse-phase high-performance liquid chromatography analysis of chloroform extract of the reaction fluid were measured to assess the enzymatic hydrolysis of the cuticle waxy layer. The pattern and extent of hydrolysis of the major cuticle constituents depended on the enzyme type and titers employed and paralleled the degree of wettability obtained. The combination of cutinase and pectin lyase resulted in a synergistic effect. The use of detergents improved enzymatic scouring. The major products released to the reaction medium by the cutinase treatment were identified by gas chromatography/mass spectrometry analysis as C:16 and C:18 saturated fatty acid chains.

  3. Structural and Functional Studies of Aspergillus oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Z.; Gosser, Y; Baker, P; Ravee, Y; Li, H; Butterfoss, G; Kong, X; Gross, R; Montclare, J; et al.

    2009-01-01

    Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

  4. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3

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    Su Lingqia

    2012-01-01

    Full Text Available Abstract Background Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3, and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. Results T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3. In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. Conclusions In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.

  5. Bioleaching of nickel by Aspergillus humicola SKP102 isolated from Indian lateritic overburden

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    Suchhanda Ghosh

    2016-01-01

    Full Text Available The lateritic deposits spread over the Eastern Ghats of Sukinda Valley, Odisha, India, produce a huge amount of overburden annually as a byproduct of chromite mining. This chromite mining overburden contains nickel, the only source of the metal in the country. During this study Aspergillus humicola SKP102, an indigenous fungus isolated from the mining overburden was employed for the leaching of nickel. About 53.89% of the nickel could be leached by the fungus when grown in batch mode using a Czapek dox medium containing 2% (w/v of the mining overburden. The parameters affecting bioleaching were optimized in order to grow the fungus and leach the metal. Of the different options of cheap carbon sources, straw infusion and molasses emerged as viable options for the growth of the fungus and the leaching of nickel. Two-step and indirect techniques were also used for this purpose, and they resulted in 53.09% and 65.04% Ni leaching respectively. Adding diluted sulfuric acid to the leaching medium resulted in 97.05% nickel recovery from the overburden pulp. A. humicola SKP102 could be a potential tool for leaching nickel from the mining overburden.

  6. Expression of fungal cutinase and swollenin in tobacco chloroplasts reveals novel enzyme functions and/or substrates.

    Directory of Open Access Journals (Sweden)

    Dheeraj Verma

    Full Text Available In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG and digalactosyldiacylglycerol (DGDG, in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

  7. Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A(2) and Humicola lanuginosa lipase

    DEFF Research Database (Denmark)

    Balashev, Konstantin; DiNardo, N. John; Callisen, Thomas H.

    2007-01-01

    at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution...

  8. Stabilizing leaf and branch compost cutinase (LCC) with glycosylation: Mechanism and effect on PET hydrolysis.

    Science.gov (United States)

    Shirke, Abhijit N; White, Christine; Englaender, Jacob A; Zwarycz, Allison Sophie; Butterfoss, Glenn L; Linhardt, Robert J; Gross, Richard A

    2018-01-12

    Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited towards these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e. 70 oC). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC's tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 oC. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration.

  9. Expression and characterization of a cutinase (AnCUT2 from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Tammar Khadijah

    2016-01-01

    Full Text Available Cutin hydrolase (EC 3.1.1.74, an extracellular polyesterase found in pollens, bacteria and fungi, is an efficient catalyst that exhibits hydrolytic activity on a variety of water-soluble esters, synthetic fibers, plastics and triglycerides. Thus, cutinase can be used in various applications such as ester synthesis, bio-scouring, food and detergent industries. Ancut2 is one of five genes encoding cutinases present in the Aspergillus niger ATCC 10574 genome. The cDNA of Ancut2 comprising of an open reading frame of 816 bp encoding a protein of 271 amino acid residues, was isolated and expressed in Pichia pastoris. The partially purified recombinant cutinase exhibited a molecular mass of approximately 40 kDa. The enzyme showed highest activity at 40°C with a preference for acidic pH (5.0-6.0. AnCUT2 showed hydrolytic activity towards various p-nitrophenyl esters with preference towards shorter chain esters such as p-nitrophenyl butyrate (C4. Scanning Electron Microscopy demonstrated that AnCUT2 was capable of modifying surfaces of synthetic polycaprolactone and polyethylene terephthalate plastics. The properties of this enzyme suggest that it may be applied in synthetic fiber modification and fruit processing industries.

  10. Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics

    Science.gov (United States)

    Masaki, Kazuo; Kamini, Numbi Ramudu; Ikeda, Hiroko; Iefuji, Haruyuki

    2005-01-01

    A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (ɛ-caprolactone), and poly(3-hydroxybutyrate). PMID:16269800

  11. Isolation of high-affinity human IgE and IgG antibodies recognising Bet v 1 and Humicola lanuginosa lipase from combinatorial phage libraries

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Bødtger, Uffe; Kristensen, Peter

    2004-01-01

    . Human IgE and IgG libraries have been created from patients allergic to birch pollen or lipase. These libraries have been used to select binders recognising the major birch pollen allergen Bet v 1 and Humicola lanuginosa lipase. A panel of allergen-specific IgE and IgG antibodies were identified...

  12. Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.

    Science.gov (United States)

    Ribitsch, Doris; Yebra, Antonio Orcal; Zitzenbacher, Sabine; Wu, Jing; Nowitsch, Susanne; Steinkellner, Georg; Greimel, Katrin; Doliska, Ales; Oberdorfer, Gustav; Gruber, Christian C; Gruber, Karl; Schwab, Helmut; Stana-Kleinschek, Karin; Acero, Enrique Herrero; Guebitz, Georg M

    2013-06-10

    A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ μM (native enzyme) to 0.21 and 0.33 s(-1)/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

  13. The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matak, Mehdi Youssefi [Department of Biophysics, Faculty of Biological Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Moghaddam, Majid Erfani, E-mail: erfani_m@modares.ac.ir [Department of Biophysics, Faculty of Biological Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2009-12-11

    Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

  14. Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources.

    Science.gov (United States)

    de Castro, Aline Machado; Carniel, Adriano; Nicomedes Junior, José; da Conceição Gomes, Absai; Valoni, Érika

    2017-06-01

    Poly(ethylene terephthalate) (PET) is one of the most consumed plastics in the world. The development of efficient technologies for its depolymerization for monomers reuse is highly encouraged, since current recycling rates are still very low. In this study, 16 commercial lipases and cutinases were evaluated for their abilities to catalyze the hydrolysis of two PET samples. Humicola insolens cutinase showed the best performance and was then used in reactions on other PET sources, solely or in combination with the efficient mono(hydroxyethyl terephthalate)-converting lipase from Candida antarctica. Synergy degrees of the final titers of up to 2.2 (i.e., more than double of the concentration when both enzymes were used, as compared to their use alone) were found, with increased terephthalic acid formation rates, reaching a maximum of 59,989 µmol/L (9.36 g/L). These findings open up new possibilities for the conversion of post-consumer PET packages into their minimal monomers, which can be used as drop in at existing industrial facilities.

  15. Evolutionary history of the ancient cutinase family in five filamentous Ascomycetes reveals differential gene duplications and losses and in Magnaporthe grisea shows evidence of sub- and neo-functionalization.

    Science.gov (United States)

    Skamnioti, Pari; Furlong, Rebecca F; Gurr, Sarah J

    2008-01-01

    * The cuticle is the first barrier for fungi that parasitize plants systematically or opportunistically. Here, the evolutionary history is reported of the multimembered cutinase families of the plant pathogenic Ascomycetes Magnaporthe grisea, Fusarium graminearum and Botrytis cinerea and the saprotrophic Ascomycetes Aspergillus nidulans and Neurospora crassa. * Molecular taxonomy of all fungal cutinases demonstrates a clear division into two ancient subfamilies. No evidence was found for lateral gene transfer from prokaryotes. The cutinases in the five Ascomycetes show significant copy number variation, they form six clades and their extreme sequence diversity is highlighted by the lack of consensus intron. The average ratio of gene duplication to loss is 2 : 3, with the exception of M. grisea and N. crassa, which exhibit extreme family expansion and contraction, respectively. * Detailed transcript profiling in vivo, categorizes the M. grisea cutinases into four regulatory patterns. Symmetric or asymmetric expression profiles of phylogenetically related cutinase genes suggest subfunctionalization and neofunctionalization, respectively. * The cutinase family-size per fungal species is discussed in relation to genome characteristics and lifestyle. The ancestry of the cutinase gene family, together with the expression divergence of its individual members provides a first insight into the drivers for niche differentiation in fungi.

  16. A strategic approach for direct recovery and stabilization of Fusarium sp. ICT SAC1 cutinase from solid state fermented broth by carrier free cross-linked enzyme aggregates.

    Science.gov (United States)

    Chaudhari, Sandeep A; Singhal, Rekha S

    2017-05-01

    The major hurdles in commercial exploitation of cutinase (having both esterolytic and lipolytic activities) with potent industrial applications are its high production cost, operational instability and reusability. Although commercially available in immobilized form, its immobilization process (synthesis of support/carrier) makes it expensive. Herein we tried to address multiple issues of production cost, stability, and reusability, associated with cutinase. Waste watermelon rinds, an agroindustrial waste was considered as a cheap support for solid state fermentation (SSF) for cutinase production by newly isolated Fusarium sp. ICT SAC1. Subsequently, carrier free cross-linked enzyme aggregates of cutinase (cut-CLEA) directly from the SSF crude broth were developed. All the process variables affecting CLEA formation along with the different additives were evaluated. It was found that 50% (w/v) of ammonium sulphate, 125μmol of glutaraldehyde, cross-linking for 1h at 30°C and broth pH of 7.0, yielded 58.12% activity recovery. All other additives (hexane, butyric acid, sodium dodecyl sulphate, Trition-X 100, Tween-20, BSA) evaluated presented negative results to our hypothesis. Kinetics and morphology studies confirmed the diffusive nature of cut-CLEA and BSA cut-CLEA. Developed CLEA showed better thermal, solvent, detergent and storage stability, making it more elegant and efficient for industrial biocatalytic process. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Synergistic chemo-enzymatic hydrolysis of poly(ethylene terephthalate) from textile waste.

    Science.gov (United States)

    Quartinello, Felice; Vajnhandl, Simona; Volmajer Valh, Julija; Farmer, Thomas J; Vončina, Bojana; Lobnik, Alexandra; Herrero Acero, Enrique; Pellis, Alessandro; Guebitz, Georg M

    2017-11-01

    Due to the rising global environment protection awareness, recycling strategies that comply with the circular economy principles are needed. Polyesters are among the most used materials in the textile industry; therefore, achieving a complete poly(ethylene terephthalate) (PET) hydrolysis in an environmentally friendly way is a current challenge. In this work, a chemo-enzymatic treatment was developed to recover the PET building blocks, namely terephthalic acid (TA) and ethylene glycol. To monitor the monomer and oligomer content in solid samples, a Fourier-transformed Raman method was successfully developed. A shift of the free carboxylic groups (1632 cm -1 ) of TA into the deprotonated state (1604 and 1398 cm -1 ) was observed and bands at 1728 and 1398 cm -1 were used to assess purity of TA after the chemo-enzymatic PET hydrolysis. The chemical treatment, performed under neutral conditions (T = 250 °C, P = 40 bar), led to conversion of PET into 85% TA and small oligomers. The latter were hydrolysed in a second step using the Humicola insolens cutinase (HiC) yielding 97% pure TA, therefore comparable with the commercial synthesis-grade TA (98%). © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

    Science.gov (United States)

    Pellis, Alessandro; Acero, Enrique Herrero; Weber, Hansjoerg; Obersriebnig, Michael; Breinbauer, Rolf; Srebotnik, Ewald; Guebitz, Georg M

    2015-09-01

    Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Indigo-cellulase interactions

    OpenAIRE

    CAMPOS,RAQUEL; Andreaus, Juergen; Gübitz, Georg M.; Paulo, Artur Cavaco

    2000-01-01

    We have studied the affinity of cellulases from different fungal origins for insoluble indigo dye. Adsorption studies have shown that "acid cellulases" from Trichoderma reesei have a higher affinity for indigo dye than "neutral cellulases" of Humicola insolens. The particle size of indigo dye agglomerates is influenced by cellulase origin and concentra tion. Evidence shows that the nonpolar residues present in higher percentages in the neutral cellulases of H. insolens seem to play an importa...

  20. Enzymatic Degradation of Poly(ethylene 2,5-furanoate Powders and Amorphous Films

    Directory of Open Access Journals (Sweden)

    Simone Weinberger

    2017-10-01

    Full Text Available Poly(ethylene 2,5-furanoate (PEF is arousing great interest as a biobased alternative to plastics like poly(ethylene terephthalate (PET due to its wide range of potential applications, such as food and beverage packaging, clothing, and in the car industry. In the present study, the hydrolysis of PEF powders of different molecular masses (Mn = 55, Mw = 104 kg/mol and Mn = 18, Mw = 29 kg/mol and various particle sizes (180 < d and 180 < d < 425 µm using cutinase 1 from Thermobifida cellulosilytica (Thc_cut1 was studied. Thereby, the effects of molecular mass, particle size and crystallinity on enzymatic hydrolysis were investigated. The results show that particles with lower molecular mass are hydrolyzed faster than those with higher masses, and that the higher the molecular mass, the lower the influence of the particle size on the hydrolysis. Furthermore, cutinases from Humicola insolens (HiC and Thc_cut1 were compared with regard to their hydrolytic activity on amorphous PEF films (measured as release of 2,5-furandicarboxylic acid (FDCA and weight loss in different reaction media (1 M KPO pH 8, 0.1 M Tris-HCl pH 7 and at different temperatures (50 °C and 65 °C. A 100% hydrolysis of the PEF films was achieved after only 72 h of incubation with a HiC in 1 M KPO pH 8 at 65 °C. Moreover, the hydrolysis reaction was monitored by LC/TOF-MS analysis of the released reaction products and by Scanning Electron Microscopy (SEM examination of the polymer surfaces. Enzymatic hydrolysis of PEF with Thc_cut1 and HiC has potential for use in surface functionalization and recycling purposes.

  1. Biological synthesis of silver nanoparticles using the fungus Humicola sp. and evaluation of their cytoxicity using normal and cancer cell lines.

    Science.gov (United States)

    Syed, Asad; Saraswati, Supriya; Kundu, Gopal C; Ahmad, Absar

    2013-10-01

    Nanoscience is a new born science of the modern era and taps into the potential of particles at nanoscale. Bulk materials reduced to nanoscale dimensions thus obtain unique properties such as electronic, optical, magnetic and chemical. As far as synthesis of nanoparticles is concerned, biological synthesis has recently sparked a great interest as compared to other available chemical and physical methods on account of its eco-friendliness and cost-effectiveness. Here we report, for the first time, the biosynthesis of silver nanoparticles by the thermophilic fungus Humicola sp. The fungus when reacted with Ag(+) ions reduces the precursor solution and leads to the formation of extracellular nanoparticles as monitored by ultra violet visible spectroscopy (UV-Vis). The morphology of nanoparticles is found to be spherical with good dispersity as revealed by transmission electron microscopy (TEM). Cell viability assays were carried out to assess the cytotoxicity of silver nanoparticles on NIH3T3 mouse embryonic fibroblast cell line and MDA-MB-231 human breast carcinoma cell line. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study.

    Science.gov (United States)

    Noinville, Sylvie; Revault, Madeleine; Baron, Marie-Hélène; Tiss, Ali; Yapoudjian, Stéphane; Ivanova, Margarita; Verger, Robert

    2002-01-01

    This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface. PMID:11964257

  3. Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system.

    Science.gov (United States)

    Tanaka, Takumi; Nakayama, Mayumi; Takahashi, Toru; Nanatani, Kei; Yamagata, Youhei; Abe, Keietsu

    2017-03-01

    Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA-CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

  4. Dependency of the hydrogen bonding capacity of the solvent anion on the thermal stability of feruloyl esterases in ionic liquid systems

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Ståhlberg, Tim; Nguyen van Buu, Olivier

    2011-01-01

    Three feruloyl esterases, EC 3.1.1.73, (FAEs), namely FAE A from Aspergillus niger (AnFaeA), FAE C from Aspergillus nidulans (AndFaeC), and the FAE activity in a commercial b-glucanase mixture from Humicola insolens (Ultraflo L) were tested for their ability to catalyse esterification of sinapic ...

  5. Mutational analysis of cutinase-like enzyme, Cut190, based on the 3D docking structure with model compounds of polyethylene terephthalate.

    Science.gov (United States)

    Kawabata, Takeshi; Oda, Masayuki; Kawai, Fusako

    2017-07-01

    The cutinase-like enzyme, Cut190, from Saccharomonospora viridis AHK190 can degrade the inner block of polyethylene terephthalate (PET) in the presence of Ca(2+), and its mutant, S226P/R228S, exhibited increased activity and higher thermostability. The crystal structures of the Cut190 S226P mutant in the absence and presence of Ca(2+) were determined, and revealed the large conformational change induced upon Ca(2+) binding. However, the substrate-bound 3D structures of Cut190 remained unknown. In this study, to determine the substrate-binding site and improve the enzyme activity, we first built 3D structures of a PET model compound bound to the crystal structures, using the distance restraints between the scissile carbonyl group of the compound and the catalytic site of the enzyme. We then mutated the putative substrate-binding site predicted from the models, and experimentally determined the enzymatic activities of the mutants for the model substrate poly(butylene succinate-co-adipate). The mutated sites with decreased activity were consistent with the putative binding sites predicted by the 3D model from the Ca(2+)-bound crystal structure, suggesting that the structure of the Ca(2+)-bound state represents the active state. Notably, we generated two mutants with significantly increased activities. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation Cinética de produção de ß-glucosidase por um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose em fermentação submersa

    Directory of Open Access Journals (Sweden)

    Syed Ali Imran Bokhari

    2008-12-01

    Full Text Available A 2-deoxyglucose-resistant mutant (M7 of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG and cellobiose (1% before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organismand M7was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45ºC and 20 g L-1 corncobs in 64 h. Both growth and product formation (17.93 U mL-1 were remarkably high at 45ºC and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g-1 dry corncobs was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.Um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose(M7 foi obtido através de exposição de conídios a raios γ, permitindo a expressão em caldo contendo 0,6% de 2-deoxiglucose (DG e celobiose (1% antes da semeadura em ágar DG esculina citrato de ferro amoniacal, da qual foram selecionadas as col

  7. Fusarium solani pisi cutinase-catalyzed synthesis of polyamides

    NARCIS (Netherlands)

    Stavila, E.; Arsyi, R. Z.; Petrovic, D. M.; Loos, K.

    Polyamides, or nylon, are widely used in fiber and engineering plastic materials, due to their good mechanical and thermal properties. Synthesis of oligomers from nylon-4,10, nylon-6,10, and nylon-8,10 were performed via polycondensation of diamines (1,4-butane-diamine, 1,6-hexanediamine, and

  8. Xyloglucan octasaccharide XXLGol derived from the seeds of hymenaea courbaril acts as a signaling molecule

    Science.gov (United States)

    Vargas-Rechia; Reicher; Rita Sierakowski M; Heyraud; Driguez; Linart

    1998-03-01

    Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-beta-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2, 4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an alpha-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced alpha-l-fucosidase.

  9. Xyloglucan Octasaccharide XXLGol Derived from the Seeds of Hymenaea courbaril Acts as a Signaling Molecule1

    Science.gov (United States)

    Vargas-Rechia, Carem; Reicher, Fany; Rita Sierakowski, Maria; Heyraud, Alain; Driguez, Hugues; Liénart, Yvette

    1998-01-01

    Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-β-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an α-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced α-l-fucosidase. PMID:9501133

  10. Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

    DEFF Research Database (Denmark)

    Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

    2017-01-01

    Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are......, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine...... substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence...

  11. Heterologous expression and biochemical studies of a thermostable glucose tolerant β-glucosidase from Methylococcus capsulatus (bath strain).

    Science.gov (United States)

    Sathe, Sneha S; Soni, Surabhi; Ranvir, Vikas P; Choudhari, Vikram G; Odaneth, Annamma A; Lali, Arvind M; Chandrayan, Sanjeev K

    2017-09-01

    Glucose inhibition of β-glucosidase (BG) is a bottleneck in biomass hydrolysis. In this study, a glucose resistant GH1 β-glucosidase gene- Mbgl from Methylococcus capsulatus (bath strain) was cloned and overexpressed in E.coli. The Ni-NTA affinity purified Mbgl displayed an optimum temperature of 70°C and optimum pH was 6.0. The calculated KM of the enzyme was 48.6mM and 0.12mM for cellobiose and 4-Nitrophenyl β-d-glucopyranoside (PNPG) respectively. PNPG hydrolysis in presence of various glucose concentrations showed that the enzyme was stimulated by ∼2.2 fold at 50mM glucose and was not inhibited up to 450-500mM glucose. Homology modeling and structural comparisons of Mbgl with a glucose tolerant β-glucosidase of Humicola insolens (HiBG) revealed that the Mbgl has a much broader active site unlike to a deep and narrow active site pocket of HiBG. The difference in active site shape reflects on an alternative mechanism of glucose tolerance in Mbgl. Supplementing a commercial cellulase enzyme mixture CTec with Mbgl in the hydrolysis of the pretreated rice straw enhanced the glucose yield by 10-15%. In addition, Mbgl was also stable in organic solvents, detergents and oxidative conditions which would be advantageous for biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Electrical activity of cellobiose dehydrogenase adsorbed on thiols: Influence of charge and hydrophobicity.

    Science.gov (United States)

    Lamberg, P; Hamit-Eminovski, J; Toscano, M D; Eicher-Lorka, O; Niaura, G; Arnebrant, T; Shleev, S; Ruzgas, T

    2017-06-01

    The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens (HiCDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5-6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the HiCDH film was studied. It was found that the presence of charged pyridinium groups was needed to successfully establish direct electron contact between the enzyme and electrode. SAMs formed from hydrophilic charged thiols achieved nearly two times higher current densities compared to hydrophobic charged thiols. Additionally, the results also indicated proportionality between HiCDH catalytic constant and water content of the enzyme film. Enzyme films on charged pyridine thiols had smaller variations in water content and viscoelastic properties than films adsorbed on the more hydrophobic thiols. This work highlights several perspectives on the underlying factors affecting performance of immobilized HiCDH. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

    Science.gov (United States)

    Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

    2011-01-01

    Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

  14. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Svendsen, A.; Langberg, H.

    1998-01-01

    reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S 146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads...

  15. Role of mechanical action in low-temperature cotton scouring with F. solani pisi cutinase and pectate lyase

    NARCIS (Netherlands)

    Agrawal, Pramod; Nierstrasz, Vincent; Warmoeskerken, Marinus

    2008-01-01

    The two principal reasons that can hinder industrial success of bioscouring are the inability to remove cotton waxes at low-temperature and that bioscouring is a slow diffusion controlled process. The main objective of this paper is to develop an improved cotton scouring process by applying

  16. Asp30 of Aspergillus oryzae cutinase CutL1 is involved in the ionic interaction with fungal hydrophobin RolA.

    Science.gov (United States)

    Terauchi, Yuki; Kim, Yoon-Kyung; Tanaka, Takumi; Nanatani, Kei; Takahashi, Toru; Abe, Keietsu

    2017-07-01

    Aspergillus oryzae hydrophobin RolA adheres to the biodegradable polyester polybutylene succinate-co-adipate (PBSA) and promotes PBSA degradation by interacting with A. oryzae polyesterase CutL1 and recruiting it to the PBSA surface. In our previous studies, we found that positively charged amino acid residues (H32, K34) of RolA and negatively charged residues (E31, D142, D171) of CutL1 are important for the cooperative ionic interaction between RolA and CutL1, but some other charged residues in the triple mutant CutL1-E31S/D142S/D171S are also involved. In the present study, on the basis of the 3D-structure of CutL1, we hypothesized that D30 is also involved in the CutL1-RolA interaction. We substituted D30 with serine and performed kinetic analysis of the interaction between wild-type RolA and the single mutant CutL1-D30S or quadruple mutant CutL1-D30S/E31S/D142S/D171S by using quartz crystal microbalance. Our results indicate that D30 is a novel residue involved in the ionic interaction between RolA and CutL1.

  17. Influence of Growth Temperature on Lipid and Soluble Carbohydrate Synthesis by Fungi Isolated from Fellfield Soil in the Maritime Antarctic

    National Research Council Canada - National Science Library

    Richard N. Weinstein; Pedro O. Montiel; Keith Johnstone

    2000-01-01

    The effects of growth temperature on soluble carbohydrate and lipid content of Humicola marvinii, Geomyces pannorum and Mortierella elongata isolated from the Antarctic (Signy Island; 60° 43′S, 45° 38′W) were investigated...

  18. Distribution of sterigmatocystin in filamentous fungi

    DEFF Research Database (Denmark)

    Rank, Christian; Nielsen, Kristian Fog; Larsen, Thomas Ostenfeld

    2011-01-01

    During the last 50y, the carcinogenic mycotoxin sterigmatocystin (ST) has been reported in several phylogenetically and phenotypically different genera: Aschersonia, Aspergillus, Bipolaris, Botryotrichum, Chaetomium, Emericella, Eurotium, Farrowia, Fusarium, Humicola, Moelleriella, Monocillium an...

  19. Isolation of Ascomycetous Fungi from a Tertiary Institution Campus ...

    African Journals Online (AJOL)

    Humicola grisea, Trichophyton rubrum, Helminthosporium cynodontis, Penicillium funiculosum, Penicillium purpurogenum, Saccharomyces cerevisiae, Trichoderma harzianum, Scopulariopsis candida.The physicochemical characteristics of soil samples was found to affect the distribution and population of fungi. The colony ...

  20. The status of some southern African nominal species of Cucumaria ...

    African Journals Online (AJOL)

    A full history of four nominal species of southern African dendrochirotid holothurians, namely Cucumaria jageri Lampert, Semperia (= Cueumaria) sykion Lampert, C. insolens Theel and C. sinorbis Cherbonnier, is given and the confusion in their taxonomic status discussed. Morphological differences and habitat selection ...

  1. Sugar cane bagasse pretreatment: An attempt to enhance the ...

    African Journals Online (AJOL)

    +1.5%NaOH. The pretreatment of bagasse with 2.0% H2O2 along with 1.5% NaOH enhanced the biosynthesis of cellulases by H. insolens. Production rate was also optimized with different parameters like thickness of fermentation medium, ...

  2. Sugar cane bagasse pretreatment: An attempt to enhance the ...

    African Journals Online (AJOL)

    Dr. J. T. Ekanem

    which made rapid rise in enzyme synthesis. With the lapse of time, the susceptible portion was completely hydrolyzed to glucose, which severely inhibited the biosynthesis of cellulolytic enzymes11,12,13. The production of cellulases by H. insolens at different pH (3.0-8.0) of fermentation medium was also studied (Fig. 2).

  3. The status of some southern African nominal species of Cucumaria ...

    African Journals Online (AJOL)

    1986-05-19

    May 19, 1986 ... and the confusion in their taxonomic status discussed. Morphological differences and habitat ... Mediterranean Oenus syraeusanus (Grube) be also classified in the new genus. A key to the three southern. African ... Deichmann's (1948) error in regarding Theel's c. insolens as a growth stage of sykion.

  4. Pseudasthenes, a new genus of ovenbird (Aves: Passeriformes: Furnariidae)

    Science.gov (United States)

    Derryberry, Elizabeth; Claramunt, Santiago; O'Quin, Kelly E.; Aleixo, Alexandre; Chesser, R. Terry; Remsen, J.V.; Brumfield, Robb T.

    2010-01-01

    Phylogenetic analysis of the family Furnariidae (Aves: Passeriformes) indicates that the genus Asthenes is polyphyletic, consisting of two groups that are not sister taxa. Pseudasthenes, a new genus of ovenbird, is described for one of these groups. The four species included in the new genus, formerly placed in Asthenes, are P. humicola, P. patagonica, P. steinbachi, and P. cactorum.

  5. Ring-Closing and Cross-Metathesis with Artificial Metalloenzymes Created by Covalent Active Site-Directed Hybridization of a Lipase

    NARCIS (Netherlands)

    Basauri-Molina, Manuel|info:eu-repo/dai/nl/328200557; Verhoeven, Dide G A|info:eu-repo/dai/nl/369416368; Van Schaik, Arnoldus J.; Kleijn, H|info:eu-repo/dai/nl/304840440; Klein Gebbink, Robertus J M|info:eu-repo/dai/nl/166032646

    2015-01-01

    A series of Grubbs-type catalysts that contain lipase-inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently

  6. Page 1 The Fungicide Kitazin and the Mycoflora of Rice 301 TABLE ...

    Indian Academy of Sciences (India)

    D. halodes (Drechs.) Subram. 'É. and Jain * 曦... 1 1 施○ 影编| 1 1. D. hawaiiensis (Bugnicourt). Subram. and Jain ... 3 . . 磁蟾1 1. 噬命○ 岭1. Alternaria tenuis Nees ... 2 * * ○ ○ 1 2 2 . . 1. Humicola fusco-atra Traaen 4 2 3 ... 1 3 1. 2 2. Sepedonium chrysospermum. (Bulliard) Fries .. * * * * 学瓷歌够1. Trichoderma viride Pers.

  7. Pathogenic fungi in selected lakes in the „Bory Tucholskie” National Park

    Directory of Open Access Journals (Sweden)

    Anna Rózga

    2014-08-01

    Full Text Available The occurrence of potentially pathogenic fungal strains in the 7 lakes of the Struga Siedmiu Jezior and in three lobelia lakes situated in the central part of the "Bory Tucholskie" National Park was investigated. Ten fungal species belonging to 4 genera: Candida (C. humicola, C. famala, C. guilliermondii, Cryptococcus (C. neoformans, C. laurentii, C. albidus, C. unigutndalus, Rhodotoorula (R. rubra and R. glutinis, and Trichosporon (T. cutaneum, were recorded and analysed in the summers of 2001 and 2002

  8. Classification of lipolytic enzymes and their biotechnological applications in the pulping industry

    CSIR Research Space (South Africa)

    Ramnath, L

    2017-03-01

    Full Text Available to release free fatty acids and glycerol in a liquid medium (Villeneuve et al. 2000). In limited liquid envi- ronments, these enzymes are capable of reversing this reaction (esterification) via acidolysis, interesterification, and alcoholysis (Villeneuve et...), Ophistoma sp. (Calero-Rueda et al. 2002), Penicillium sp. (Horne et al. 2002), Aspergillus sp. (Giuliani et al. 2001), Humicola sp. (Hatzakis et al. 2003), Sporotrichum sp. (Topakas et al. 2003), Saccharomyces sp. (Lomolino et al. 2003), Candida sp. (Ghosh...

  9. New and Interesting Records of South African Fungi, Part VI

    Directory of Open Access Journals (Sweden)

    G. C. A. van der Westhuizen

    1969-12-01

    Full Text Available Six species of fungi, recorded for the first time in South Africa, are described. The species are Gelasinospora cerealis Dowding from roots of  Eucalyptus saligna; Spegazzinia tessarthra (B. & C. Saccardo from  Zea mays; Melampsora larici-populina Klebahn from  Populus deltoides; Saccobolus depauperatus (Berk. & Hr. Phill. from horse dung;  Humicola stellata Bunce from grass hay and  Chaetomium cochliodes Palliser from garden soil.

  10. Direct site-directed photocoupling of proteins onto surfaces coated with β-cyclodextrins

    DEFF Research Database (Denmark)

    Städe, Lars W; Wimmer, Reinhard; Stensballe, Allan

    2010-01-01

    . Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A molecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion......A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag...... is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli...

  11. Effect of pH on the solubilization of brewers' spent grain by microbial carbohydrases and proteases.

    Science.gov (United States)

    Faulds, Craig B; Robertson, James A; Waldron, Keith W

    2008-08-27

    The potential for enzymatic solubilization of brewers' spent grain by carbohydrases and proteases was examined over a broad pH range (pH 3.2-11.2). Enzymes from Trichoderma (Depol 686) were most efficient at a lower pH, while enzymes from the Humicola preparation (Depol 740) were the best performer over the whole range. Profiling of key glycoside hydrolase, esterase and protease activities across the pH range demonstrated that solubilization of spent grain by the Trichoderma enzymes corresponded to the range of maximum activities. This was not the case with the Humicola enzymes, where maximum solubilization of the substrate occurred at pH 9.1, at which pH the determined activities were low. Protease activity in Depol 740 was associated with a high solubilization, but inhibition of proteolytic activity resulted in only a 5% decrease in spent grain solubilization. These results suggest that while enzymes can be used to exploit agro-industrials byproduct, the use of high pH increases the extent of hydrolysis and an unidentified factor produced by Humicola improves the enzyme-catalyzed solubilization of lignocellulosic material.

  12. Manganese tolerance in yeasts involves polyphosphate, magnesium, and vacuolar alterations.

    Science.gov (United States)

    Ryazanova, Lubov; Zvonarev, Anton; Rusakova, Tatiana; Dmitriev, Vladimir; Kulakovskaya, Tatiana

    2016-07-01

    Basidiomycetous and ascomycetous yeast species were tested for manganese tolerance. Basidiomycetous Cryptococcus humicola, Cryptococcus terricola, Cryptococcus curvatus and ascomycetous Candida maltosa, Kluyveromyces marxianus, Kuraishia capsulata, Lindnera fabianii and Sacharomyces cerevisiae were able to grow at manganese excess (2.5 mmol/L), while the growth of basidiomycetous Rhodotorula bogoriensis was completely suppressed. The lag phase duration increased and the exponential growth rate decreased at manganese excess. The increase of cell size and enlargement of vacuoles were characteristics for the cells grown at manganese excess. The alterations in inorganic polyphosphate content and cellular localization were studied. L. fabianii, K. capsulata, C. maltosa, and Cr. humicola accumulated the higher amounts of inorganic polyphosphates, while Cr. terricola and Cr. curvatus demonstrated no such accumulation. The polyphosphate content in the cell wall tested by DAPI staining increased in all species under the study; however, this effect was more pronounced in Cr. terricola and Cr. curvatus. The accumulation of Mg(2+) in the cell wall under Mn(2+) excess was observed in Cr. humicola, Cr. curvatus and Cr. terricola. The accumulation of polyphosphate and magnesium in the cell wall was supposed to be a factor of manganese tolerance in yeasts.

  13. Functional analysis of FarA transcription factor in the regulation of the genes encoding lipolytic enzymes and hydrophobic surface binding protein for the degradation of biodegradable plastics in Aspergillus oryzae.

    Science.gov (United States)

    Garrido, Sharon Marie; Kitamoto, Noriyuki; Watanabe, Akira; Shintani, Takahiro; Gomi, Katsuya

    2012-05-01

    FarA is a Zn(II)(2)Cys(6) transcription factor which upregulates genes required for growth on fatty acids in filamentous fungi like Aspergillus nidulans. FarA is also highly similar to the cutinase transcription factor CTF1α of Fusarium solani which binds to the cutinase gene promoter in this plant pathogen. This study determines whether FarA transcriptional factor also works in the regulation of genes responsible for the production of cutinase for the degradation of a biodegradable plastic, poly-(butylene succinate-co-adipate) (PBSA), in Aspergillus oryzae. The wild-type and the farA gene disruption strains were grown in minimal agar medium with emulsified PBSA, and the wild-type showed clear zone around the colonies while the disruptants did not. Western blot analysis revealed that the cutinase protein CutL1 and a hydrophobic surface binding protein such as HsbA were produced by the wild-type but not by the disruptants. In addition, the expressions of cutL1, triacylglycerol lipase (tglA), and mono- and di-acylglycerol lipase (mdlB) genes as well as the hsbA gene were significantly lower in the disruptants compared to the wild-type. These results indicated that the FarA transcriptional factor would be implicated in the expression of cutL1 and hsbA genes that are required for the degradation of PBSA as well as lipolytic genes such as mdlB and tglA for lipid hydrolysis. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Fungus mediated synthesis of biomedically important cerium oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Shadab Ali; Ahmad, Absar, E-mail: a.ahmad@ncl.res.in

    2013-10-15

    Graphical abstract: - Highlights: • First time biological synthesis of cerium oxide oxide nanoparticles using fungus Humicola sp. • Complete characterization of cerium oxide nanoparticles. • Biosynthesis of naturally protein capped, luminescent and water dispersible CeO{sub 2} nanoparticles. • Biosynthesized CeO{sub 2} nanoparticles can be used for many biomedical applications. - Abstract: Nanomaterials can be synthesized by chemical, physical and the more recently discovered biological routes. The biological routes are advantageous over the chemical and physical ones as unlike these, the biological synthesis protocols occur at ambient conditions, are cheap, non-toxic and eco-friendly. Although purely biological and bioinspired methods for the synthesis of nanomaterials are environmentally benign and energy conserving processes, their true potential has not been explored yet and attempts are being made to extend the formation of technologically important nanoparticles using microorganisms like fungi. Though there have been reports on the biosynthesis of oxide nanoparticles by our group in the past, no attempts have been made to employ fungi for the synthesis of nanoparticles of rare earth metals or lanthanides. Here we report for the first time, the bio-inspired synthesis of biomedically important cerium oxide (CeO{sub 2}) nanoparticles using the thermophilic fungus Humicola sp. The fungus Humicola sp. when exposed to aqueous solutions of oxide precursor cerium (III) nitrate hexahydrate (CeN{sub 3}O{sub 9}·6H{sub 2}O) results in the extracellular formation of CeO{sub 2} nanoparticles containing Ce (III) and Ce (IV) mixed oxidation states, confirmed by X-ray Photoemission Spectroscopy (XPS). The formed nanoparticles are naturally capped by proteins secreted by the fungus and thus do not agglomerate, are highly stable, water dispersible and are highly fluorescent as well. The biosynthesized nanoparticles were characterized by UV–vis spectroscopy

  15. Yeasts in pigeon feacal droppings in Lisbon - Portugal, 1994

    Directory of Open Access Journals (Sweden)

    Herminia Maria Lourdes Martins

    1997-10-01

    Full Text Available In this work, the results of a preliminary survey held in city of Lisbon. Eighty faecal samples were examined between Summer and Autumn, 1994, from twelve different urban areas, mainly near churchs and monuments where birds nest, rest or eat. From each sample 1 g was weighted and suspensed in 10 ml of destilled sterilized water and consecutive decimal diluitions were executed. Yeasts were enumerated and grouped by species, based on morphological types. On eighty faecal samples the most prevalent yeasts identified were: Candida humicola (51.5%, Candida albicans (48.7%, Cryptococcus neoformans (5% and Trichosporon cutaneum (37.5%.

  16. PRODUÇÃO, CARACTERIZAÇÃO E AVALIAÇÃO DE ENZIMAS FIBROLÍTICAS NA DIGESTIBILIDADE DA FORRAGEM DE MILHO

    Directory of Open Access Journals (Sweden)

    Cristine dos Santos Settimi Cysneiros

    2013-12-01

    Full Text Available The objectives of this research were to produce and characterize an enzyme complex (EC, using the fungus Humicola grisea, and evaluate its effect on true digestibility of forage maize dry matter. We observed that the fungus produced cellulase, b-glucosidase and xylanase enzymes. The cellulase and xylanase activities were high at the temperature of 50° C. The optimum temperature of β-glucosidase was between 50 and 60° C. The optimum pH of cellulase and xylanase enzyme was 6.0. As for β-glucosidase, the enzyme showed higher activity at pH 6.5. Cellulase remained stable for 60 minutes at 39° C. Xylanase and β-glucosidase maintained 99.2 and 88.2% of their activity at 50° C for 240 minutes, respectively. The treatments were as follows: control (10 mL of sterile water, level 1 (2.5 mL EC, level 2 (5.0 mL EC and level 3 (10 mL EC. In the digestibility experiment, there was interaction between enzyme levels and time of incubation in the rumen. The addition of 10 mL of the fibrolytic enzymes improved the digestibility at 10.58; 12.52; 9.05 and 6.81% compared to control for 12; 24; 48 and 96 hours of incubation, respectively. The fungus Humicola grisea is an enzyme producer that is important in ruminant feed.

  17. Antarctic Yeasts: Biodiversity and Potential Applications

    Science.gov (United States)

    Shivaji, S.; Prasad, G. S.

    This review is an attempt in cataloguing the diversity of yeasts in Antarctica, highlight their biotechnological potential and understand the basis of adaptation to low temperature. As of now several psychrophilic and psychrotolerant yeasts from Antarctic soils and marine waters have been characterized with respect to their growth characteristics, ecological distribution and taxonomic significance. Interestingly most of these species belonged to basidiomycetous yeasts which as a group are known for their ability to circumvent and survive under stress conditions. Simultaneously their possible role as work horses in the biotechnological industry was recognized due to their ability to produce novel enzymes and biomolecules such as agents for the breakdown of xenobiotics, and novel pharmaceutical chemi cals. The high activity of psychrophilic enzymes at low and moderate temperatures offers potential economic benefits. As of now lipases from Pseudozyma antarctica have been extensively studied to understand their unique thermal stability at 90°C and also because of its use in the pharmaceutical, agriculture, food, cosmetics and chemical industry. A few of the other enzymes which have been studied include extracellular alpha-amylase and glucoamylase from the yeast Pseudozyma antarctica (Candida antarctica), an extra-cellular protease from Cryptococcus humicola, an aspartyl proteinase from Cryptococcus humicola, a novel extracellular subtilase from Leucosporidium antarcticum, and a xylanase from Cryptococcus adeliensis

  18. Microbiological Assessment of Poultry Feeds within Ilorin, Nigeria

    Directory of Open Access Journals (Sweden)

    Ismaila Olawale SULE

    2017-03-01

    Full Text Available The poultry feeds were obtained from 20 different poultry pens and their microbial contents were assessed. The antibiotics resistance patterns of the bacterial isolates were also determined. The bacterial count ranged from 5.0 × 103 to 1.76 × 106 cfu/g while the fungal count ranged from 3.5 × 104 to 1.9 × 105 cfu/g. The bacterial species isolated were Streptococcus salivarius, Streptococcus pyogenes, Micrococcus luteus, Micrococcus varians, Micrococcus roseus, Staphylococcus aureus, Staphylococcus saprophyticus and Staphylococcus hominis, while the fungal species isolated were Saccharomyces cerevisisae, Fusarium oxysporum, Penicillium sp., Humicola grisea, Aspergillus fumigatus, Hansenula sp. and Humicola fuscoatra. All the bacterial isolates were resistant to ceftazidime and cefuroxime and all the isolates were resistant to at least three antibiotics. Ofloxacin produced the highest zone of inhibition, followed by gentamicin, and then erythromycin. The presence of some pathogenic microorganisms in the poultry feeds revealed high level of contaminations. It is recommended that poultry feeds should be made from good quality grains and it should be prevented from environmental or other contamination.

  19. Further studies on Egyptian soil fungi: succession of sugar and osmophilic fungi in soil amended with five organic substrates.

    Science.gov (United States)

    Shaban, G M

    1996-01-01

    The sugar and osmophilic fungal composition of soils amended with five organic substrates (newspaper, orange peel, bromegrass leaves, wheat straw and wood sawdust) was estimated after 2, 4, 6, 8 and 10 weeks using the dilution plate method on glucose and 50% sucrose Czapek's agar media. Wheat straw was the best substrate for total counts of both sugar and osmophilic fungi followed by newspaper, bromegrass leaves, wood sawdust and orange peel. Wood sawdust supported the highest average counts of total sugar fungi, Fusarium, Mucor, Scopulariopsis, Trichoderma and Trimmatostroma spp.; Newspaper, of Aspergillus (8 spp.), Penicillium (4 spp.) and Chaetomium sp.; bromegrass leaves of Cladosporium sp., Humicola sp. and Sporotrichum sp.; orange peel, of Alternaria sp., Circinella sp. and Stachybotrys sp.; and wheat straw, of Botryotrichum sp. and Myrothecium sp. Bromegrass leaves and orange peel supported the highest average counts of total osmophilic fungi, Aspergillus (10 spp.), Cladosporium sp. Paecillomyces sp. and Rhizopus sp.; and of Stemphylium sp., Trichoderma sp., Humicola sp. and Circinella sp. respectively; wheat straw, of Epicoccum sp., Scopulariopsis sp. and Trichothecium sp.; newspaper, of Penicillium (4 spp.) and Alternaria sp.; and wood sawdust of Curvularia sp. and Fusarium (3 spp.). The best colonizers throughout the experimental periods were Aspergilus and Penicillium spp.

  20. Genomics of Aspergillus oryzae: Learning from the History of Koji Mold and Exploration of Its Future

    Science.gov (United States)

    Machida, Masayuki; Yamada, Osamu; Gomi, Katsuya

    2008-01-01

    At a time when the notion of microorganisms did not exist, our ancestors empirically established methods for the production of various fermentation foods: miso (bean curd seasoning) and shoyu (soy sauce), both of which have been widely used and are essential for Japanese cooking, and sake, a magical alcoholic drink consumed at a variety of ritual occasions, are typical examples. A filamentous fungus, Aspergillus oryzae, is the key organism in the production of all these traditional foods, and its solid-state cultivation (SSC) has been confirmed to be the secret for the high productivity of secretory hydrolases vital for the fermentation process. Indeed, our genome comparison and transcriptome analysis uncovered mechanisms for effective degradation of raw materials in SSC: the extracellular hydrolase genes that have been found only in the A. oryzae genome but not in A. fumigatus are highly induced during SSC but not in liquid cultivation. Also, the temperature reduction process empirically adopted in the traditional soy-sauce fermentation processes has been found to be important to keep strong expression of the A. oryzae-specific extracellular hydrolases. One of the prominent potentials of A. oryzae is that it has been successfully applied to effective degradation of biodegradable plastic. Both cutinase, responsible for the degradation of plastic, and hydrophobin, which recruits cutinase on the hydrophobic surface to enhance degradation, have been discovered in A. oryzae. Genomic analysis in concert with traditional knowledge and technology will continue to be powerful tools in the future exploration of A. oryzae. PMID:18820080

  1. Production of a biodegradable plastic-degrading enzyme from cheese whey by the phyllosphere yeast Pseudozyma antarctica GB-4(1)W.

    Science.gov (United States)

    Watanabe, Takashi; Shinozaki, Yukiko; Suzuki, Ken; Koitabashi, Motoo; Yoshida, Shigenobu; Sameshima-Yamashita, Yuka; Kuze Kitamoto, Hiroko

    2014-08-01

    Cheese whey is a by-product of cheese production and has high concentrations of lactose (about 5%) and other nutrients. Pseudozyma antarctica produces a unique cutinase-like enzyme, named PaE, that efficiently degrades biodegradable plastics. A previous study showed that a combination of 1% oil and 0.5% lactose increased cutinase-like enzyme production by another species of yeast. In this study, to produce PaE from cheese whey, we investigated the effects of soybean oil on PaE production (expressed as biodegradable plastic-degrading activity) by P. antarctica growing on lactose or cheese whey. In flask cultures, the final PaE activity was only 0.03 U/ml when soybean oil was used as the sole carbon source, but increased to 1.79 U/ml when a limited amount of soybean oil (under 0.5%) was combined with a relatively high concentration of lactose (6%). Using a 5-L jar fermentor with lactose fed-batch cultivation and periodic soybean oil addition, about 14.6 U/ml of PaE was obtained after 5 days of cultivation. When the lactose was replaced with cheese whey, PaE production was 10.8 U/ml after 3 days of cultivation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Enzymatic hydrolysis of poly(ethyleneterephthalate) used for and analysed by pore modification of track-etched membranes.

    Science.gov (United States)

    Gamerith, Caroline; Gajda, Martyna; Ortner, Andreas; Acero, Enrique Herrero; Guebitz, Georg M; Ulbricht, Mathias

    2017-10-25

    The potential of limited enzymatic poly(ethylene terephthalate) (PET) surface hydrolysis for the modification of track-etched (TE) membranes was investigated. Cutinases 1 and 2 from Thermobifida cellulosilytica as well as a fusion protein of cutinase 1 with the polymer binding module from the polyhydroxyalkanoate depolymerase of Alcaligenes faecalis (Thc_Cut1_PBM) were shown to hydrolyse highly crystalline PET TE membranes with a pore diameter of ∼120nm at very narrow size distribution. Furthermore the effects of surface chemistry were investigated by comparison of enzymatic hydrolysis by Thc_Cut1_PBM of "as received" PET TE membranes with two surface functionalized versions towards a "hydrophilic" and a more "hydrophobic" surface. The effects of adsorbed protein and the efficacy of cleaning steps after enzymatic treatment were elucidated by complementary methods for surface analysis and membrane characterization. With the optimized cleaning protocol, all adsorbed protein could be removed from the enzyme-treated membranes and effects of chemical surface functionalization of the PET TE membranes were demonstrated. The highest efficiency of enzymatic surface hydrolysis was observed for the original PET TE membranes, leading to an 0.36% weight loss corresponding to a removal of ∼3nm PET from the entire surface of the porous membrane. This correlates very well with the measured increase of barrier pore diameter by 4nm (a radius reduction? of 2nm), leading to about a two-fold increased water permeability. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods

    Science.gov (United States)

    Tang, Pei-Ling; Hassan, Osman; Maskat, Mohamad Yusof; Badri, Khairiah

    2015-01-01

    In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17 ± 49.44 ppm hydroxybenzoic acid, 5.67 ± 0.25 ppm p-hydroxybenzaldehyde, 25.57 ± 1.64 ppm vanillic acid, 168.68 ± 23.23 ppm vanillin, 75.44 ± 6.71 ppm syringic acid, 815.26 ± 41.77 ppm syringaldehyde, 15.21 ± 2.19 ppm p-coumaric acid, and 44.75 ± 3.40 ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about 642.83 ± 14.45 ppm hydroxybenzoic acid, 70.19 ± 3.31 ppm syringaldehyde, 22.80 ± 1.04 ppm vanillin, 27.06 ± 1.20 ppm p-coumaric acid, and 50.19 ± 2.23 ppm ferulic acid were produced. PMID:26798644

  4. Diversity and dynamics of the microbial community on decomposing wheat straw during mushroom compost production.

    Science.gov (United States)

    Zhang, Xi; Zhong, Yaohua; Yang, Shida; Zhang, Weixin; Xu, Meiqing; Ma, Anzhou; Zhuang, Guoqiang; Chen, Guanjun; Liu, Weifeng

    2014-10-01

    The development of communities of three important composting players including actinobacteria, fungi and clostridia was explored during the composting of wheat straw for mushroom production. The results revealed the presence of highly diversified actinobacteria and fungal communities during the composting process. The diversity of the fungal community, however, sharply decreased in the mature compost. Furthermore, an apparent succession of both actinobacteria and fungi with intensive changes in the composition of communities was demonstrated during composting. Notably, cellulolytic actinomycetal and fungal genera represented by Thermopolyspora, Microbispora and Humicola were highly enriched in the mature compost. Analysis of the key cellulolytic genes revealed their prevalence at different composting stages including several novel glycoside hydrolase family 48 exocellulase lineages. The community of cellulolytic microbiota also changed substantially over time. The prevalence of the diversified cellulolytic microorganisms holds the great potential of mining novel lignocellulose decomposing enzymes from this specific ecosystem. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Extracellular biosynthesis of gadolinium oxide (Gd2O3 nanoparticles, their biodistribution and bioconjugation with the chemically modified anticancer drug taxol

    Directory of Open Access Journals (Sweden)

    Shadab Ali Khan

    2014-03-01

    Full Text Available As a part of our programme to develop nanobioconjugates for the treatment of cancer, we first synthesized extracellular, protein-capped, highly stable and well-dispersed gadolinium oxide (Gd2O3 nanoparticles by using thermophilic fungus Humicola sp. The biodistribution of the nanoparticles in rats was checked by radiolabelling with Tc-99m. Finally, these nanoparticles were bioconjugated with the chemically modified anticancer drug taxol with the aim of characterizing the role of this bioconjugate in the treatment of cancer. The biosynthesized Gd2O3 nanoparticles were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM, X-ray diffraction (XRD and X-ray photoemission spectroscopy (XPS. The Gd2O3–taxol bioconjugate was confirmed by UV–vis spectroscopy and fluorescence microscopy and was purified by using high performance liquid chromatography (HPLC.

  6. Current progress in crystallographic studies of new lipases from filamentous fungi.

    Science.gov (United States)

    Derewenda, U; Swenson, L; Green, R; Wei, Y; Yamaguchi, S; Joerger, R; Haas, M J; Derewenda, Z S

    1994-04-01

    Lipases from filamentous fungi have been studied extensively over many years. They exhibit properties attractive for industrial applications, e.g. in laundry detergents, tanning and paper industries and stereospecific organic synthesis. Enzymes from the fungi Rhizomucor miehei and Geotrichum candidum have been among the first neutral lipases to be characterized structurally by X-ray diffraction methods. In this paper we report a preliminary account of crystallographic studies of three other fungal lipases homologous to that from R. miehei and obtained from Humicola lanuginosa, Penicillium camembertii and Rhizopus delemar. These newly characterized structures have important implications for our understanding of structure-function relationships in lipases in general and the molecular basis of interfacial activation.

  7. Black yeast-like fungi in skin and nail

    DEFF Research Database (Denmark)

    Saunte, D M; Tarazooie, B; Arendrup, M C

    2011-01-01

    Black yeast-like fungi are rarely reported from superficial infections. We noticed a consistent prevalence of these organisms as single isolations from mycological routine specimens. To investigate the prevalence of black yeast-like fungi in skin, hair and nail specimens and to discuss the probab......Black yeast-like fungi are rarely reported from superficial infections. We noticed a consistent prevalence of these organisms as single isolations from mycological routine specimens. To investigate the prevalence of black yeast-like fungi in skin, hair and nail specimens and to discuss...... prevalent species were Phialophora europaea (n = 29), Coniosporium epidermidis (n = 12), Ochroconis cf. humicola (n = 6) and Cladophialophora boppii (n = 4). These are not common saprobes and thus less likely to be coincidental colonizers. In 10/30 cases, discolouration of nail/skin had been noticed...

  8. Pilot project at Hazira, India, for capture of carbon dioxide and its biofixation using microalgae.

    Science.gov (United States)

    Yadav, Anant; Choudhary, Piyush; Atri, Neelam; Teir, Sebastian; Mutnuri, Srikanth

    2016-11-01

    The objective of the present study was to set up a small-scale pilot reactor at ONGC Hazira, Surat, for capturing CO 2 from vent gas. The studies were carried out for CO 2 capture by either using microalgae Chlorella sp. or a consortium of microalgae (Scenedesmus quadricauda, Chlorella vulgaris and Chlorococcum humicola). The biomass harvested was used for anaerobic digestion to produce biogas. The carbonation column was able to decrease the average 34 vol.% of CO 2 in vent gas to 15 vol.% of CO 2 in the outlet gas of the carbonation column. The yield of Chlorella sp. was found to be 18 g/m 2 /day. The methane yield was 386 l CH 4 /kg VS fed of Chlorella sp. whereas 228 l CH 4 /kg VS fed of the consortium of algae.

  9. Phenotypical characterization of Candida spp. isolated from crop of parrots (Amazona spp. Caracterização fenotípica de Candida spp. isoladas de inglúvio de papagaios (Amazona spp.

    Directory of Open Access Journals (Sweden)

    Renata G. Vieira

    2009-06-01

    Full Text Available The purpose of this study was to characterize Candida isolates from crop of parrots. Forty baby parrots of genus Amazona, species aestiva and amazonica that were apprehended from wild animal traffic were used: 18 presented ingluvitis and 22 other alterations, but showing general debilitation. Samples were seeded on Sabouraud dextrose agar with chloramphenicol after be obtained by the introduction of urethral probe through the esophagus. Based on morphology and biochemical reactions (API 20C Candida was confirmed; it was still searched the production of proteinase and phospholipase, virulence factors for Candida species. Candida spp. were isolated from 57.5% parrots, being 72.2% from birds with ingluvitis and 45.5% from without ones. Twenty-five strains of Candida were isolated, 60% and 40%, respectively from parrots with and without ingluvitis, and were speciated: 28% C. humicola, 24% C. parapsilosis, 20% C. guilliermondii, 20% C. famata, and 8% C. albicans. These results demonstrate that C. albicans is not the most frequent species isolated, and it is the first report that shows C. guilliermondii, C. famata, and C. humicola causing infection in parrots. Many isolates presented filamentation (76%, 100% produced proteinase and 68% phospholipase. The observation of Candida spp. producing virulence factors reinforce the pathogenic role of these yeasts in the cases studied.O objetivo do presente trabalho foi caracterizar cepas de Candida spp. isoladas de inglúvio de papagaios. Foram utilizados 40 papagaios do gênero Amazona, espécies aestiva e amazonica, apreendidos de tráfico de animais selvagens: 18 apresentavam ingluvite e 22 outras alterações, mas todos mostrando sinais de debilitação geral. Colheram-se as amostras clínicas através da introdução de sonda uretral no esôfago dos animais e estas foram semeadas em ágar Sabouraud dextrose acrescido de cloranfenicol. A identificação das espécies de Candida foi baseada em caracter

  10. Hyphomycetes from soil of an area affected by copper mining activities in the State of Bahia, Brazil Hyphomycetes de solo de uma área de mineração de cobre no Estado da Bahia, Brasil

    Directory of Open Access Journals (Sweden)

    Isabella P.M. Wanderley Costa

    2006-09-01

    Full Text Available With the aim of observing the impact produced by copper-mining activities on soil fungi, samples were collected from an area at the Caraíba Mining, in the State of Bahia, Brazil. This area was divided in six sub-areas: one had native vegetation and was used as control, while the others varied according to degrees of impact. The samples, collected during the dry and the rainy seasons, were submitted to serial dilutions and placed on Petri dishes with Sabouraud medium plus antibiotic. Sixty five species and 16 genera of Hyphomycetes were identified: Acremonium, Acrophialophora, Aspergillus, Cladosporium, Chrysosporium, Curvularia, Fusarium, Humicola, Malbranchea, Myrothecium, Paecilomyces, Penicillium, Scolecobasidium, Staphylotrichum, Stilbella and Trichoderma.Acrophialophora levis, Crhysosporium merdarium, Curvularia verruculosa, Malbranchea chrysosporoidea, Penicillium adametzii, Staphylotrichum coccosporum and Stilbella sebacea were isolated for the first time in Brazil.Com o objetivo de observar o impacto produzido pelas atividades da mineração de cobre em fungos do solo, amostras foram coletadas de uma área da Mineração Caraíba no Estado da Bahia, Brasil. Esta área foi dividida em seis sub-áreas: uma com vegetação nativa, usada como controle enquanto as outras variavam de acordo com os graus de impacto. As amostras, coletadas durante os períodos de estiagem e chuvoso, foram submetidas a diluições sucessivas e colocadas em placas de Petri contendo meio Sabouraud acrescido de antibiótico. Sessenta e cinco espécies e 16 gêneros de Hyphomycetes foram identificados: Acremonium, Acrophialophora, Aspergillus, Cladosporium, Chrysosporium, Curvularia, Fusarium, Humicola, Malbranchea, Myrothecium, Paecilomyces, Penicillium, Scolecobasidium, Staphilotricum, Stilbella e Trichoderma.Acrophialophora levis, Chrysosporium merdarium, Curvularia verruculosa, Malbranchea chrysosporoidea, Penicillium adametzii, Staphylotrichum coccosporum e

  11. An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

    Directory of Open Access Journals (Sweden)

    Rohe Peter

    2012-10-01

    Full Text Available Abstract Background High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. Results To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. Conclusions The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput

  12. An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

    Science.gov (United States)

    2012-01-01

    Background High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. Results To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. Conclusions The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and

  13. An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform.

    Science.gov (United States)

    Rohe, Peter; Venkanna, Deepak; Kleine, Britta; Freudl, Roland; Oldiges, Marco

    2012-10-31

    High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved

  14. Morphological and physiological comparison of taxa comprising the Sporothrix schenckii complex.

    Science.gov (United States)

    Zhao, Ming-dan; Zhou, Xun; Liu, Ting-ting; Yang, Zhi-bang

    2015-11-01

    Based on recent molecular data, it has been suggested that Sporothrix globosa is the main causal agent of sporotrichosis in China. The objective of this study was to compare the morphology, growth characteristics, patterns of carbon source usage, and susceptibility to antifungal agents among Sporothrix strains. A total of 15 clinical strains confirmed to be S. globosa, from three different regions of China, and 11 ex-type strains from the CBS-KNAW biodiversity center were obtained. The elongated conidia of S. pallida, S. variecibatus, S. schenckii, and S. schenckii luriei were clearly different from the subglobose and globose conidia of S. globosa strains. S. schenckii is able to assimilate sucrose, raffinose, and ribitol. Susceptibility profiles of these Sporothrix species were evaluated by measuring minimum inhibitory concentrations (MICs). Fluconazole, itraconazole, terbinafine, and amphotericin B showed good activity against most S. globosa clinical isolates from China. Potassium iodide also showed a low MIC against S. pallida, while fluconazole showed a high MIC for S. mexicana, S. humicola, S. globosa, S. schenckii, and S. inflata; these strains might be considered tolerant. The species showed differences in susceptibility to antifungal drugs and should therefore be properly identified during diagnosis prior to designing therapeutic strategies.

  15. Morphological and physiological comparison of taxa comprising the Sporothrix schenckii complex*

    Science.gov (United States)

    ZHAO, Ming-dan; ZHOU, Xun; LIU, Ting-ting; YANG, Zhi-bang

    2015-01-01

    Based on recent molecular data, it has been suggested that Sporothrix globosa is the main causal agent of sporotrichosis in China. The objective of this study was to compare the morphology, growth characteristics, patterns of carbon source usage, and susceptibility to antifungal agents among Sporothrix strains. A total of 15 clinical strains confirmed to be S. globosa, from three different regions of China, and 11 ex-type strains from the CBS-KNAW biodiversity center were obtained. The elongated conidia of S. pallida, S. variecibatus, S. schenckii, and S. schenckii luriei were clearly different from the subglobose and globose conidia of S. globosa strains. S. schenckii is able to assimilate sucrose, raffinose, and ribitol. Susceptibility profiles of these Sporothrix species were evaluated by measuring minimum inhibitory concentrations (MICs). Fluconazole, itraconazole, terbinafine, and amphotericin B showed good activity against most S. globosa clinical isolates from China. Potassium iodide also showed a low MIC against S. pallida, while fluconazole showed a high MIC for S. mexicana, S. humicola, S. globosa, S. schenckii, and S. inflata; these strains might be considered tolerant. The species showed differences in susceptibility to antifungal drugs and should therefore be properly identified during diagnosis prior to designing therapeutic strategies. PMID:26537212

  16. Endophytic bacterial and fungal communities transmitted from cotyledons and germs in peanut (Arachis hypogaea L.) sprouts.

    Science.gov (United States)

    Huang, Yali; Kuang, Zaoyuan; Deng, Zujun; Zhang, Ren; Cao, Lixiang

    2017-07-01

    Seed-borne endophytes could be transmitted into sprouts. Whether this happened in peanuts and the difference between microbial taxa in peanut germs and cotyledons remain unknown. In this research, Illumina-based sequencing was employed to investigate the microbial taxa in peanut germs, cotyledons, and sprouts. Sulfur-oxidizing bacteria was isolated and inoculated into peanut sprouts, and then, the growth of peanut seedlings was measured. The results illustrated that diverse bacteria and fungi were detected in peanut germs, cotyledons, and sprouts. The number of bacterial OTUs declined with the germination from germs and cotyledons to sprouts. However, the number of fungal OTUs increased during the seedling procedure. Seed-borne dominant bacterial genera Halothiobacillus and Synechococcus and fungal genera Humicola, Emericella, and Penicillium were detected in sprouts. Based on the endophytic community information, the Halothiobacillus strains were isolated from sprouts. Pot experiments that illustrated the growth of peanut seedlings inoculated with the strain were promoted. These results provide new understanding into plant-microbe interactions in peanut and suggest that the selection for biocontrol agents based on mycobiome and bacteriome analysis is reliable and feasible compared with the present greenhouse selection.

  17. Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant Azadirachta indica A. Juss., and Impact of Culture Media on their Isolation.

    Science.gov (United States)

    Verma, Vijay C; Gond, Surendra K; Kumar, Anuj; Kharwar, Ravindra N; Boulanger, Lori-Ann; Strobel, Gary A

    2011-10-01

    Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.

  18. Antimicrobial Activity of Extracts of the Oyster Culinary Medicinal Mushroom Pleurotus ostreatus (Higher Basidiomycetes) and Identification of a New Antimicrobial Compound.

    Science.gov (United States)

    Younis, Ahmed M; Wu, Fang-Sheng; El Shikh, Hussien H

    2015-01-01

    Pleurotus ostreatus is an edible mushroom that also has high medicinal values. In this study, P. ostreatus was tested for its ability to inhibit the growth of fungi and bacteria. The freeze-dried fruiting body, broth from submerged culture, and mycelial biomass of P. ostreatus were extracted using alcohols and water as solvents. The extracts were then tested for their antimicrobial activity against the growth of fungi and bacteria. It was observed that the water extract from fruiting bodies had the strongest effect in inhibiting the growth of most fungi. The most sensitive test microfungi to the inhibition were Candida albicans, Cryptococcus humicola, and Trichosporon cutaneum, and the most sensitive test bacteria were Staphylococcus aureus followed by Escherichia coli. Water extracts from culture broth or mycelial biomass were moderately inhibitive to the growth of fungi and bacteria. The alcohol-based solvents from all samples had much less antimicrobial activity against most test microorganisms. An antimicrobial compound was purified from the water extracts of fruiting bodies with Sephadex G 100 column chromatography and characterized by infrared absorption spectrum (IR), nuclear magnetic resonance (NMR), and mass spectroscopic analysis. We have identified this compound to be 3-(2-aminopheny1thio)-3-hydroxypropanoic acid. This purified compound had a minimum inhibitory concentration of 30 µg/mL and 20 µg/mL against the growth of fungi and bacteria, respectively.

  19. Different drying technologies and alternation of mycobiots in the raw material of Hyssopus officinalis L.

    Science.gov (United States)

    Raila, Algirdas; Lugauskas, Albinas; Kemzūraite, Aurelija; Zvicevicius, Egidijus; Ragazinskiene, Ona; Railiene, Marija

    2009-01-01

    Contamination of medicinal plant mass with mycobiots is one of the negative factors deteriorating the quality of raw material. In order to evaluate the impact of the yield processing technologies upon the changes of mycobiots in raw material, the mycobiotic conditions of herb hyssop (Hyssopus officinalis L.) raw material were evaluated under various regimes of active ventilation and optimization of the drying parameters. The impact of ventilation intensity and temperature of drying agent upon the changes and abundance of mycobiota species in medicinal raw material was determined. Irrespective of the temperature of the airflow, the strongest suppressive effect upon the mycobiotic contamination in Hyssopi herba was produced by the 5,000 m3 x (t x h)(-1) airflow. Analysis of the isolated fungi revealed the prevalence of Penicillium, Aspergillus, Alternaria, Cladosporium, Mucor, Rhizopus species in the raw material. In separate samples Botrytis cinerea, Sclerotinia sclerotiorum, Aureobasidium pullulans, Chrysosporium merdarium, Cladorrhinum foecundissimum, Ulocladium consortiale, Trichoderma hamatum, T. harzianum, Gilmaniella humicola, Talaromyces flavus, Rhizomucor pusillus, Hansfordia ovalispora, Verticicladium trifi dum, Trichosporiella cerebriformis micromycetes were also rather abundant. Detection of the above-mentioned micromycetes in herb hyssop samples differed, and partially depended upon the medium used for their isolation.

  20. Soil bacterial and fungal communities respond differently to various isothiocyanates added for biofumigation

    Directory of Open Access Journals (Sweden)

    Ping eHu

    2015-01-01

    Full Text Available The meals from many oilseed crops have potential for biofumigation due to their release of biocidal compounds such as isothiocyanates (ITCs. Various ITCs are known to inhibit numerous pathogens; however, much less is known about how the soil microbial community responds to the different types of ITCs released from oilseed meals (SMs. To simulate applying ITC-releasing SMs to soil, we amended soil with 1% flax SM (contains no biocidal chemicals along with four types of ITCs (allyl, butyl, phenyl, and benzyl ITC in order to determine their effects on soil fungal and bacterial communities in a replicated microcosm study. Microbial communities were analyzed based on the ITS region for fungi and 16S rRNA gene for bacteria using qPCR and tag-pyrosequencing with 454 GS FLX titanium technology. A dramatic decrease in fungal populations (~85% reduction was observed after allyl ITC addition. Fungal community compositions also shifted following ITC amendments (e.g., Humicola increased in allyl and Mortierella in butyl ITC amendments. Bacterial populations were less impacted by ITCs, although there was atransient increase in the proportion of Firmicutes, related to bacteria know to be antagonistic to plant pathogens, following amendment with allyl ITC. Our results indicate that the type of ITC released from SMs can result in differential impacts on soil microorganisms. This information will aid selection and breeding of plants for biofumigation-based control of soil-borne pathogens while minimizing the impacts on non-target microorganisms.

  1. Studies on the solubilization of German coal by fungi

    Energy Technology Data Exchange (ETDEWEB)

    Reiss, J. (Grahamhaus Stadt kg, Bad Kreuznach (Germany))

    1992-09-01

    The capability of seven basidiomycetes (Trametes versicolor, Poria placenta, Pleurotus florida, P. ostreatus, P. sajor-caju, P. eryngii, Stropharia sp.), one ascomycete (Chaetomium globosum) and five hyphomycetes and moulds (Humicola grisea, Trichoderma viride, Aspergillus terreus, Paecilomyces varioti, Papulaspora immersa) to solubilize medium and high volatile bituminous coals (types A and B) as well as four types of lignite B from Germany was tested in surface cultures. The intensity of bioliquefaction was determined by estimating the rate of droplet formation and by measuring the loss of weight of the coal granules gravimetrically. The bituminous coals with a relative high degree of coalification were only moderately converted by Trametes versicolor, Pleurotus florida, P. ostreatus and P. sajor-caju. The three species of Pleurotus caused the greatest rate of biosolubilization of lignite, yielding a loss of weight of the coal granules of more than 5.8% with a maximum of 7.6% with P. florida. The non-basidiomycetes proved to be less active with a liquefaction rate of up to 3.5% with Trichoderma viride. In general, the geologically younger lignite coals were more effectively solubilized than the older hard coals. The volatile matter and the oxygen content proved to be the principal factors influencing the intensity of bioconversion.

  2. Long-term no-till: A major driver of fungal communities in dryland wheat cropping systems.

    Directory of Open Access Journals (Sweden)

    Dipak Sharma-Poudyal

    Full Text Available In the dryland Pacific Northwest wheat cropping systems, no-till is becoming more prevalent as a way to reduce soil erosion and fuel inputs. Tillage can have a profound effect on microbial communities and soilborne fungal pathogens, such as Rhizoctonia. We compared the fungal communities in long-term no-till (NT plots adjacent to conventionally tilled (CT plots, over three years at two locations in Washington state and one location in Idaho, US. We used pyrosequencing of the fungal ITS gene and identified 422 OTUs after rarefication. Fungal richness was higher in NT compared to CT, in two of the locations. Humicola nigrescens, Cryptococcus terreus, Cadophora spp. Hydnodontaceae spp., and Exophiala spp. were more abundant in NT, while species of Glarea, Coniochaetales, Mycosphaerella tassiana, Cryptococcus bhutanensis, Chaetomium perlucidum, and Ulocladium chartarum were more abundant in CT in most locations. Other abundant groups that did not show any trends were Fusarium, Mortierella, Penicillium, Aspergillus, and Macroventuria. Plant pathogens such as Rhizoctonia (Ceratobasidiaceae were not abundant enough to see tillage differences, but Microdochium bolleyi, a weak root pathogen, was more abundant in NT. Our results suggest that NT fungi are better adapted at utilizing intact, decaying roots as a food source and may exist as root endophytes. CT fungi can utilize mature plant residues that are turned into the soil with tillage as pioneer colonizers, and then produce large numbers of conidia. But a larger proportion of the fungal community is not affected by tillage and may be niche generalists.

  3. Fungal agents isolated from cancer patients.

    Science.gov (United States)

    Alvarez Gasca, M A; Argüero Licea, B; Pliego Castañeda, A; García Tena, S

    1998-01-01

    With the aim to know the frequency of mycotic agents in patients with different types of cancer, samples were obtained from 81 patients from the Hospital de Oncología, Centro Médico Nacional Siglo XXI, IMSS from May 1995 through May 1996. In a conventional grouping seven (7) ambulatory patients were found in early stages, twenty seven (27) occasionally hospitalized patients were found in intermediate stage and forty seven (47) hospitalized patients in terminal stage of cancer. The different samples were processed through routine mycologycal methods and the following fungi species were isolated and identified: fifty four strains (58%) of Candida albicans followed by eleven strains (11.8%) of Candida tropicalis, six strains (6.45%) of Candida parapsilosis, five strains (5.37%) of Candida krusei, four strains (4.3%) of Candida humicola and five strains (5.37%) of Rodothorula rubra. From medical devices like catheter tips, drainage catheters (Pen rouse, Foley) and gallbladder catheters; four (4) strains of C. albicans, three (3) strains of Rodothorula rubra and two (2) strains of Aspergillus sp were isolated. Of the Candida non albicans it was relevant to find C. krusei more frequently than Rodothorula rubra, Aspergillus sp and Penicillum sp. The frequency of the presence of fungi increases commensurately to the advancement of the clincal stage of the cancer.

  4. Synthesis of magneto-sensitive iron-containing nanoparticles by yeasts.

    Science.gov (United States)

    Vainshtein, Mikhail; Belova, Natalia; Kulakovskaya, Tatiana; Suzina, Natalia; Sorokin, Vladimir

    2014-04-01

    Industrial production of magneto-sensitive nanoparticles, which can be used in the production of target drug delivery carriers, is a subject of interest for biotechnology and microbiology. Synthesis of these nanoparticles by microorganisms has been described only for bacterial species. At the same time, it is well known that yeasts can form various metal-containing nanoparticles used, for instance, in semiconductors, etc. This paper describes the first results of the biosynthesis of magneto-sensitive nanoparticles by yeasts. The organisms we used-Saccharomyces cerevisiae and Cryptococcus humicola-represented two different genera. Magneto-sensitive nanoparticles were synthesized at room temperature in bench-scale experiments. The study included transmission electron microscopy of the yeast cells and their energy dispersive spectrum analyses and revealed the presence of iron-containing nanoparticles. Both yeast cultures synthesized nanoparticles at high concentrations of dissolved iron. Electron microscopy showed that nanoparticles were associated mainly with the yeast cell wall. Formation of magneto-sensitive nanoparticles was studied under conditions of applied magnetic fields; a possible stimulating role of magnetic field is suggested. On the whole, the paper reports a novel approach to green biosynthesis of magneto-sensitive nanoparticles.

  5. The preliminary assessment and isolation of entomopathogenic fungi to be used in biological control with twospotted spider mite [Tetranychus urticae (acari, tetranychidae)] from East Anatolia

    Science.gov (United States)

    Örtücü, Serkan; Algur, Ömer Faruk

    2017-04-01

    This study was conducted to isolation entomopathogenic fungi for possible use in biocontrol of two-spotted spider mite Tetranychus urticae Koch. and to determine their pathogenicity. For this purpose, plant leaves infected with T. urticae were collected from Erzurum, Kars and Ardahan. At laboratory, the internal and external mycoflora of T.urticae individuals on plant leaves were determined. As a result of isolation, twenty-five different fungi species belonging to the genera Acremonium, Alternaria, Aspergillus, Beauveria, Cladosporium, Gliocladium, Humicola, Penicillium, Trichoderma, Isaria, Ulocladium and Verticillium were obtained. Pathogenicity of this forty-five isolate belonging to twenty-five species were evaluated. As a test organism, T. urticae was used and suspensions (1 × 108conidia ml-1) were prepared in Tween 80. 2ml suspension of a single dose was sprayed onto down side of bean leaf discs using hand sprayer. Mortality was recorded daily for 7 days. A total of twelve isolates belonging to three species were determined to be pathogen against T.urticae. According to scale used: AT020 Isaria farinosa and AT025 Cladosporium cladosporioides were determined as least pathogen, AT037 and AT101 Beauveria bassiana, and AT019 and AT026 C. cladosporioides, and AT035 and AT036 I. farinosa as moderate pathogen, AT007, AT021, AT034 and AT076 B. bassiana as highly pathogen. The other thirty-three isolates found that not pathogenic against T.urticae.

  6. Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

    Science.gov (United States)

    Miyazawa, Ken; Yoshimi, Akira; Zhang, Silai; Sano, Motoaki; Nakayama, Mayumi; Gomi, Katsuya; Abe, Keietsu

    2016-09-01

    Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.

  7. A novel protein elicitor (SsCut) from Sclerotinia sclerotiorum induces multiple defense responses in plants.

    Science.gov (United States)

    Zhang, Huajian; Wu, Qun; Cao, Shun; Zhao, Tongyao; Chen, Ling; Zhuang, Peitong; Zhou, Xiuhong; Gao, Zhimou

    2014-11-01

    In this study, we report the cloning of the SsCut gene encoding cutinase from Sclerotinia sclerotiorum. We isolated a 609-bp cDNA encoding a polypeptide of 202 amino acids with a molecular weight of 20.4 kDa. Heterologous expression of SsCut in Escherichia coli (His-SsCut) caused the formation of lesions in tobacco that closely resembled hypersensitive response lesions. Mutational analysis identified the C-terminal-half peptide and the same amino acids indispensable for both enzyme and elicitor activity. His-SsCut was caused cell death in Arabidopsis, soybean (Glycine max), oilseed rape (Brassica napus), rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum), indicating that both dicot and monocot species are responsive to the elicitor. Furthermore, the elicitation of tobacco was effective in the induction of the activities of hydrogen peroxide, phenylalanine ammonia-lyase, peroxides, and polyphenol oxidase. His-SsCut-treated plants exhibited enhanced resistance as indicated by a significant reduction in the number and size of S. sclerotiorum, Phytophthora sojae, and P. nicotianae lesions on leaves relative to controls. Real-time PCR results indicated that the expression of defense-related genes and genes involved in signal transduction were induced by His-SsCut. Our results demonstrate that SsCut is an elicitor that triggers defense responses in plants and will help to clarify its relationship to downstream signaling pathways that induce defense responses.

  8. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

    Energy Technology Data Exchange (ETDEWEB)

    Payne, K.; Sun, Q.; Sacchettini, J.; Hatfull, G.F.; (Pitt)

    2010-08-27

    Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an {alpha}/{beta} hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles {Delta}lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

  9. A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET and Polylactic Acid (PLA

    Directory of Open Access Journals (Sweden)

    Georg Steinkellner

    2012-02-01

    Full Text Available A new esterase from Thermobifida halotolerans (Thh_Est was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA and polyethylene terephthalate (PET. Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 85–87% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethylterephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme while no higher oligomers like bis-(2-hydroxyethyl terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8° and 75.5° to 50.4° and to a complete spread of the water drop on the surface, respectively.

  10. A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, Akhil [Univ. of Hawaii, Manoa, HI (United States); Ohm, Robin A. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Oxiles, Lindsay [Univ. of Hawaii, Manoa, HI (United States); Brooks, Fred [Univ. of Hawaii, Manoa, HI (United States); Lawrence, Christopher B. [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Grigoriev, Igor V. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Cho, Yangrae [Univ. of Hawaii, Manoa, HI (United States)

    2011-10-26

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19- reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

  11. Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

    Science.gov (United States)

    Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

    2017-01-01

    Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases. © 2017 Elsevier Inc. All rights reserved.

  12. Fragrance Release from the Surface of Branched Poly (Amide S

    Directory of Open Access Journals (Sweden)

    T. Youngs

    2005-01-01

    Full Text Available Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased.

  13. Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

    Directory of Open Access Journals (Sweden)

    Juliane Schmidt

    2017-02-01

    Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

  14. Yeasts associated with an abandoned mining area in Pernek and their tolerance to different chemical elements.

    Science.gov (United States)

    Vadkertiová, Renáta; Molnárová, Jana; Lux, Alexander; Vaculík, Marek; Lišková, Desana

    2016-05-01

    Four plants, Cirsium arvense (creeping thistle), Equisetum arvense (field horsetail), Oxalis acetosella (wood sorrel) and Phragmites australis (common reed), which grew in an abandoned Sb-mining area in Pernek (Malé Karpaty Mts., Slovakia), were investigated for the yeast species. Yeasts were isolated from both the leaves of the plants and the soil adjacent to the plants. In total, 65 yeast cultures, belonging to 11 ascomycetous and 5 basidiomycetous yeast species, were isolated. The species most frequently isolated from both the soil and leaf samples were Trichosporon porosum, Galactomyces candidus and Candida solani, whereas Aureobasidium pullulans, Candida tsuchiyae and Sporidiobolus metaroseus were isolated exclusively from the plant leaves. All the yeast species isolated were tested for their tolerance to two heavy metals (Cd, Zn) and three metalloids (As, Sb and Si). The yeasts isolated from both the leaves and soils exhibited a high tolerance level to both As and Sb, present in elevated concentrations at the locality. Among the yeast species tested, Cryptococcus musci, a close relative to Cryptococcus humicola, was the species most tolerant to all the chemical elements tested, with the exception of Si. It grew in the presence of 200 mmol/L Zn, 200 mmol/L Cd, 60 mmol/L As and 50 mmol/L Sb, and therefore, it can be considered as a multi-tolerant species. Some of the yeast species were tolerant to the individual chemical elements. The yeast-like species Trichosporon laibachii exhibited the highest tolerance to Si of all yeasts tested, and Cryptococcus flavescens and Lindnera saturnus showed the same tolerance as Cryptococcus musci to Zn and As, respectively. The majority of the yeasts showed a notably low tolerance to Cd (not exceeded 0.5 mmol/L), which was present in small amounts in the soil. However, Candida solani, isolated from the soil, exhibited a higher tolerance to Cd (20 mmol/L) than to As (2 mmol/L).

  15. Fungi colonising the above-ground parts of fodder galega (Galega orientalis Lam. cultivated in pure sowing and mixed with smooth brome-grass (Bromus inermis Leyss.

    Directory of Open Access Journals (Sweden)

    Bożena Cwalina-Ambroziak

    2012-12-01

    Full Text Available Field experiments were carried out in 1999-2001 in the experimental field in Knopin near Dobre Miasto to determine the intensity of fodder galega diseases cultivated in pure sowing and mixed with smooth brome-grass (the Hillstrand and Auld' s modified scale, 1982. The fungi colonising the phyllosphere of fodder galega were analysed in a laboratory (Chruoeciak , 1974. The following symptoms were observed in fodder galega: ascochyta blight (Ascochyta sp., gray mould (Botrytis cinerea and plant wilting (Fusarium oxysporum.. The climatic conditions had an effect on the development of diseases. The greatest intensity of gray mould (Ii = 24.3% and plant wilting (17.9% of plants with the disease symptoms were observed in 2001. Ascochyta blight occurred with the lowest intensity and the highest infection index in 1999 in the cultivation of fodder galega mixed with smooth brome-grass was only 12.1%. The type of cultivation also modified fodder galega disease intensity. Gray mould and plant wilting developed better in pure sowing than in mixed sowing with smooth brome-grass. Throughout the entire experiment period the average infection index was 22.8% and 15.9% of plants with the wilt symptoms. Ascochyta blight found better conditions for development in plants cultivated in a mix with smooth brome-grass (average infection index - 10.0%. The fodder galega phyllosphere provided 4149 fungal isolates represented by 17 species and yeast-like fungi. Yeast-like fungi dominated (75.6% of the total isolates. The following species were less numerous: Botrytis cinerea, Humicola brevis, Acremonium strictum and Cladosporium cladosporioides. From the leaves of fodder galega cultivated in pure sowing, 3.8% more fungi were obtained than from the leaves of plants cultivated with a mix of smooth brome-grass, including more frequently isolated pathogenic fungi representing the genera of Fusarium and the species of Botrytis cinerea.

  16. Potentially pathogenic yeasts from soil of children’s recreational areas in the city of Łódź (Poland

    Directory of Open Access Journals (Sweden)

    Anna Wójcik

    2013-06-01

    Full Text Available Objectives: Yeasts may become potential human and animal pathogens, particularly for individuals with a depressed immune system. Their presence in the environment, especially in soil, may favour their spread into human ontocenoses. Materials and Methods: Eighty-four soil samples obtained from 21 children's recreational sites in Łódź in autumn 2010 and spring 2011 were evaluated. The yeasts were isolated by classical microbiological methods and identified on the basis of morphological and biochemical features. Results: The fungi were found in 73.8% and in 69.0% of the examined samples collected in autumn and spring, respectively. Among 97 isolates of yeasts, the species potentially pathogenic to humans and animals were Candida colliculosa, C. guilliermondii, C. humicola, C. inconspicua, C. lambica, C. lusitaniae, C. pelliculosa, C. tropicalis, Cryptococcus albidus, C. laurentii, C. neoformans, C. terreus, Kloeckera japonica, Geotrichum candidum, G. penicillatum, Rhodotorula mucilaginosa, R. glutinis, Saccharomyces cerevisiae, Sporobolomyces salmonicolor and Trichosporon cutaneum. The most frequently isolated fungi included the genus Cryptococcus (38 isolates and two species: Rhodotorula glutinis (15, Trichosporon cutaneum (14. C. neoformans, an etiological factor of cryptococcal meningitis, was present in the sandpits of 3 kindergartens. The Candida species were identified from park playgrounds and school sports fields mainly in autumn 2010 (14 isolates, in spring 2011 - only 1 isolate. The concentration of fungal species in particular samples varied considerably, but in the majority of samples, fungi were present at concentration of up to 1×102 CFU/1 g of soil. Conclusions: Yeasts were present in the soil of parks, schools and kindergarten recreational areas; the fact may pose a health risk to humans, especially to children, and this type of biological pollution should be regarded as a potential public health concern.

  17. Microbial ice nucleators scavenged from the atmosphere during simulated rain events

    Science.gov (United States)

    Hanlon, Regina; Powers, Craig; Failor, Kevin; Monteil, Caroline L.; Vinatzer, Boris A.; Schmale, David G.

    2017-08-01

    Rain and snow collected at ground level have been found to contain biological ice nucleators. These ice nucleators have been proposed to have originated in clouds, where they may have participated in the formation of precipitation via ice phase nucleation. We conducted a series of field experiments to test the hypothesis that at least some of the microbial ice nucleators (prokaryotes and eukaryotes) present in rain may not originate in clouds but instead be scavenged from the lower atmosphere by rainfall. Thirty-three simulated rain events were conducted over four months off the side of the Smart Road Bridge in Blacksburg, VA, USA. In each event, sterile water was dispensed over the side of the bridge and recovered in sterile containers in an open fallow agricultural field below (a distance of ∼55 m). Microbes scavenged from the simulated rain events were cultured and their ice nucleation activity was examined. Putative microbial ice nucleators were cultured from 94% (31/33) of the simulated rain events, and represented 1.5% (121/8331) of the total colonies assayed. Putative ice nucleators were subjected to additional droplet freezing assays, and those confirmed through these repeated assays represented 0.4% (34/8331) of the total. Mean CFUs scavenged by simulated rain ranged from 2 to 267 CFUs/mL. Scavenged ice nucleators belong to a number of taxa including the bacterial genera Pseudomonas, Pantoea, and Xanthomonas, and the fungal genera Fusarium, Humicola, and Mortierella. An ice-nucleating strain of the fungal genus Penicillium was also recovered from a volumetric air sampler at the study site. This work expands our knowledge of the scavenging properties of rainfall, and suggests that at least some ice nucleators in natural precipitation events may have been scrubbed from the atmosphere during rainfall, and thus are not likely to be involved in precipitation.

  18. 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.

  19. Hormone profiles in microalgae: gibberellins and brassinosteroids.

    Science.gov (United States)

    Stirk, W A; Bálint, P; Tarkowská, D; Novák, O; Strnad, M; Ördög, V; van Staden, J

    2013-09-01

    Endogenous gibberellins and brassinosteroids were quantified in 24 axenic microalgae strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae and Charophyceae microalgae strains after 4 days in culture. This is the first report of endogenous gibberellins being successfully detected in microalgae. Between 18 and 20 gibberellins were quantified in all strains with concentrations ranging from 342.7 pg mg(-1) DW in Raphidocelis subcapitata MACC 317-4746.1 pg mg(-)(1) DW in Scotiellopsis terrestris MACC 44. Slower growing strains (S. terrestris MACC 44, Gyoerffyana humicola MACC 334, Nautococcus mamillatus MACC 716 and Chlorococcum ellipsoideum MACC 712) exhibited the highest gibberellin contents while lowest levels of gibberellins were found in faster growing strains (R. subcapitata MACC 317 and Coelastrum excentrica MACC 504). In all strains, the active gibberellin detected in the highest concentration was GA6, the predominant intermediates were GA15 and GA53 and the main biosynthetic end products were GA13 and GA51. Gibberellin profiles were similar in all strains except for the presence/absence of GA12 and GA12ald. To date this is the second report of endogenous brassinosteroids in microalgae. Brassinosteroids were detected in all 24 strains with concentrations ranging from 117.3 pg mg(-)(1) DW in R. subcapitata MACC 317-977.8 pg mg(-)(1) DW in Klebsormidium flaccidum MACC 692. Two brassinosteroids, brassinolide and castasterone were determined in all the strains. Generally, brassinolide occurred in higher concentrations than castasterone. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  20. Fungi isolated from phyllosphere of fodder galega (Galega orientalis

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    Bożena Cwalina-Ambrozik

    2013-12-01

    Full Text Available The object of the experiment was fodder galega (Galega orientalis Lam. cultivated in 2001-2003 as field crop on three plots: 1. without fertilization, 2. 40 kg P2O5 × ha-1 and 80 kg K2O × ha-1, 3. 80 kg P2O5 × ha-1 and 160 kg K2O × ha-1. During the dry and warm vegetation season of 2002 almost two times fewer isolates were obtained from the leaves than in 2003 that was the most abundant in fungi. Yeasts-like fungi (30% of the total number of isolates and saprotrophic fungi with dominated species: Acremonium strictum (8.5%, genus Epicoccum (7.8%, Humicola (9.5% and Penicillium (18.9% were the fungi most frequently populating the leaves of galega. The share of pathogens in the total number of isolates obtained from the phyllosphere was 10.6%. They were represented by fungi of Ascochyta spp., Botrytis cinerea, genus Fusarium, Phoma medicaginis and Sclerotinia sclerotiorum. Reduction by 1.9 to 4.6% in the number of fungi isolated from the phyllosphere of galega without fertilization as compared to galega cultivated in combinations with fertilization was recorded. Generally, the smallest number of pathogens was recovered from galega fertilized with 40 kg P2O5 × ha-1 and 80 kg K2O × ha-1. B. cinerea most frequently populated galega in combination without fertilization, genus Fusarium fungi in combination without fertilization and with fertilization with 80 kg P2O5 × ha-1 and 160 kg K2O × ha-1, while Ascochyta spp. were isolated from galega with fertilization only.

  1. An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum

    Science.gov (United States)

    Colombres, Marcela; Garate, José A.; Lagos, Carlos F.; Araya-Secchi, Raúl; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Pérez-Acle, Tomas; Eyzaguirre, Jaime

    2008-01-01

    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 Å resolution (PDB 1G66). The enzyme possesses the α/β hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

  2. Proteomic analysis of Fusarium solani isolated from the Asian longhorned beetle, Anoplophora glabripennis.

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    Erin D Scully

    Full Text Available Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle, and that a fungal species, Fusarium solani (ATCC MYA 4552, is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent

  3. Proteomic analysis of Fusarium solani isolated from the Asian longhorned beetle, Anoplophora glabripennis.

    Science.gov (United States)

    Scully, Erin D; Hoover, Kelli; Carlson, John; Tien, Ming; Geib, Scott M

    2012-01-01

    Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were

  4. Biochemical Control of Fungal Biomass and Enzyme Production During Native Hawaiian Litter Degradation

    Science.gov (United States)

    Amatangelo, K. L.; Cordova, T. P.; Vitousek, P. M.

    2007-12-01

    Microbial growth and enzyme production during decomposition is controlled by the availability of carbon substrates, essential elements, and the ratios of these (such as lignin:N). We manipulated carbon:nutrient stoichiometry during decomposition using a natural fertility gradient in Hawaii and litter of varying initial biochemistry. We collected freshly senesced litter of seven biochemically distinct species from three sites offering differing levels of N, P, cations, and 15N , but similar yearly rainfall and temperature patterns. Litter types were decomposed at both the sites they were collected, and at the other site(s) that species was found. Litter was collected at multiple time points, and after one year of decomposition, calculated K constants varied an order of magnitude, from 0.276 to 2.76. Decomposition rates varied significantly with both litter site of origin and deployment, except at the oldest, P-limited site, where litter site of origin was not significantly correlated with decomposition within species. As microbial exocellular enzymes provide the catalyst for the breakdown of organic molecules including phenols, cellulose, and cutin, we assayed polyphenol oxidase, cellobiohydrolase, cutinase, chitinase, and lignin peroxidase to evaluate the breakdown sequence of different litter types. To measure the fungal biomass accumulating during decomposition, we extracted (22E)-Ergosta-5,7,22-trien-3beta- ol (ergosterol) on a subset of samples. The production of particular exocellular enzymes on litter species responded distinctly to origin and decomposition sites: after six months, chitinase and cellobiohydrolase were significantly affected by origin site, whereas polyphenol oxidase activity was controlled by deployment site. We conclude that site characteristics can alter the interaction between litter carbon:nutrient ratios and decomposition rate, mediated through microbial biomass and enzyme production.

  5. Genome Assembly of the Fungus Cochliobolus miyabeanus, and Transcriptome Analysis during Early Stages of Infection on American Wildrice (Zizania palustris L..

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    Claudia V Castell-Miller

    Full Text Available The fungus Cochliobolus miyabeanus causes severe leaf spot disease on rice (Oryza sativa and two North American specialty crops, American wildrice (Zizania palustris and switchgrass (Panicum virgatum. Despite the importance of C. miyabeanus as a disease-causing agent in wildrice, little is known about either the mechanisms of pathogenicity or host defense responses. To start bridging these gaps, the genome of C. miyabeanus strain TG12bL2 was shotgun sequenced using Illumina technology. The genome assembly consists of 31.79 Mbp in 2,378 scaffolds with an N50 = 74,921. It contains 11,000 predicted genes of which 94.5% were annotated. Approximately 10% of total gene number is expected to be secreted. The C. miyabeanus genome is rich in carbohydrate active enzymes, and harbors 187 small secreted peptides (SSPs and some fungal effector homologs. Detoxification systems were represented by a variety of enzymes that could offer protection against plant defense compounds. The non-ribosomal peptide synthetases and polyketide synthases (PKS present were common to other Cochliobolus species. Additionally, the fungal transcriptome was analyzed at 48 hours after inoculation in planta. A total of 10,674 genes were found to be expressed, some of which are known to be involved in pathogenicity or response to host defenses including hydrophobins, cutinase, cell wall degrading enzymes, enzymes related to reactive oxygen species scavenging, PKS, detoxification systems, SSPs, and a known fungal effector. This work will facilitate future research on C. miyabeanus pathogen-associated molecular patterns and effectors, and in the identification of their corresponding wildrice defense mechanisms.

  6. Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

    Science.gov (United States)

    2010-01-01

    Background Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. Conclusions Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae. PMID:20626842

  7. A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity.

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    Floriane L'Haridon

    2011-07-01

    Full Text Available Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS, including H(2O(2 and O(2 (-, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI or catalase. H(2O(2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA. Accordingly, ABA biosynthesis mutants (aba2 and aba3 were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending

  8. Genomic insight into pathogenicity of dematiaceous fungus Corynespora cassiicola

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    Hong Keat Looi

    2017-01-01

    Full Text Available Corynespora cassiicola is a common plant pathogen that causes leaf spot disease in a broad range of crop, and it heavily affect rubber trees in Malaysia (Hsueh, 2011; Nghia et al., 2008. The isolation of UM 591 from a patient’s contact lens indicates the pathogenic potential of this dematiaceous fungus in human. However, the underlying factors that contribute to the opportunistic cross-infection have not been fully studied. We employed genome sequencing and gene homology annotations in attempt to identify these factors in UM 591 using data obtained from publicly available bioinformatics databases. The assembly size of UM 591 genome is 41.8 Mbp, and a total of 13,531 (≥99 bp genes have been predicted. UM 591 is enriched with genes that encode for glycoside hydrolases, carbohydrate esterases, auxiliary activity enzymes and cell wall degrading enzymes. Virulent genes comprising of CAZymes, peptidases, and hypervirulence-associated cutinases were found to be present in the fungal genome. Comparative analysis result shows that UM 591 possesses higher number of carbohydrate esterases family 10 (CE10 CAZymes compared to other species of fungi in this study, and these enzymes hydrolyses wide range of carbohydrate and non-carbohydrate substrates. Putative melanin, siderophore, ent-kaurene, and lycopene biosynthesis gene clusters are predicted, and these gene clusters denote that UM 591 are capable of protecting itself from the UV and chemical stresses, allowing it to adapt to different environment. Putative sterigmatocystin, HC-toxin, cercosporin, and gliotoxin biosynthesis gene cluster are predicted. This finding have highlighted the necrotrophic and invasive nature of UM 591.

  9. Proteomic Analysis of Fusarium solani Isolated from the Asian Longhorned Beetle, Anoplophora glabripennis

    Science.gov (United States)

    Scully, Erin D.; Hoover, Kelli; Carlson, John; Tien, Ming; Geib, Scott M.

    2012-01-01

    Wood is a highly intractable food source, yet many insects successfully colonize and thrive in this challenging niche. Overcoming the lignin barrier of wood is a key challenge in nutrient acquisition, but full depolymerization of intact lignin polymers has only been conclusively demonstrated in fungi and is not known to occur by enzymes produced by insects or bacteria. Previous research validated that lignocellulose and hemicellulose degradation occur within the gut of the wood boring insect, Anoplophora glabripennis (Asian longhorned beetle), and that a fungal species, Fusarium solani (ATCC MYA 4552), is consistently associated with the larval stage. While the nature of this relationship is unresolved, we sought to assess this fungal isolate's ability to degrade lignocellulose and cell wall polysaccharides and to extract nutrients from woody tissue. This gut-derived fungal isolate was inoculated onto a wood-based substrate and shotgun proteomics using Multidimensional Protein Identification Technology (MudPIT) was employed to identify 400 expressed proteins. Through this approach, we detected proteins responsible for plant cell wall polysaccharide degradation, including proteins belonging to 28 glycosyl hydrolase families and several cutinases, esterases, lipases, pectate lyases, and polysaccharide deacetylases. Proteinases with broad substrate specificities and ureases were observed, indicating that this isolate has the capability to digest plant cell wall proteins and recycle nitrogenous waste under periods of nutrient limitation. Additionally, several laccases, peroxidases, and enzymes involved in extracellular hydrogen peroxide production previously implicated in lignin depolymerization were detected. In vitro biochemical assays were conducted to corroborate MudPIT results and confirmed that cellulases, glycosyl hydrolases, xylanases, laccases, and Mn- independent peroxidases were active in culture; however, lignin- and Mn- dependent peroxidase activities were

  10. Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

    Science.gov (United States)

    Su, Lingqia; Jiang, Qi; Yu, Lingang; Wu, Jing

    2017-02-08

    Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

  11. Comparative Genomics Including the Early-Diverging Smut Fungus Ceraceosorus bombacis Reveals Signatures of Parallel Evolution within Plant and Animal Pathogens of Fungi and Oomycetes.

    Science.gov (United States)

    Sharma, Rahul; Xia, Xiaojuan; Riess, Kai; Bauer, Robert; Thines, Marco

    2015-08-27

    Ceraceosorus bombacis is an early-diverging lineage of smut fungi and a pathogen of cotton trees (Bombax ceiba). To study the evolutionary genomics of smut fungi in comparison with other fungal and oomycete pathogens, the genome of C. bombacis was sequenced and comparative genomic analyses were performed. The genome of 26.09 Mb encodes for 8,024 proteins, of which 576 are putative-secreted effector proteins (PSEPs). Orthology analysis revealed 30 ortholog PSEPs among six Ustilaginomycotina genomes, the largest groups of which are lytic enzymes, such as aspartic peptidase and glycoside hydrolase. Positive selection analyses revealed the highest percentage of positively selected PSEPs in C. bombacis compared with other Ustilaginomycotina genomes. Metabolic pathway analyses revealed the absence of genes encoding for nitrite and nitrate reductase in the genome of the human skin pathogen Malassezia globosa, but these enzymes are present in the sequenced plant pathogens in smut fungi. Interestingly, these genes are also absent in cultivable oomycete animal pathogens, while nitrate reductase has been lost in cultivable oomycete plant pathogens. Similar patterns were also observed for obligate biotrophic and hemi-biotrophic fungal and oomycete pathogens. Furthermore, it was found that both fungal and oomycete animal pathogen genomes are lacking cutinases and pectinesterases. Overall, these findings highlight the parallel evolution of certain genomic traits, revealing potential common evolutionary trajectories among fungal and oomycete pathogens, shaping the pathogen genomes according to their lifestyle. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars.

    Science.gov (United States)

    Verma, Dheeraj; Kanagaraj, Anderson; Jin, Shuangxia; Singh, Nameirakpam D; Kolattukudy, Pappachan E; Daniell, Henry

    2010-04-01

    It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

  13. Changes in soil microbial communities after 10 years of winter wheat cultivation versus fallow in an organic-poor soil in the Loess Plateau of China

    Science.gov (United States)

    Tian, Hui; Wang, Hui; Hui, Xiaoli; Wang, Zhaohui; Drijber, Rhae A.

    2017-01-01

    Agricultural management methods, such as cultivation or fallowing, have led to significant changes in soil fertility and hence, crop yield. Such changes may have stemmed from changes in soil microbial communities and associated biogeochemical processes. This phenomenon is particularly true in organic-poor soil in the Loess Plateau of China. In this study, we examined three existing soil management regimes as part of a 10-year field experiment and evaluated their effects on fungal and bacterial community structures by performing high-throughput 454 pyrosequencing. These management regimes were (i) fertilized winter wheat (Triticum aestivum L.) (FW), (ii) continuous natural fallow with weeds but without crop grown (NF), and (iii) continuous bare fallow without weeds or crop grown (BF). After 10 years, soil organic carbon (SOC), microbial biomass carbon (MBC), and available potassium (K) concentrations were highest in NF. Soil N behaved differently, with BF obtaining the highest nitrate nitrogen (N). Meanwhile, slight differences in total N (TN) were observed among FW, NF, and BF. Available phosphorus (P) was highest and available K was lowest in FW. Microbial communities were dominated by Ascomycota (59.1% of fungal sequences), and Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria (75.7% of bacterial sequences) in FW, NF and BF at the phylum level. Soil management regimes did not affect the fungal and bacterial richness and diversity but significantly modified their community compositions. Compared with FW, the abundances of Ascomycota (fungi phylum) and Alternaria, Gibberella, and Emericella (fungi genus) were increased by NF, whereas the values of Chaetomium, Humicola, and Cryptococcus (fungi genus) were decreased by BF. The abundances of Verrucomicrobia (bacteria phylum), and Steroidobacter (bacteria genus) were increased by NF, and Bacteroides (bacteria genus) was increased by BF. Canonical correspondence analysis showed that SOC

  14. Changes in soil microbial communities after 10 years of winter wheat cultivation versus fallow in an organic-poor soil in the Loess Plateau of China.

    Science.gov (United States)

    Tian, Hui; Wang, Hui; Hui, Xiaoli; Wang, Zhaohui; Drijber, Rhae A; Liu, Jinshan

    2017-01-01

    Agricultural management methods, such as cultivation or fallowing, have led to significant changes in soil fertility and hence, crop yield. Such changes may have stemmed from changes in soil microbial communities and associated biogeochemical processes. This phenomenon is particularly true in organic-poor soil in the Loess Plateau of China. In this study, we examined three existing soil management regimes as part of a 10-year field experiment and evaluated their effects on fungal and bacterial community structures by performing high-throughput 454 pyrosequencing. These management regimes were (i) fertilized winter wheat (Triticum aestivum L.) (FW), (ii) continuous natural fallow with weeds but without crop grown (NF), and (iii) continuous bare fallow without weeds or crop grown (BF). After 10 years, soil organic carbon (SOC), microbial biomass carbon (MBC), and available potassium (K) concentrations were highest in NF. Soil N behaved differently, with BF obtaining the highest nitrate nitrogen (N). Meanwhile, slight differences in total N (TN) were observed among FW, NF, and BF. Available phosphorus (P) was highest and available K was lowest in FW. Microbial communities were dominated by Ascomycota (59.1% of fungal sequences), and Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria (75.7% of bacterial sequences) in FW, NF and BF at the phylum level. Soil management regimes did not affect the fungal and bacterial richness and diversity but significantly modified their community compositions. Compared with FW, the abundances of Ascomycota (fungi phylum) and Alternaria, Gibberella, and Emericella (fungi genus) were increased by NF, whereas the values of Chaetomium, Humicola, and Cryptococcus (fungi genus) were decreased by BF. The abundances of Verrucomicrobia (bacteria phylum), and Steroidobacter (bacteria genus) were increased by NF, and Bacteroides (bacteria genus) was increased by BF. Canonical correspondence analysis showed that SOC

  15. Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

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    Raquel M Cadete

    Full Text Available BACKGROUND: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. METHODOLOGY/PRINCIPAL FINDINGS: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g and productivities (0.62 g/L · h to 0.75 g/L · h. Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g, with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g and productivity (0.2 g/L · h, while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

  16. Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis

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    Wynne James W

    2012-03-01

    to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.