Sample records for thermobacillus xylanilyticus gh-51

  1. The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth

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    Rakotoarivonina Harivony


    Full Text Available Abstract Background Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization, were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth. Results Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran. Conclusion This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided

  2. Streptomyces xylanilyticus sp. nov., isolated from soil. (United States)

    Moonmangmee, Duangtip; Kanchanasin, Pawina; Phongsopitanun, Wongsakorn; Tanasupawat, Somboon; Moonmangmee, Somporn


    A novel actinomycete, strain SR2-123 T , belonging to the genus Streptomyces, was isolated from a soil sample collected from the Sakaerat Environmental Research Station, Thailand Institute of Scientific and Technological Research, Nakhon Ratchasima Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic study. Strain SR2-123 T contained ll-diaminopimelic acid, glucose, mannose and ribose in whole-cell hydrolysates. The N-acyl type of muramic acid was acetyl. Menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and iso-C17 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, an unknown phospholipid, unknown glycolipids, an unknown aminophospholipid, unknown lipids and an unknown aminolipid. The DNA G+C content was 74.8 mol%. The strain was closely related to Streptomyces coeruleorubidus JCM 4359 T (98.5 %), Streptomyces flavofungini JCM 4753 T (98.5 %), Streptomyces coerulescens NBRC 12758 T (98. 5 %) and Streptomyces alboflavus JCM 4615 T (98.4 %), based on 16S rRNA gene sequence similarities. The novel strain exhibited low DNA-DNA relatedness values with the type strains (11.4-25.0 %) of closely related species. On the basis of phenotypic and genotypic characteristics, strain SR2-123 T could be distinguished from closely related species of the genus Streptomyces and represents a novel species of the genus Streptomyces for which the name Streptomyces xylanilyticus sp. nov. is proposed. The type strain is SR2-123 T (=TISTR 2493 T =KCTC 39909 T ).

  3. The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans. (United States)

    Bouraoui, Hanen; Desrousseaux, Marie-Laure; Ioannou, Eleni; Alvira, Pablo; Manaï, Mohamed; Rémond, Caroline; Dumon, Claire; Fernandez-Fuentes, Narcis; O'Donohue, Michael J


    Conceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. THSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4-7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (k cat/K M = 1050 s(-1) mM(-1)) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg(-1), respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg(-1)). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality. The discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the

  4. Sequential and simultaneous strategies for biorefining of wheat straw using room temperature ionic liquids, xylanases and cellulases. (United States)

    Husson, Eric; Auxenfans, Thomas; Herbaut, Mickael; Baralle, Manon; Lambertyn, Virginie; Rakotoarivonina, Harivoni; Rémond, Caroline; Sarazin, Catherine


    Sequential and simultaneous strategies for fractioning wheat straw were developed in combining 1-ethyl-3-methyl imidazolium acetate [C2mim][OAc], endo-xylanases from Thermobacillus xylanilyticus and commercial cellulases. After [C2mim][OAc]-pretreatment, hydrolysis catalyzed by endo-xylanases of wheat straw led to efficient xylose production with very competitive yield (97.6 ± 1.3%). Subsequent enzymatic saccharification allowed achieving a total degradation of cellulosic fraction (>99%). These high performances revealed an interesting complementarity of [C2mim][OAc]- and xylanase-pretreatments for increasing enzymatic digestibility of cellulosic fraction in agreement with the structural and morphological changes of wheat straw induced by each of these pretreatment steps. In addition a higher tolerance of endo-xylanases from T. xylaniliticus to [C2mim][AcO] until 30% v/v than cellulases from T. reesei was observed. Based on this property, a simultaneous strategy combining [C2mim][OAc]- and endo-xylanases as pretreatment in a one-batch produced xylose with similar yield than those obtained by the sequential strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Effect of lignin content on a GH11 endoxylanase acting on glucuronoarabinoxylan-lignin nanocomposites. (United States)

    Boukari, Imen; Rémond, Caroline; O'Donohue, Michael; Chabbert, Brigitte


    The effects of lignin content on the activity and action pattern of GH11 endoxylanase from Thermobacillus xylanilyticus were investigated using in vitro reconstituted non-covalent glucuronoarabinoxylan-model lignin (GAX-DHP) nanocomposites. Four types of nanocomposites were prepared, each displaying different lignin contents. Variations in the DHP (model lignin) polymerization process were induced by increasing the coniferyl alcohol concentration. Examination of the morphology of the nanocomposites revealed globular particles enrobed in a matrix. The size of these particles increased in line with the lignin concentration. Physicochemical characterization of the in vitro reconstituted GAX-DHPs strongly suggested that increased particle size is directly related to the solubility and reactivity of coniferyl alcohol, as reflected by changes in the amount of β-O-4 linkages. Evaluation of the impact of the GH11 endoxylanase on the GAX-DHP nanocomposites revealed a negative correlation between the proportion and organization patterns of DHP in the nanocomposites and enzyme activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Bacterial Community Structure and Biochemical Changes Associated With Composting of Lignocellulosic Oil Palm Empty Fruit Bunch

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    Mohd Huzairi Mohd Zainudin


    Full Text Available Bacterial community structure and biochemical changes during the composting of lignocellulosic oil palm empty bunch (EFB and palm oil mill effluent (POME anaerobic sludge were studied by examining the succession of the bacterial community and its association with changes in lignocellulosic components by denaturing gradient gel electrophoresis (DGGE and the 16S rRNA gene clone library. During composting, a major reduction in cellulose after 10 days from 50% to 19% and the carbon content from 44% to 27% towards the end of the 40-day composting period were observed. The C/N ratio also decreased. A drastic change in the bacterial community structure and diversity throughout the composting process was clearly observed using PCR-DGGE banding patterns. The bacterial community drastically shifted between the thermophilic and maturing stages. 16s rRNA clones belonging to the genera Bacillus, Exiguobacterium, Desemzia, and Planococcus were the dominant groups throughout composting. The species closely related to Solibacillus silvestris were found to be major contributors to changes in the lignocellulosic component. Clones identified as Thermobacillus xylanilyticus, Brachybacterium faecium, Cellulosimicrobium cellulans, Cellulomonas sp., and Thermobifida fusca, which are known to be lignocellulosic-degrading bacteria, were also detected and are believed to support the lignocellulose degradation.

  7. Biodegradation of Low-Density Polyethylene (LDPE) by Mixed Culture of Lysinibacillus xylanilyticus and Aspergillus niger in Soil (United States)

    Esmaeili, Atefeh; Pourbabaee, Ahmad Ali; Alikhani, Hossein Ali; Shabani, Farzin; Esmaeili, Ensieh


    In this study, two strains of Aspergillus sp. and Lysinibacillus sp. with remarkable abilities to degrade low-density polyethylene (LDPE) were isolated from landfill soils in Tehran using enrichment culture and screening procedures. The biodegradation process was performed for 126 days in soil using UV- and non-UV-irradiated pure LDPE films without pro-oxidant additives in the presence and absence of mixed cultures of selected microorganisms. The process was monitored by measuring the microbial population, the biomass carbon, pH and respiration in the soil, and the mechanical properties of the films. The carbon dioxide measurements in the soil showed that the biodegradation in the un-inoculated treatments were slow and were about 7.6% and 8.6% of the mineralisation measured for the non-UV-irradiated and UV-irradiated LDPE, respectively, after 126 days. In contrast, in the presence of the selected microorganisms, biodegradation was much more efficient and the percentages of biodegradation were 29.5% and 15.8% for the UV-irradiated and non-UV-irradiated films, respectively. The percentage decrease in the carbonyl index was higher for the UV-irradiated LDPE when the biodegradation was performed in soil inoculated with the selected microorganisms. The percentage elongation of the films decreased during the biodegradation process. The Fourier transform infra-red (FT-IR), x-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to determine structural, morphological and surface changes on polyethylene. These analyses showed that the selected microorganisms could modify and colonise both types of polyethylene. This study also confirmed the ability of these isolates to utilise virgin polyethylene without pro-oxidant additives and oxidation pretreatment, as the carbon source. PMID:24086254

  8. Biodegradation of low-density polyethylene (LDPE by mixed culture of Lysinibacillus xylanilyticus and Aspergillus niger in soil.

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    Atefeh Esmaeili

    Full Text Available In this study, two strains of Aspergillus sp. and Lysinibacillus sp. with remarkable abilities to degrade low-density polyethylene (LDPE were isolated from landfill soils in Tehran using enrichment culture and screening procedures. The biodegradation process was performed for 126 days in soil using UV- and non-UV-irradiated pure LDPE films without pro-oxidant additives in the presence and absence of mixed cultures of selected microorganisms. The process was monitored by measuring the microbial population, the biomass carbon, pH and respiration in the soil, and the mechanical properties of the films. The carbon dioxide measurements in the soil showed that the biodegradation in the un-inoculated treatments were slow and were about 7.6% and 8.6% of the mineralisation measured for the non-UV-irradiated and UV-irradiated LDPE, respectively, after 126 days. In contrast, in the presence of the selected microorganisms, biodegradation was much more efficient and the percentages of biodegradation were 29.5% and 15.8% for the UV-irradiated and non-UV-irradiated films, respectively. The percentage decrease in the carbonyl index was higher for the UV-irradiated LDPE when the biodegradation was performed in soil inoculated with the selected microorganisms. The percentage elongation of the films decreased during the biodegradation process. The Fourier transform infra-red (FT-IR, x-ray diffraction (XRD and scanning electron microscopy (SEM were used to determine structural, morphological and surface changes on polyethylene. These analyses showed that the selected microorganisms could modify and colonise both types of polyethylene. This study also confirmed the ability of these isolates to utilise virgin polyethylene without pro-oxidant additives and oxidation pretreatment, as the carbon source.

  9. Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy

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    Song Letian


    Full Text Available Abstract Background Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second-generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn. Results Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilized arabinoxylans that were not modified by the parental enzyme. The most active mutants, S27T and Y111T, increased the solubilization of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold, respectively, in comparison to the wild-type enzyme. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%tot. xyl (wild-type Tx-Xyn to 18.6% to 20.4%tot. xyl. Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500, thus procuring increases in overall wheat straw hydrolysis. Conclusions Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to (i improved ligand binding in a putative secondary binding site, (ii the diminution of surface hydrophobicity, and/or (iii the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organization of the plant cell wall and the lignin barrier.

  10. The use of thermostable bacterial hemicellulases improves the conversion of lignocellulosic biomass to valuable molecules. (United States)

    Rakotoarivonina, Harivony; Revol, Pierre-Vincent; Aubry, Nathalie; Rémond, Caroline


    The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60 °C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.

  11. Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting (United States)

    Lemos, Leandro N.; Pereira, Roberta V.; Quaggio, Ronaldo B.; Martins, Layla F.; Moura, Livia M. S.; da Silva, Amanda R.; Antunes, Luciana P.; da Silva, Aline M.; Setubal, João C.


    Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the São Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new Thermobacillus species; the first Bacillus thermozeamaize genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as Geobacillus thermoglucosidasius and Caldibacillus debilis. The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new Thermobacillus species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built

  12. Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates. (United States)

    Chen, Yu; Gao, Hongwei; Zhang, Yanming; Deng, Mingjun; Wu, Zhenxing; Zhu, Laihua; Duan, Qing; Xu, Biao; Liang, Chengzhu; Yue, Zhiqin; Xiao, Xizhi


    Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

  13. Lysinibacillus louembei sp. nov., a spore-forming bacterium isolated from Ntoba Mbodi, alkaline fermented leaves of cassava from the Republic of the Congo

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    Ouoba, Labia Irène I.; Mbozo, Alain B. Vouidibio; Thorsen, Line


    . xylanilyticus DSM 23493T and L. odysseyi DSM 18869T was 41%, 16% and 15%, respectively. The internal transcribed spacer-PCR profile of the isolate was different from those of closely related bacteria. The cellwall peptidoglycan type was A4α, L-Lys-D-Asp and the major fatty acids were iso-C15:0, anteiso-C15......Investigation of the microbial diversity of Ntoba Mbodi, an African food made from the alkaline fermentation of cassava leaves, revealed the presence of a Gram-positive, catalase-positive, aerobic, motile and rod-shaped endospore-forming bacterium (NM73) with unusual phenotypic and genotypic......:0, anteiso-C17:0 and iso-C17:0 and iso-C17:1ω10c. The polar lipids included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphoaminolipid, aminolipid, two phospholipids and two unknown lipids. The predominant menaquinones were MK-7 and MK-6.Ribose was the only whole-cell sugar...

  14. Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer. (United States)

    Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi


    In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

  15. Genomics of aerobic cellulose utilization systems in actinobacteria.

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    Iain Anderson

    Full Text Available Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms.

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

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


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

  17. Plant surface cues prime Ustilago maydis for biotrophic development.

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    Daniel Lanver


    Full Text Available Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the sho1/msb2-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime U. maydis for biotrophic development.

  18. [Diversity of Bacillus species inhabiting on the surface and endophyte of lichens collected from Wuyi Mountain]. (United States)

    Ge, Cibin; Liu, Bo; Che, Jianmei; Chen, Meichun; Liu, Guohong; Wei, Jiangchun


    The present work reported the isolation, identification and diversity of Bacillus species colonizing on the surface and endophyte in lichens collected from Wuyi Mountain. Nine lichen samples of Evernia, Stereocaulon, Menegazzia and other 6 genera belonging to 7 families were collected from Wuyi mountain nature reserve. The bacillus-like species colonizing on the surface and endophyte in these lichens were isolated and identified by 16S rRNA gene sequence analysis. There was no bacillus-like species isolated from Evernia, Ramalina and Lecarona. A total of 34 bacillus-like bacteria were isolated from another 6 lichen samples. These bacteria were identified as 24 species and were classified into Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus and Viridiibacillus. Paenibacillus and Bacillus are the dominant genera, and accounting for 41. 2% and 35. 3% of all isolated bacteria respectively. Brevibacillus, Lysinibacillus and Viridiibacillu were first reported being isolated from lichens. There were different species and quantity of bacillus colonizing on the surface and endophyte in different lichens. The quantity of bacillus colonizing on the surface of Physcia was more than 3.85 x 10(6) cfu/g and was the largest in the isolated bacteria, while the species of bacillus colonizing on the surface and endophyte in Stereocaulon was the most abundant. Most of the isolated bacteria were colonizing on (in) one lichen genera, but Paenibacillus taichungensis, Paenibacillus odorifer, Brevibacillus agri, Lysinibacillus xylanilyticus was respectively colonizing on (in) 2-3 lichen genera and Bacillus mycoides was colonizing on (in) Menegazzia, Cladonia Physcia, and Stereocaulon. There are species and quantity diversity of bacillus colonizing on (in) lichens.

  19. Respiratory clearance of aerosolized radioactive solutes of varying molecular weight

    International Nuclear Information System (INIS)

    Huchon, G.J.; Montgomery, A.B.; Lipavsky, A.; Hoeffel, J.M.; Murray, J.F.


    To determine the influence of varying molecular weight (mol wt) on respiratory clearance of aerosolized solutes, we studied eight radiopharmaceuticals, each administered to four dogs: sodium /sup 99m/Tc pertechnetate (TcO4), /sup 99m/Tc glucoheptonate ([/sup 99m/Tc]GH), 51Cr-ethylenedinitrotetraacetate ([51Cr]EDTA), /sup 99m/Tc diethylenetriaminepentaacetate ([99mTc] DTPA), 111 In diethylenetriaminepentaacetate ([ 111 In]DTPA), 67 Ga desferoxaminemesylate ([ 67 Ga]DFOM), /sup 99m/Tc dextran ([/sup 99m/Tc]DX) and 111 In transferrin ([ 111 In]TF). After aerosolization (0.8 m MMD, 2.4 GSD), clearance was determined for 30 min and then corrected by intravenous injection for nonairspace radioactivity. In-TF clearance (0.11 +/- 0.10%/min) was lower than TcO4 (6.32 +/- 0.62%/min), [/sup 99m/Tc]GH (1.50 +/- 0.37%/min), [ 51 Cr]EDTA (2.38 +/- 1.02%/min), [/sup 99m/Tc]DTPA (3.51 +/- 0.40%/min), [ 111 In]DTPA (2.35 +/- 0.42%/min), [ 67 Ga] DFOM (1.99 +/- 0.49%/min) and [/sup 99m/Tc]DX (1.81 +/- 0.75%/min) clearances (p less than 0.001). TcO4 clearance was higher than others (p less than 0.001). Technetium binding to DX was unsatisfactory; aerosolization caused unbinding from DTPA. We conclude that respiratory clearance of large mol wt solutes within 30 min is negligible and, that clearance of molecules between 347-5099 daltons differs greatly, suggesting that binding and/or intrapulmonary retention affect transfer

  20. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

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    Wu Jiajie


    Full Text Available Abstract Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice, the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar. To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model and Sorghum bicolor (sorghum. We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51, we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets

  1. Analysis of the arabinoxylan arabinofuranohydrolase gene family in barley does not support their involvement in the remodelling of endosperm cell walls during development. (United States)

    Laidlaw, Hunter K C; Lahnstein, Jelle; Burton, Rachel A; Fincher, Geoffrey B; Jobling, Stephen A


    Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.

  2. Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation. (United States)

    Kozlova, Liudmila V; Gorshkov, Oleg V; Mokshina, Natalia E; Gorshkova, Tatyana A


    Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.