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Gaudin, Christian, Anne Belaich, Stéphanie Champ y Jean-Pierre Belaich. "CelE, a Multidomain Cellulase fromClostridium cellulolyticum: a Key Enzyme in the Cellulosome?" Journal of Bacteriology 182, n.º 7 (1 de abril de 2000): 1910–15. http://dx.doi.org/10.1128/jb.182.7.1910-1915.2000.
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ABSTRACT CelE, one of the three major proteins of the cellulosome ofClostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active onp-nitrophenyl–cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.
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Krauss, Jan, Vladimir V. Zverlov y Wolfgang H. Schwarz. "In VitroReconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose". Applied and Environmental Microbiology 78, n.º 12 (20 de abril de 2012): 4301–7. http://dx.doi.org/10.1128/aem.07959-11.
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ABSTRACTArtificial cellulase complexes active on crystalline cellulose were reconstitutedin vitrofrom a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of aC. thermocellum cipAmutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. Thein vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities forin vitrocomplex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.
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Hetzler, Stephan, Daniel Bröker y Alexander Steinbüchel. "Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains". Applied and Environmental Microbiology 79, n.º 17 (21 de junio de 2013): 5159–66. http://dx.doi.org/10.1128/aem.01214-13.
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ABSTRACTThe noncellulolytic actinomyceteRhodococcus opacusstrain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation inR. opacusPD630, we engineered strains that episomally expressed six different cellulase genes fromCellulomonas fimiATCC 484 (cenABC,cex,cbhA) andThermobifida fuscaDSM43792 (cel6A), thereby enablingR. opacusPD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose byR. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabledR. opacusPD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain ofR. opacusPD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
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Lynd, Lee R., Paul J. Weimer, Willem H. van Zyl y Isak S. Pretorius. "Microbial Cellulose Utilization: Fundamentals and Biotechnology". Microbiology and Molecular Biology Reviews 66, n.º 3 (septiembre de 2002): 506–77. http://dx.doi.org/10.1128/mmbr.66.3.506-577.2002.
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SUMMARY Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
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Caspi, Jonathan, Yoav Barak, Rachel Haimovitz, Diana Irwin, Raphael Lamed, David B. Wilson y Edward A. Bayer. "Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode". Applied and Environmental Microbiology 75, n.º 23 (9 de octubre de 2009): 7335–42. http://dx.doi.org/10.1128/aem.01241-09.
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ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
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Kudanga, T. y E. Mwenje. "Extracellular cellulase production by tropical isolates of Aureobasidium pullulans". Canadian Journal of Microbiology 51, n.º 9 (1 de septiembre de 2005): 773–76. http://dx.doi.org/10.1139/w05-053.
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Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and α-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 µmol glucose·(mg protein)–1·h–1, while activities on α-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 µmol glucose·(mg protein)–1·day–1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.Key words: Aureobasidium pullulans, plant cell wall, cellulases, endoglucanase, exoglucanase.
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Wang, Hongliang, Fabio Squina, Fernando Segato, Andrew Mort, David Lee, Kirk Pappan y Rolf Prade. "High-Temperature Enzymatic Breakdown of Cellulose". Applied and Environmental Microbiology 77, n.º 15 (17 de junio de 2011): 5199–206. http://dx.doi.org/10.1128/aem.00199-11.
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ABSTRACTCellulose is an abundant and renewable biopolymer that can be used for biofuel generation; however, structural entrapment with other cell wall components hinders enzyme-substrate interactions, a key bottleneck for ethanol production. Biomass is routinely subjected to treatments that facilitate cellulase-cellulose contacts. Cellulases and glucosidases act by hydrolyzing glycosidic bonds of linear glucose β-1,4-linked polymers, producing glucose. Here we describe eight high-temperature-operating cellulases (TCel enzymes) identified from a survey of thermobacterial and archaeal genomes. Three TCel enzymes preferentially hydrolyzed soluble cellulose, while two preferred insoluble cellulose such as cotton linters and filter paper. TCel enzymes had temperature optima ranging from 85°C to 102°C. TCel enzymes were stable, retaining 80% of initial activity after 120 h at 85°C. Two modes of cellulose breakdown, i.e., with endo- and exo-acting glucanases, were detected, and with two-enzyme combinations at 85°C, synergistic cellulase activity was observed for some enzyme combinations.
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Zhou, Qingxin, Jintao Xu, Yanbo Kou, Xinxing Lv, Xi Zhang, Guolei Zhao, Weixin Zhang, Guanjun Chen y Weifeng Liu. "Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose". Eukaryotic Cell 11, n.º 11 (21 de septiembre de 2012): 1371–81. http://dx.doi.org/10.1128/ec.00170-12.
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ABSTRACTAppropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolyticHypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist inH. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displayingin vitrotransglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion ofcel1aandcel1bsignificantly compromised the efficient gene expression of the major cellulase gene,cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation inH. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.
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Liu, Wenjin, Xiao-Zhou Zhang, Zuoming Zhang y Y. H. Percival Zhang. "Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability". Applied and Environmental Microbiology 76, n.º 14 (28 de mayo de 2010): 4914–17. http://dx.doi.org/10.1128/aem.00958-10.
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ABSTRACT A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.
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Murashima, Koichiro, Akihiko Kosugi y Roy H. Doi. "Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans". Journal of Bacteriology 184, n.º 18 (15 de septiembre de 2002): 5088–95. http://dx.doi.org/10.1128/jb.184.18.5088-5095.2002.
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ABSTRACT Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.
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Blair, Benjie G. y Kevin L. Anderson. "Regulation of cellulose-inducible structures of Clostridium cellulovorans". Canadian Journal of Microbiology 45, n.º 3 (1 de marzo de 1999): 242–49. http://dx.doi.org/10.1139/w99-004.
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Scanning electron microscopy was used to detect ultrastructural protuberances on the cellulolytic anaerobe Clostridium cellulovorans. Numerous ultrastructural protuberances were observed on cellulose-grown cells, but few were detected on glucose-, fructose-, cellobiose-, or carboxymethylcellulose (CMC)-grown cells. Formation of these protuberances was detected within 2 h of incubation in cellulose medium, but 4 h incubation was required before numerous structures were observed on the cells. When a soluble carbohydrate or CMC was mixed with cellulose-grown cells, the ultrastructural protuberances could no longer be detected. In fact, no protuberances were observed within 5 min following the addition of glucose, cellobiose, or methylglucose to cellulose-grown cells. The presence of these protuberances corresponded with the binding of the Bandeiraea simplicifolia BSI-B4 isolectin to the cell. Cellulose-grown cells had a greater level of observable lectin binding than cellobiose-grown cells, and lectin binding was not detected on glucose- or fructose-grown cells. In addition, lectin binding ability was lost by cellulose-grown cells following the addition of glucose, fructose, or methylglucose to the cellulose medium. A cellulose-affinity protein fraction expressing cellulase activity was also detected in cell extracts of cellobiose- or cellulose-grown cultures. However, this protein fraction was not detected in extracts of glucose-grown cultures, and was rapidly lost (within 5 min) following the addition of glucose to cellulose-grown cultures. The ability of C. cellulovorans to adhere to cellulose was also affected by the energy substrate, but not in the same manner as the protuberance formation or the cellulase-containing protein fraction. Rather, cellobiose-, cellulose-, and CMC-grown cultures adhered to cellulose, but this adherence was not affected by addition of glucose to the medium. This is the first report that soluble carbohydrates caused the rapid loss of some cellulose-inducible systems of C. cellulovorans.Key words: cellulolytic bacteria, bacterial ultrastructure, polycellulosome, scanning electron microscope, lectin binding, cellulosome.
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Yadav, Vikas, Bruce J. Paniliatis, Hai Shi, Kyongbum Lee, Peggy Cebe y David L. Kaplan. "Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus". Applied and Environmental Microbiology 76, n.º 18 (23 de julio de 2010): 6257–65. http://dx.doi.org/10.1128/aem.00698-10.
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ABSTRACT Despite excellent biocompatibility and mechanical properties, the poor in vitro and in vivo degradability of cellulose has limited its biomedical and biomass conversion applications. To address this issue, we report a metabolic engineering-based approach to the rational redesign of cellular metabolites to introduce N-acetylglucosamine (GlcNAc) residues into cellulosic biopolymers during de novo synthesis from Gluconacetobacter xylinus. The cellulose produced from these engineered cells (modified bacterial cellulose [MBC]) was evaluated and compared with cellulose produced from normal cells (bacterial cellulose [BC]). High GlcNAc content and lower crystallinity in MBC compared to BC make this a multifunctional bioengineered polymer susceptible to lysozyme, an enzyme widespread in the human body, and to rapid hydrolysis by cellulase, an enzyme commonly used in biomass conversion. Degradability in vivo was demonstrated in subcutaneous implants in mice, where modified cellulose was completely degraded within 20 days. We provide a new route toward the production of a family of tailorable modified cellulosic biopolymers that overcome the longstanding limitation associated with the poor degradability of cellulose for a wide range of potential applications.
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Murashima, Koichiro, Akihiko Kosugi y Roy H. Doi. "Synergistic Effects of Cellulosomal Xylanase and Cellulases from Clostridium cellulovorans on Plant Cell Wall Degradation". Journal of Bacteriology 185, n.º 5 (1 de marzo de 2003): 1518–24. http://dx.doi.org/10.1128/jb.185.5.1518-1524.2003.
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ABSTRACT Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.
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Bae, Jungu, Kouichi Kuroda y Mitsuyoshi Ueda. "Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface". Applied and Environmental Microbiology 81, n.º 1 (10 de octubre de 2014): 59–66. http://dx.doi.org/10.1128/aem.02864-14.
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ABSTRACTProximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on theSaccharomyces cerevisiaecell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.
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Mohand-Oussaid, O., S. Payot, E. Guedon, E. Gelhaye, A. Youyou y H. Petitdemange. "The Extracellular Xylan Degradative System inClostridium cellulolyticum Cultivated on Xylan: Evidence for Cell-Free Cellulosome Production". Journal of Bacteriology 181, n.º 13 (1 de julio de 1999): 4035–40. http://dx.doi.org/10.1128/jb.181.13.4035-4040.1999.
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ABSTRACT In this study, we demonstrate that the cellulosome ofClostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticumis able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.
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Mingardon, Florence, Angélique Chanal, Chantal Tardif, Edward A. Bayer y Henri-Pierre Fierobe. "Exploration of New Geometries in Cellulosome-Like Chimeras". Applied and Environmental Microbiology 73, n.º 22 (28 de septiembre de 2007): 7138–49. http://dx.doi.org/10.1128/aem.01306-07.
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ABSTRACT In this study, novel cellulosome chimeras exhibiting atypical geometries and binding modes, wherein the targeting and proximity functions were directly incorporated as integral parts of the enzyme components, were designed. Two pivotal cellulosomal enzymes (family 48 and 9 cellulases) were thus appended with an efficient cellulose-binding module (CBM) and an optional cohesin and/or dockerin. Compared to the parental enzymes, the chimeric cellulases exhibited improved activity on crystalline cellulose as opposed to their reduced activity on amorphous cellulose. Nevertheless, the various complexes assembled using these engineered enzymes were somewhat less active on crystalline cellulose than the conventional designer cellulosomes containing the parental enzymes. The diminished activity appeared to reflect the number of protein-protein interactions within a given complex, which presumably impeded the mobility of their catalytic modules. The presence of numerous CBMs in a given complex, however, also reduced their performance. Furthermore, a “covalent cellulosome” that combines in a single polypeptide chain a CBM, together with family 48 and family 9 catalytic modules, also exhibited reduced activity. This study also revealed that the cohesin-dockerin interaction may be reversible under specific conditions. Taken together, the data demonstrate that cellulosome components can be used to generate higher-order functional composites and suggest that enzyme mobility is a critical parameter for cellulosome efficiency.
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López-Contreras, Ana M., Krisztina Gabor, Aernout A. Martens, Bernadet A. M. Renckens, Pieternel A. M. Claassen, John van der Oost y Willem M. de Vos. "Substrate-Induced Production and Secretion of Cellulases by Clostridium acetobutylicum". Applied and Environmental Microbiology 70, n.º 9 (septiembre de 2004): 5238–43. http://dx.doi.org/10.1128/aem.70.9.5238-5243.2004.
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ABSTRACT Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of β-1,3- and β-1,4-linked β-d-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.
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Yi-Heng, Zhang Percival y Lee R. Lynd. "Regulation of Cellulase Synthesis in Batch and Continuous Cultures of Clostridium thermocellum". Journal of Bacteriology 187, n.º 1 (1 de enero de 2005): 99–106. http://dx.doi.org/10.1128/jb.187.1.99-106.2005.
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ABSTRACT Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.
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Mingardon, Florence, Ang�lique Chanal, Ana M. L�pez-Contreras, Cyril Dray, Edward A. Bayer y Henri-Pierre Fierobe. "Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes". Applied and Environmental Microbiology 73, n.º 12 (27 de abril de 2007): 3822–32. http://dx.doi.org/10.1128/aem.00398-07.
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ABSTRACT Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.
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Somkuti, G. A. "Synthesis of Cellulase by Mucor pusillus and Mucor miehei". Microbiology 81, n.º 1 (1 de enero de 2000): 1–6. http://dx.doi.org/10.1099/00221287-81-1-1.
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Strains of Mucor pusillus and M. miehei were found to synthesize β-1,4-glucan glucanohydrolase (cellulase) in a complex medium. The inducible enzyme complex hydrolysed carboxymethylcellulose, acid-swollen cellulose and unmodified cellulose.
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Schmoll, Monika, André Schuster, Roberto do Nascimento Silva y Christian P. Kubicek. "The G-Alpha Protein GNA3 of Hypocrea jecorina (Anamorph Trichoderma reesei) Regulates Cellulase Gene Expression in the Presence of Light". Eukaryotic Cell 8, n.º 3 (9 de enero de 2009): 410–20. http://dx.doi.org/10.1128/ec.00256-08.
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ABSTRACT Although the enzymes enabling Hypocrea jecorina (anamorph Trichoderma reesei) to degrade the insoluble substrate cellulose have been investigated in some detail, little is still known about the mechanism by which cellulose signals its presence to the fungus. In order to investigate the possible role of a G-protein/cyclic AMP signaling pathway, the gene encoding GNA3, which belongs to the adenylate cyclase-activating class III of G-alpha subunits, was cloned. gna3 is clustered in tandem with the mitogen-activated protein kinase gene tmk3 and the glycogen phosphorylase gene gph1. The gna3 transcript is upregulated in the presence of light and is almost absent in the dark. A strain bearing a constitutively activated version of GNA3 (gna3QL) exhibits strongly increased cellulase transcription in the presence of the inducer cellulose and in the presence of light, whereas a gna3 antisense strain showed delayed cellulase transcription under this condition. However, the gna3QL mutant strain was unable to form cellulases in the absence of cellulose. The necessity of light for stimulation of cellulase transcription by GNA3 could not be overcome in a mutant which expressed gna3 under control of the constitutive gpd1 promoter also in darkness. We conclude that the previously reported stimulation of cellulase gene transcription by light, but not the direct transmission of the cellulose signal, involves the function and activation of GNA3. The upregulation of gna3 by light is influenced by the light modulator ENVOY, but GNA3 itself has no effect on transcription of the light regulator genes blr1, blr2, and env1. Our data for the first time imply an involvement of a G-alpha subunit in a light-dependent signaling event in fungi.
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Mba Medie, Felix, Iskandar Ben Salah, Michel Drancourt y Bernard Henrissat. "Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms". Microbiology 156, n.º 5 (1 de mayo de 2010): 1468–75. http://dx.doi.org/10.1099/mic.0.037812-0.
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The genome of the tuberculosis agent Mycobacterium tuberculosis encodes a putative cellulose-binding protein (CBD2), one candidate cellulase (Cel12), and one fully active cellulase (Cel6). This observation is puzzling, because cellulose is a major component of plant cell walls, whereas M. tuberculosis is a human pathogen without known contact with plants. In order to investigate the biological role of such cellulose-targeting genes in M. tuberculosis we report here the search for and transcription analysis of this set of genes in the genus Mycobacterium. An in silico search for cellulose-targeting orthologues found that only 2.5 % of the sequenced bacterial genomes encode the Cel6, Cel12 and CBD2 gene set simultaneously, including those of the M. tuberculosis complex (MTC) members. PCR amplification and sequencing further demonstrated the presence of these three genes in five non-sequenced MTC bacteria. Among mycobacteria, the combination of Cel6, Cel12 and CBD2 was unique to MTC members, with the exception of Mycobacterium bovis BCG Pasteur, which lacked CBD2. RT-PCR in M. tuberculosis H37Rv indicated that the three cellulose-targeting genes were transcribed into mRNA. The present work shows that MTC organisms are the sole mycobacteria among very few organisms to encode the three cellulose-targeting genes CBD2, Cel6 and Cel12. Our data point toward a unique, yet unknown, relationship with non-plant cellulose-producing hosts such as amoebae.
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Demain, Arnold L., Michael Newcomb y J. H. David Wu. "Cellulase, Clostridia, and Ethanol". Microbiology and Molecular Biology Reviews 69, n.º 1 (marzo de 2005): 124–54. http://dx.doi.org/10.1128/mmbr.69.1.124-154.2005.
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SUMMARY Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.
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Rincón, Marco T., Sheila I. McCrae, James Kirby, Karen P. Scott y Harry J. Flint. "EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain". Applied and Environmental Microbiology 67, n.º 10 (1 de octubre de 2001): 4426–31. http://dx.doi.org/10.1128/aem.67.10.4426-4431.2001.
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ABSTRACT The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase geneendB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression inEscherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.
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Aro, Nina, Marja Ilmén, Anu Saloheimo y Merja Penttilä. "ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression". Applied and Environmental Microbiology 69, n.º 1 (enero de 2003): 56–65. http://dx.doi.org/10.1128/aem.69.1.56-65.2003.
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ABSTRACT We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.
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Cunha, Eva S., Christine L. Hatem y Doug Barrick. "Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity". Applied and Environmental Microbiology 79, n.º 21 (23 de agosto de 2013): 6684–96. http://dx.doi.org/10.1128/aem.02121-13.
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ABSTRACTDegradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8;Clostridium thermocellum) and Cel12A (C12A, GH12;Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12Acatalyticdomain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.
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Gupta, Pratima, Kalpana Samant y Avinash Sahu. "Isolation of Cellulose-Degrading Bacteria and Determination of Their Cellulolytic Potential". International Journal of Microbiology 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/578925.
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Eight isolates of cellulose-degrading bacteria (CDB) were isolated from four different invertebrates (termite, snail, caterpillar, and bookworm) by enriching the basal culture medium with filter paper as substrate for cellulose degradation. To indicate the cellulase activity of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose Congo Red agar media were measured. CDB 8 and CDB 10 exhibited the maximum zone of clearance around the colony with diameter of 45 and 50 mm and with the hydrolytic value of 9 and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and cellulase (endoglucanase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. All the cultures were also further tested for their capacity to degrade filter paper by gravimetric method. The maximum filter paper degradation percentage was estimated to be 65.7 for CDB 8. Selected bacterial isolates CDB 2, 7, 8, and 10 were co-cultured withSaccharomyces cerevisiaefor simultaneous saccharification and fermentation. Ethanol production was positively tested after five days of incubation with acidified potassium dichromate.
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Han, Sung O., Hideaki Yukawa, Masayuki Inui y Roy H. Doi. "Effect of carbon source on the cellulosomal subpopulations of Clostridium cellulovorans". Microbiology 151, n.º 5 (1 de mayo de 2005): 1491–97. http://dx.doi.org/10.1099/mic.0.27605-0.
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Clostridium cellulovorans produces a cellulase enzyme complex called the cellulosome. When cells were grown on different carbon substrates such as Avicel, pectin, xylan, or a mixture of all three, the subunit composition of the cellulosomal subpopulations and their enzymic activities varied significantly. Fractionation of the cellulosomes (7–11 fractions) indicated that the cellulosome population was heterogeneous, although the composition of the scaffolding protein CbpA, endoglucanase EngE and cellobiohydrolase ExgS was relatively constant. One of the cellulosomal fractions with the greatest endoglucanase activity also showed the highest or second highest cellulase activity under all growth conditions tested. The cellulosomal fractions produced from cells grown on a mixture of carbon substrates showed the greatest cellulase activity and contained CbpA, EngE/EngK, ExgS/EngH and EngL. High xylanase activity in cellulose, pectin and mixed carbon-grown cells was detected with a specific cellulosomal fraction which had relatively larger amounts of XynB, XynA and unknown proteins (35–45 kDa). These results in toto indicate that the assembly of cellulosomes occurs in a non-random fashion.
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Jahn, Courtney E., Dija A. Selimi, Jeri D. Barak y Amy O. Charkowski. "The Dickeya dadantii biofilm matrix consists of cellulose nanofibres, and is an emergent property dependent upon the type III secretion system and the cellulose synthesis operon". Microbiology 157, n.º 10 (1 de octubre de 2011): 2733–44. http://dx.doi.org/10.1099/mic.0.051003-0.
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Dickeya dadantii is a plant-pathogenic bacterium that produces cellulose-containing biofilms, called pellicles, at the air–liquid interface of liquid cultures. D. dadantii pellicle formation appears to be an emergent property dependent upon at least three gene clusters, including cellulose synthesis, type III secretion system (T3SS) and flagellar genes. The D. dadantii cellulose synthesis operon is homologous to that of Gluconacetobacter xylinus, which is used for industrial cellulose production, and the cellulose nanofibres produced by D. dadantii were similar in diameter and branching pattern to those produced by G. xylinus. Salmonella enterica, an enterobacterium closely related to D. dadantii, encodes a second type of cellulose synthesis operon, and it produced biofilm strands that differed in width and branching pattern from those of D. dadantii and G. xylinus. Unlike any previously described cellulose fibre, the D. dadantii cellulose nanofibres were decorated with bead-like structures. Mutation of the cellulose synthesis operon genes resulted in loss of cellulose synthesis and production of a cellulase-resistant biofilm. Mutation of other genes required for pellicle formation, including those encoding FliA (a sigma factor that regulates flagella production), HrpL (a sigma factor that regulates the T3SS), and AdrA, a GGDEF protein, affected both biofilm and cell morphology. Mutation of the cellulose synthase bcsA or of bcsC resulted in decreased accumulation of the T3SS-secreted protein HrpN.
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Cai, Shichun, Jiabao Li, Fen Ze Hu, Kegui Zhang, Yuanming Luo, Benjamin Janto, Robert Boissy, Garth Ehrlich y Xiuzhu Dong. "Cellulosilyticum ruminicola, a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes". Applied and Environmental Microbiology 76, n.º 12 (16 de abril de 2010): 3818–24. http://dx.doi.org/10.1128/aem.03124-09.
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ABSTRACT Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.
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Xu, Feng, Hanshu Ding y Ani Tejirian. "Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase". Enzyme and Microbial Technology 45, n.º 3 (septiembre de 2009): 203–9. http://dx.doi.org/10.1016/j.enzmictec.2009.06.002.
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Han, Sung Ok, Hideaki Yukawa, Masayuki Inui y Roy H. Doi. "Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans". Journal of Bacteriology 185, n.º 20 (15 de octubre de 2003): 6067–75. http://dx.doi.org/10.1128/jb.185.20.6067-6075.2003.
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ABSTRACT The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.
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Kosugi, Akihiko, Yoshihiko Amano, Koichiro Murashima y Roy H. Doi. "Hydrophilic Domains of Scaffolding Protein CbpA Promote Glycosyl Hydrolase Activity and Localization of Cellulosomes to the Cell Surface of Clostridium cellulovorans". Journal of Bacteriology 186, n.º 19 (1 de octubre de 2004): 6351–59. http://dx.doi.org/10.1128/jb.186.19.6351-6359.2004.
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ABSTRACT CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.
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Derda, Monika, Jadwiga Winiecka-Krusnell, Markus B. Linder y Ewert Linder. "Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues". Applied and Environmental Microbiology 75, n.º 21 (4 de septiembre de 2009): 6827–30. http://dx.doi.org/10.1128/aem.01555-09.
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ABSTRACT Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.
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DAS, ARPAN, TANMAY PAUL, SUMAN KUMAR HALDER, CHIRANJIT MAITY, PRADEEP KUMAR DAS MOHAPATRA, BIKASH RANJAN PATI y KESHAB CHANDRA MONDAL. "Study on Regulation of Growth and Biosynthesis of Cellulolytic Enzymes from Newly Isolated Aspergillus fumigatus ABK9". Polish Journal of Microbiology 62, n.º 1 (2013): 31–43. http://dx.doi.org/10.33073/pjm-2013-004.
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This study was aimed to evaluate the pattern of cellulase biosynthesis from Aspergillusfumigatus ABK9 under submerged fermentation. Production was increased concomitantly with fungal growth up to 72 h and reached maximum (Xmax -6.72 g/l) with specific growth rate (mu max) of 0.126/h. Highest specific rate of enzyme production (q ) was found at initial medium pH of 5.0 and incubation temperature of 30 degrees C. At the same time, in the presence of 2-deoxy-D-glucose concentration of 0.5 mg/ml, the production of cellulolytic enzymes, viz, carboxymethyl cellulase activity (CMCase), filter paper degrading activity (FPase) and P-glucosidase activity reached maximum of 132.2, 21.3 and 28.9 U/ml, respectively. Cellulase biosynthesis was induced in respect to higher volumetric production rate (Qp), specific rate of enzymes production (qp, U/g biomass/h) and enzyme/biomass yield (YE/X) when grown in carboxymethyl cellulose in comparison to other saccharides as sole carbon source. Induction ratios (IR) of cellulases were between 12.3 and 24.4 in the presence of 1.5% (w/v) CMC in the culture media. The strain was quite resistant to catabolic repression by glucose up to 0.4% (w/v). Cellulases production was greatly influenced in the presence of yeast extract and potassium dihydrogen phosphate (KH2POA) as nitrogen and phosphate sources in the culture media. C/N ratio of 10.0 and C/P ratio of 4.0 proved to be the best for the production of enzyme cocktail. Along with the high production yield, the crude enzymes showed a promising cellulose hydrolyzing efficiency of rice straw, indicating the enzyme could be beneficial for its large scale industrial exploitation.
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Wen, Fei, Jie Sun y Huimin Zhao. "Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol". Applied and Environmental Microbiology 76, n.º 4 (18 de diciembre de 2009): 1251–60. http://dx.doi.org/10.1128/aem.01687-09.
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ABSTRACT By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.
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Patel, Milind A., Mark S. Ou, Roberta Harbrucker, Henry C. Aldrich, Marian L. Buszko, Lonnie O. Ingram y K. T. Shanmugam. "Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid". Applied and Environmental Microbiology 72, n.º 5 (mayo de 2006): 3228–35. http://dx.doi.org/10.1128/aem.72.5.3228-3235.2006.
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ABSTRACT Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50�C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [13C]lactate accounted for more than 90% of the total 13C-labeled products from [13C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.
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Doud, Devin F. R., Robert M. Bowers, Frederik Schulz, Markus De Raad, Kai Deng, Angela Tarver, Evan Glasgow et al. "Function-driven single-cell genomics uncovers cellulose-degrading bacteria from the rare biosphere". ISME Journal 14, n.º 3 (21 de noviembre de 2019): 659–75. http://dx.doi.org/10.1038/s41396-019-0557-y.
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AbstractAssigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.
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Chen, Xin-ai, Nobuhiro Ishida, Nemuri Todaka, Risa Nakamura, Jun-ichi Maruyama, Haruo Takahashi y Katsuhiko Kitamoto. "Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1". Applied and Environmental Microbiology 76, n.º 8 (19 de febrero de 2010): 2556–61. http://dx.doi.org/10.1128/aem.02499-09.
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ABSTRACT Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.
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Gold, Nicholas D. y Vincent J. J. Martin. "Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis". Journal of Bacteriology 189, n.º 19 (20 de julio de 2007): 6787–95. http://dx.doi.org/10.1128/jb.00882-07.
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ABSTRACT A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium Clostridium thermocellum, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.
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Lochner, Adriane, Richard J. Giannone, Miguel Rodriguez, Manesh B. Shah, Jonathan R. Mielenz, Martin Keller, Garabed Antranikian, David E. Graham y Robert L. Hettich. "Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis". Applied and Environmental Microbiology 77, n.º 12 (15 de abril de 2011): 4042–54. http://dx.doi.org/10.1128/aem.02811-10.
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ABSTRACTThe extremely thermophilic, Gram-positive bacteriaCaldicellulosiruptor besciiandCaldicellulosiruptor obsidiansisefficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses fromC. besciiandC. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations:C. obsidiansisexpressed the COB47_1671 protein with GH10/5 domains, whileC. besciiexpressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. TheC. obsidiansispreparation, however, had higher cellulase specific activity and greater thermostability. TheC. besciiculture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.
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Cann, Isaac K. O., Svetlana Kocherginskaya, Michael R. King, Bryan A. White y Roderick I. Mackie. "Molecular Cloning, Sequencing, and Expression of a Novel Multidomain Mannanase Gene from Thermoanaerobacterium polysaccharolyticum". Journal of Bacteriology 181, n.º 5 (1 de marzo de 1999): 1643–51. http://dx.doi.org/10.1128/jb.181.5.1643-1651.1999.
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ABSTRACT The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli. The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative −35 (TTCGC) and −10 (TAAAAT) promoter sequence. The ManA of T. polysaccharolyticum is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of theBacillus-Clostridium cluster. The ManA of T. polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.
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OGAWA, KIHACHIRO, DAISUKE TOYAMA y NOBORU FUJII. "Microcrystalline cellulose-hydrolyzing cellulase (endo-cellulase) from Trichoderma reesei CDU-11." Journal of General and Applied Microbiology 37, n.º 3 (1991): 249–59. http://dx.doi.org/10.2323/jgam.37.249.
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Chérif, Mohamed, Nicole Benhamou y Richard R. Bélanger. "Occurrence of cellulose and chitin in the hyphal walls of Pythium ultimum: a comparative study with other plant pathogenic fungi". Canadian Journal of Microbiology 39, n.º 2 (1 de febrero de 1993): 213–22. http://dx.doi.org/10.1139/m93-030.
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Exoglucanase, a β-1, 4-glucan cellobiohydrolase with specific affinity for β-1, 4-linked glucans, and wheat germ agglutinin, a lectin with N-acetylglucosamine-binding specifity, were used for localizing cellulosic β-1, 4-glucans and chitin in the cell walls of six plant pathogenic fungi. Both chitin and cellulose were found to occur in the cell walls of the oomycete fungus Pythium ultimum whereas only cellulose was present in those of Phythophthora parasitica var. nicotianae, another oomycete fungus. The two compounds were also simultaneously detected in the cell walls of two ascomycetes, Ophiostoma ulmi and Colletotrichum lindemuthianum. Finally, only chitin could be detected in the cell walls of the ascomycete Fusarium oxysporum f.sp. radicis-lycopersici (FORL) and the basidiomycete Rhizoctonia solani. The dual occurrence of chitin and cellulose in Pythium ultimum cell walls was further confirmed by enzymatic extraction performed on isolated cell walls. Treatment of Pythium ultimum cell walls with hen egg white lysozyme, an enzyme with strong chitinolitic activity, prior to labeling with wheat germ agglutinin - ovomucoid - gold complex, resulted in a near abolition of labeling. Similar results were obtained with FORL cell walls used as positive controls to assess the validity of the enzymatic extraction process. When cell walls of Pythium ultimum were treated with a mixture of hen egg white lysozyme and cellulase, both chitin and cellulose were hydrolysed as shown by the considerable reduction of labeling. Thus, these data provide evidence for the presence of chitin in Pythium ultimum cell walls and suggest that classification of Oomycetes as a cellulose-glucan group may be reconsidered.Key words: Pythium ultimum, chitin, cellulose.
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VIJAYARAGHAVAN, PONNUSWAMY y S. G. PRAKASH VINCENT. "Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field". Polish Journal of Microbiology 61, n.º 1 (2012): 51–55. http://dx.doi.org/10.33073/pjm-2012-006.
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A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.
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Park, Yoen Ju y Jinru Chen. "Inactivation of Shiga Toxin-Producing Escherichia coli (STEC) and Degradation and Removal of Cellulose from STEC Surfaces by Using Selected Enzymatic and Chemical Treatments". Applied and Environmental Microbiology 77, n.º 24 (14 de octubre de 2011): 8532–37. http://dx.doi.org/10.1128/aem.06450-11.
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ABSTRACTSome Shiga toxin-producingEscherichia coli(STEC) strains produce extracellular cellulose, a long polymer of glucose with β-1-4 glycosidic bonds. This study evaluated the efficacies of selected enzymatic and chemical treatments in inactivating STEC and degrading/removing the cellulose on STEC surfaces. Six cellulose-producing STEC strains were treated with cellulase (0.51 to 3.83 U/15 ml), acetic and lactic acids (2 and 4%), as well as an acidic and alkaline sanitizer (manufacturers' recommended concentrations) under appropriate conditions. Following each treatment, residual amounts of cellulose and surviving populations of STEC were determined. Treatments with acetic and lactic acids significantly (P< 0.05) reduced the populations of STEC, and those with lactic acid also significantly decreased the amounts of cellulose on STEC. The residual amounts of cellulose on STEC positively correlated to the surviving populations of STEC after the treatments with the organic acids (r= 0.64 to 0.94), and the significance of the correlations ranged from 83 to 99%. Treatments with cellulase and the sanitizers both degraded cellulose. However, treatments with cellulase had no influence on the fate of STEC, and those with the sanitizers reduced STEC cell populations to undetectable levels. Thus, the correlations between the residual amounts of cellulose and the surviving populations of STEC caused by these two treatments were not observed. The results suggest that the selected enzymatic and chemical agents degraded and removed the cellulose on STEC surfaces, and the treatments with organic acids and sanitizers also inactivated STEC cells. The amounts of cellulose produced by STEC strains appear to affect their susceptibilities to certain sanitizing treatments.
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Gilad, Rachel, Larisa Rabinovich, Sima Yaron, Edward A. Bayer, Raphael Lamed, Harry J. Gilbert y Yuval Shoham. "CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose". Journal of Bacteriology 185, n.º 2 (15 de enero de 2003): 391–98. http://dx.doi.org/10.1128/jb.185.2.391-398.2003.
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ABSTRACT The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.
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Jun, Hyun-Sik, Meng Qi, Joshua Gong, Emmanuel E. Egbosimba y Cecil W. Forsberg. "Outer Membrane Proteins of Fibrobacter succinogenes with Potential Roles in Adhesion to Cellulose and in Cellulose Digestion". Journal of Bacteriology 189, n.º 19 (20 de julio de 2007): 6806–15. http://dx.doi.org/10.1128/jb.00560-07.
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ABSTRACT Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.
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Woodward, Jonathan. "Immobilized cellulases for cellulose utilization". Journal of Biotechnology 11, n.º 4 (septiembre de 1989): 299–311. http://dx.doi.org/10.1016/0168-1656(89)90015-1.
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Qi, Meng, Hyun-Sik Jun y Cecil W. Forsberg. "Cel9D, an Atypical 1,4-β-d-Glucan Glucohydrolase from Fibrobacter succinogenes: Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases". Journal of Bacteriology 190, n.º 6 (18 de enero de 2008): 1976–84. http://dx.doi.org/10.1128/jb.01667-07.
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ABSTRACT The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to β-1→4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k cat /K m values for cello-oligosaccharides with higher degrees of polymerization. The k cat /K m value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-β-d-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.