Letteratura scientifica selezionata sul tema "Cellulose – Microbiology"
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Articoli di riviste sul tema "Cellulose – Microbiology"
1
Gaudin, Christian, Anne Belaich, Stéphanie Champ e Jean-Pierre Belaich. "CelE, a Multidomain Cellulase fromClostridium cellulolyticum: a Key Enzyme in the Cellulosome?" Journal of Bacteriology 182, n. 7 (1 aprile 2000): 1910–15. http://dx.doi.org/10.1128/jb.182.7.1910-1915.2000.
Testo completoAbstract (sommario):
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.
2
Krauss, Jan, Vladimir V. Zverlov e Wolfgang H. Schwarz. "In VitroReconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose". Applied and Environmental Microbiology 78, n. 12 (20 aprile 2012): 4301–7. http://dx.doi.org/10.1128/aem.07959-11.
Testo completoAbstract (sommario):
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.
3
Hetzler, Stephan, Daniel Bröker e Alexander Steinbüchel. "Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains". Applied and Environmental Microbiology 79, n. 17 (21 giugno 2013): 5159–66. http://dx.doi.org/10.1128/aem.01214-13.
Testo completoAbstract (sommario):
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.
4
Lynd, Lee R., Paul J. Weimer, Willem H. van Zyl e Isak S. Pretorius. "Microbial Cellulose Utilization: Fundamentals and Biotechnology". Microbiology and Molecular Biology Reviews 66, n. 3 (settembre 2002): 506–77. http://dx.doi.org/10.1128/mmbr.66.3.506-577.2002.
Testo completoAbstract (sommario):
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.
5
Caspi, Jonathan, Yoav Barak, Rachel Haimovitz, Diana Irwin, Raphael Lamed, David B. Wilson e 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. 23 (9 ottobre 2009): 7335–42. http://dx.doi.org/10.1128/aem.01241-09.
Testo completoAbstract (sommario):
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.
6
Kudanga, T., e E. Mwenje. "Extracellular cellulase production by tropical isolates of Aureobasidium pullulans". Canadian Journal of Microbiology 51, n. 9 (1 settembre 2005): 773–76. http://dx.doi.org/10.1139/w05-053.
Testo completoAbstract (sommario):
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.
7
Wang, Hongliang, Fabio Squina, Fernando Segato, Andrew Mort, David Lee, Kirk Pappan e Rolf Prade. "High-Temperature Enzymatic Breakdown of Cellulose". Applied and Environmental Microbiology 77, n. 15 (17 giugno 2011): 5199–206. http://dx.doi.org/10.1128/aem.00199-11.
Testo completoAbstract (sommario):
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.
8
Zhou, Qingxin, Jintao Xu, Yanbo Kou, Xinxing Lv, Xi Zhang, Guolei Zhao, Weixin Zhang, Guanjun Chen e Weifeng Liu. "Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose". Eukaryotic Cell 11, n. 11 (21 settembre 2012): 1371–81. http://dx.doi.org/10.1128/ec.00170-12.
Testo completoAbstract (sommario):
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.
9
Liu, Wenjin, Xiao-Zhou Zhang, Zuoming Zhang e Y. H. Percival Zhang. "Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability". Applied and Environmental Microbiology 76, n. 14 (28 maggio 2010): 4914–17. http://dx.doi.org/10.1128/aem.00958-10.
Testo completoAbstract (sommario):
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.
10
Murashima, Koichiro, Akihiko Kosugi e Roy H. Doi. "Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans". Journal of Bacteriology 184, n. 18 (15 settembre 2002): 5088–95. http://dx.doi.org/10.1128/jb.184.18.5088-5095.2002.
Testo completoAbstract (sommario):
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.
Tesi sul tema "Cellulose – Microbiology"
1
Du, Plessis Lisa. "Co-expression of cellulase genes in Saccharomyces cerevisiae for cellulose degradation". Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/1818.
Testo completo
2
Porter, Suzanne L. "Evidence of multiple cellulase forms in Trichoderma harzianum E58 and their significance in cellulose hydrolysis". Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5829.
Testo completoAbstract (sommario):
The occurrence of multiple cellulase components of Trichoderma harzianum E58 and the implications of their existence on the hydrolysis of cellulose were examined. A single commercial enzyme preparation, Novo-Celluclast, showed different extents of hydrolysis of several cellulosic substrates over time. The filter paper activities of six batches of T. harzianum E58 showed poor correlation with the ability of these enzymes to hydrolyze other cellulosic substrates over extended periods of time. Hydrolysis of a single substrate by a single enzyme preparation resulted in similar slopes in reducing sugar production with enzyme concentration, between one-hour and twenty-four-hour hydrolyses. The multiplicity of the cellulase components of T. harzianum E58 was examined, and the number of endoglucanase components and their specificities towards $\beta$-1,4-linkages were studied. Several types of endoglucanases were produced by the fungus. The role of the exoglucanase was examined using sub-saturation concentrations of T. harzianum E58 cellulase. No significant increase in hydrolysis was observed when purified exoglucanase was added to the cellulase mixture. The high proportion of non-specific endoglucanases and the need for an efficient endoglucanase-to-exoglucanase ratio are discussed in terms of a modified model for cellulose hydrolysis. (Abstract shortened by UMI.)
3
Mokatse, Khomotso. "Production, characterization and evaluation of fungal cellulases for effective digestion of cellulose". Thesis, University of Limpopo (Turfloop Campus), 2013. http://hdl.handle.net/10386/1129.
Testo completoAbstract (sommario):
Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2013The production of cellulase is a key factor in the hydrolysis of cellulosic materials and it is essential to make the process economically viable. Cellulases are the most studied multi- enzyme complex and comprise of endo-glucanases (EG), cellobiohydrolases (CBH) and β- glucosidases (BGL). The complete cellulase system; comprising CBH, EG and BGL components thus acts synergistically to convert crystalline cellulose to glucose. Cellulases are currently the third largest industrial enzyme worldwide. This is due to their wide applications in cotton processing, paper recycling, juice extraction, as detergent enzymes and additives in animal feed. In this study, production of cellulase by five fungal isolates (BTU 251-BTU 255) isolated from mushrooms, was investigated and optimised. Internal transcribed spacer regions (ITS1 and ITS4) were applied to identify the five fungal microorganisms. Isolates were identified as follows: BTU 251 as Aspegillus niger,BTU 253 as Penicillium polonicum, and BTU 255 as Penicillium polonicum. Cellulase was produced in shake flask cultures using Mandel’s mineral solution medium and Avicel as a carbon source. Cellulase activity was tested using 3, 5-Dinitrosalicylic acid assay and zymography, A. niger BTU 251 showed five activity bands ranging from 25- 61 kDa had an average nkat of 7000. Cultures from BTU 252 were the least active with an average nkat/ml of 200 and one activity band of 25 kDa. P. polonicum BTU 253 showed three activity bands ranging between 45 and 60 kDa and had an average nkat/ml of 2200. BTU 254 showed five activity bands ranging from 22- 116 kDa and had average nkat of 350. P. polonicum BTU 255 produced the highest cellulase activity of 8000 nkat/ml and with three activity bands estimated at 45-60 kDa on zymography. The optimal temperature for activity of the cellulases was between 55-70°C and enzymes were most active within a pH range of 4-6. Optimal pH for production of cellulases by P. polonicum BTU 255, P. polonicum BTU 253 and A. niger BTU 251 was 4 while optimal temperature for production of the cellulases was between 50-55°C. Total cellulase activity was determined using Whatman No.1 filter paper as a substrate and β- glucosidase production was determined in polyacrylamide gels using esculin as a substrate. In the hydrolysis of crystalline cellulose (Avicel), a combination of A. niger BTU 251 and P. polonicum BTU 255 (1:1), (1:9), (1:3), and (1:2) produced maximum glucose as follows: 1:1 (0.83g/L), 1:9 (10.4g/L), 1:3 (0.77g/L) and 1:2 (0.73g/L). Cellulases from P. polonicum BTU 255 were partially purified using affinity precipitation and analysed using MALDI- TOF/TOF. Peptide sequences of P. polonicum obtained from MALDI-TOF/TOF analysis were aligned by multiple sequence alignment with C. pingtungium. Conserved regions were identified using BLAST anaylsis as sequences of cellobiohydrolases. More research is required in producing a variety of cellulases that are capable of hydrolysing crystalline cellulose, the current study contributes to possible provision of locally developed combinations of cellulases that can be used in the production of bioethanol.
4
Helle, Steve. "Biosurfactants & cellulose hydrolysis". Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61308.
Testo completoAbstract (sommario):
The action of many antimicrobial agents is dependent on their ability to interact with biological membranes. A group of polypeptide antibiotics was found to have surface activite properties. One of them, gramicidinS, produced a minimum in the surface tension curve, which was attributed to instabilities in the intra-molecular hydrogen bonds. Biosurfactants were found to have a great effect on the two phase hydrolysis of cellulose by cellulase. Seven times as much sugar was produced by the hydrolysis of Sigmacell 100 when the biosurfactant sophorolipid was present. The surfactant affects the adsorption of cellulase onto cellulose, and prevents the cellulase from binding irreversibly to the cellulose and becoming inactive.
5
Van, Rooyen Ronel 1976. "Genetic engineering of the yeast Saccharomyces cerevisiae to ferment cellobiose". Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/19455.
Testo completoAbstract (sommario):
Dissertation (PhD)--Stellenbosch University, 2007.PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en
ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology.
AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC 3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek of dit op te neem en intrasellulêr te benut. Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/ transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen 0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te fermenteer. Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1 ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium. Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende anaerobe geassosieer word. Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut. Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg word.
6
Houghton, James. "Molecular diversity and functional composition of cellulose degrading communities in anoxic environments". Thesis, University of Liverpool, 2013. http://livrepository.liverpool.ac.uk/14933/.
Testo completoAbstract (sommario):
The major fraction of microbial communities cannot be cultivated by artificial means in the laboratory. In order to access the full diversity of microbial life in the open environment it is necessary to employ culture independent methods. Molecular biology and now metagenomics have enabled the phylogenetic and functional investigation of microbial communities without isolation and cultivation of organisms and has led to a new appreciation of the breadth of diversity of microbes on Earth and to the discovery and characterisation of new enzymes. Here, molecular biological techniques have been applied to the study of microbial communities specifically in anaerobic environments and with an emphasis on those involved in the primary degradation of plant cellulosic biomass. Quantitative PCR was used to assess the presence of cellulolytic bacteria both in landfill leachate and specifically in association with cotton cellulose “baits” maintained in leachate microcosms. Lineages of clostridia previously associated with cellulose degrading strains were detected in all five of the landfill leachate samples, and Fibrobacter spp. were detected at low abundance (2.3% of total bacteria) in one sample. Clostridia Group III and Fibrobacter spp. were enriched on the surface of a bait (17% and 29% of total bacteria, respectively) that was rapidly degraded by the colonising community and were present in low abundance (< 1%) and absent, respectively, on another colonised by a community which did not exhibit any degradation of the cellulose. The observed correlation between high levels of cellulose degradation and presence Fibrobacter spp. demonstrates a cellulolytic role outside of the gut environment for these organisms the first time. A metatranscriptome was prepared from a set of cotton cellulose baits maintained in a lake sediment for 2-8 weeks, and Illumina sequencing was used to generated ca. 7 million paired-end reads. Just under one million putative protein coding sequences were identified and of these, MEGAN analysis determined that 40% had no blast hit to the NCBI NR database suggesting that a large number of unknown sequences were present. Analysis of this metatranscriptome and a metagenome produced from the same site revealed that bacteria accounted for 75% of the protein coding sequences and 97% of the metagenome. Genes with matches to cellulolytic lineages of clostridia were found to be present and Fibrobacter sequences were also detected in both of these datasets further demonstrating their presence in the wider environment as probable cellulose degraders ORF prediction and HMM searching were used to search for expressed cellulases in the metatranscriptome and identified 503 sequences with high similarity to glycoside hydrolase protein families, representing carbohydrate active enzymes with possible cellulolytic activity. Of these 112 were also found to have representatives in the metagenome with 100% sequence similarity. All of these sequences had a low level of identity to entries in the NCBI NR database indicating the discovery of previously unknown genes. A fosmid library was produced from the same DNA used to generate the metatranscriptome and it is possible that full-length copies of the expressed genes identified in silico will have been captured. This fosmid library can be interrogated accordingly using probe and PCR primer sequences designed using the curated metatranscriptome dataset. In this way, potentially novel cellulases can be discovered for biochemical characterisation, genetic manipulation and biotechnological exploitation.
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Shaw, Paul B. "Studies of the alkaline degradation of cellulose and the isolation of isosaccharinic acids". Thesis, University of Huddersfield, 2013. http://eprints.hud.ac.uk/id/eprint/19266/.
Testo completoAbstract (sommario):
Cellulosic materials are expected to form a significant proportion of the waste proposed for disposal in underground repositories being designed for the storage of radioactive waste. Under the alkaline conditions of these facilities, cellulose degrades by a so called „peeling‟ reaction resulting in the production of a complex mixture of products (CDPs), the major components being α- and β isosaccharinic acid (α and β-ISA). A significant amount of research has been performed on ISA as part of the safety assessment for the development of these underground repositories due to the ability of ISA to complex with, and increase the solubility of radioactive isotopes. Until now, the vast majority of this research has involved the readily-available α-ISA, only a limited number of studies have involved β-ISA because no simple procedure is available for its isolation. Therefore, in this project, a method for the synthesis and isolation of β-ISA was developed. Cellulose degradation experiments which were performed to maximise solution concentrations of β-ISA are described in chapter 3. Microcrystalline cellulose was degraded under anaerobic conditions at either RT, 50 °C or 90 °C and comparisons were made between the use of NaOH and Ca(OH)2 as the base catalyst. As expected, the major products of all degradation reactions were α- and β-ISA, in addition, small amounts of free metasaccharinic acid (MSA) was detected in the Ca(OH)2 reactions. The largest solution concentrations of β-ISA were produced when cellulose was degraded at 90 °C using NaOH; after 24 hrs of reaction, solution concentrations of 12.7 g L-1 were achieved, whereas, in the equivalent Ca(OH)2 reaction, after 4 days a maximum concentration of only 5.1 g L-1was produced. For this reason, cellulose was degraded at 90 °C using NaOH to produce degradation solutions to be used in procedures to isolate β ISA. An additional finding was that significant amounts of ISA were being removed from degradation solutions due to absorption on to unreacted cellulose fibres; in the NaOH reaction, absorption was occurring rapidly and the percentage of ISA in both the solution and solid phases were very similar. In the Ca(OH)2 reaction, the absorption was a slow process and the percentage of ISA on the solid phase (61 %) was lower than the percentage of ISA in the solution phase (84 %) suggesting that solid Ca(OH)2 was affecting both the rate at which absorption was occurring and the composition of the absorbed species; this was possibly due to solid Ca(OH)2 physically obstructing the access of ISA to the cellulose fibres and also catalysing the oxidation of some of the ISA into smaller fragmentation products. Methods which were developed to isolate β-ISA are described in chapter 4. Isolation of β-ISA was initially achieved by eluting crude cellulose degradation solutions directly through a column of anion exchange resin. Using an automated system, a large throughput of material was possible resulting in the accumulation of relatively large amounts of β-ISA; after repeating the column 17 times, 1 g of pure β-ISA was isolated. However, using this method, the crude solutions severely fouled the anion exchange resin, concluding that anion exchange was more suited to small scale isolations of β-ISA. A final isolation procedure was developed which involved the elution of mixtures of benzoylated CDPs through normal phase silica columns. It was determined that prior to elution, coloured impurities could be efficiently removed by passing the derivatised mixture through a wide bed of silica. Slow elution of the resulting clean syrup through a large silica column allowed up to 7 g of tribenzoylated β-ISAL to be isolated and following de-benzoylation procedures, 2.6 g of β-ISA was isolated from a single column. The large protecting groups also allowed single crystals of both α- and β-tribenzoate to be produced and the resulting X-ray structures confirmed the absolute configuration of tribenzoylated β-ISAL as being 2R, 4S. Additional NMR analysis of collected fractions allowed several other polyhydroxylated compounds to be identified, also present as their perbenzoylated esters, these being: 3,4-dihydroxybutanoic acid, 2,5-dihydroxypentanoic acid, 2,3-dideoxypentanoic acid and 2,4,5-trihydroxypentanoic acid. The isolation of large amounts of β-ISA allowed several solution phase physical properties of β ISA to be measured and these are reported in chapter 5, including the aqueous pKa (3.61) which was determined using NMR methods. The rate constants for the inter-conversion between ISAH and ISAL were also studied for both α- and β-ISA. In acidic environments, ISAH undergoes an acid catalysed lactonisation to generate isosaccharino-1,4-lactone (ISAL), conversely in basic environments, ISAL undergoes a base catalysed ring-opening to produce ISAH. Using pH-stat autotitration, the second-order rate constants for the lactone hydrolysis reaction were determined, to which values of 25.3 M-1 s-1 for β-ISAL and 97.0 M-1 s-1 for α-ISAL were observed. The acid catalysed lactonisation of ISAH was studied using 1H NMR spectroscopy; the second-order rate constant for the lactonisation of β-ISAH (3.10 x 10-3 M-1 s-1) was larger than the second order rate constant for the lactonisation of α-ISAH (7.04 x 10-4 M-1 s-1).
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Sadie, Christa J. (Christiena Johanna). "Expression and characterization of an intracellular cellobiose phosphorylase in Saccharomyces cerevisiae". Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/19862.
Testo completoAbstract (sommario):
Thesis (MSc)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer on earth. Agricultural waste is rich in cellulose and exploiting these renewable sources as a substrate for ethanol production can assist in producing enough bioethanol as a cost-effective replacement for currently used decreasing fossil fuels. Saccharomyces cerevisiae is an excellent fermentative organism of hexoses; however the inability of the yeast to utilize cellulose as a carbon source is a major obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or cellobiose phosphorylase, the latter having a possible metabolic advantage over -glucosidase. Recently, it has been showed that S. cerevisiae is able to transport cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has the advantage that end-product inhibition of the extracellular cellulases by glucose and cellobiose is relieved. Furthermore, the extracellular glucose concentration remains low and the possibility of contamination is decreased. In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium was cloned and expressed under transcriptional control of the constitutive PGK1 promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The enzyme was expressed intracellulary and thus required the transport of cellobiose into the cell. The fur1 gene was disrupted for growth of the recombinant strain on complex media without the loss of the plasmid. The recombinant strain, S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram cellobiose consumed. The recombinant enzyme had activity optima of 60°C and pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69 and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant protein was localized in the membrane fraction and no activity was present in the intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher CepA activity than whole cells indicating that the transport of cellobiose was inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass formation. Cellobiose utilization increased when combined with other sugars (glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This is possibly due to more ATP being available for the cell for cellobiose transport. However, no cellobiose was utilized when grown with fructose indicating catabolite repression by this sugar. To our knowledge this is the first report of a heterologously expressed cellobiose phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report also reaffirms previous data that cellobiose can be utilized intracellularly in S. cerevisiae.
AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol. Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut, het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag gehou en die moontlikheid van kontaminasie beperk. In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en ‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S. cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers (glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose was nie benut nie. Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het. Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.
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Fugelstad, Johanna. "Functional characterization of cellulose and chitin synthase genes in Oomycetes". Doctoral thesis, KTH, Glykovetenskap, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-34012.
Testo completoAbstract (sommario):
Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica. Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.QC 20110531
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Ferdinand, Pierre-Henri. "Adhérence et colonisation des fibres de cellulose par la bactérie cellulolytique Clostridium cellulolyticum. : étude du rôle des protéines CipC et HycP". Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4729.
Testo completoAbstract (sommario):
Clostridium cellulolyticum est une bactérie anaérobie stricte et cellulolytique qui produit des complexes multienzymatiques (cellulosomes) très performants pour la dégradation des polysaccharides de la paroi végétale. C. cellulolyticum adhère à la cellulose et ce phénomène intervient dès les premiers stades de croissance. Pour de nombreuses bactéries cellulolytiques, les cellulosomes semblent impliqués dans le processus d'adhérence et alors que les mécanismes moléculaires mis en jeu pour l'adhérence à la cellulose sont connus ou proposés, celui ou ceux de C. cellulolyticum sont inconnus.Mon projet de thèse a consisté à étudier l'adhérence et la colonisation des fibres de cellulose par C. cellulolyticum et d'identifier le ou les facteurs moléculaires impliqués dans l'adhérence. J'ai ainsi mis en œuvre deux stratégies distinctes. D'une part, une approche par mutagénèse aléatoire qui a permis d'isoler deux clones à l'adhérence diminuée et d'autre part, une approche par mutagénèse ciblée visant à inactiver des gènes candidats, susceptibles d'intervenir dans l'adhérence.J'ai aussi étudié la colonisation des fibres de cellulose par C. cellulolyticum et observé que les cellules adhèrent avec une haute spécificité et affinité à la cellulose. La colonisation des fibres se ferait en mono-couche cellulaire et par successions d'événements d'adhésion-relarguage-réadhésion. Un mutant d'inactivation de CipC, la protéine d'échafaudage des cellulosomes, a mis en évidence l'implication de cette protéine dans l'adhérence, mais aussi que l'adhérence à la cellulose pourrait être multifactorielle. Enfin, j'ai étudié le rôle de HycP, une protéine à CBM3 dont la fonction est inconnueClostridium cellulolyticum is a strict anaerobe, cellulolytic bacteria. It produces multienzymatic complexes, called cellulosomes, which are able to efficiently degrade the plant cell wall polysaccharides. Cellulolytic bacteria, including C. cellulolyticum do binds to cellulose since early growth stage. For most of the studied cellulolytic bacteria, adherence to cellulose seems to be mediated by their cellulosomes. However, molecular factors involved in C. cellulolyticum adherence to cellulose remain unknown.My Ph.D. aimed to implement different but complementary strategies to study adhesion and colonization of cellulose fibers by C. cellulolyticum and to identify the molecular mechanism(s) by which the bacteria bind to cellulose. In order to identify some proteins encoding genes involved in adhesion, I firstly developed random mutagenesis and isolated two adhesion deficient mutants. I also used a targeted mutagenesis tool to inactivate some candidate genes.My studies highlight C. cellulolyticum adheres with both high specificity and affinity to cellulose. Colonization of cellulose fibers by C. cellulolyticum forms a mono-layer of segregated cells on cellulose surface and may occur through cycles of adhesion-release-re-adhesion to substrate. Inactivation of the CipC encoding gene led to a short decrease of the mutant strain's adherence level. This result suggests some other proteins may be involved in C. cellulolyticum adhesion to cellulose. Finally, I studied HycP, a produced and secreted CBM3 encoding protein of unknown function. HycP is a unique protein among databases and may have a phagic origin
Libri sul tema "Cellulose – Microbiology"
1
Simončič, Barbara. Biodegradation of cellulose fibers. New York: Nova Science Publishers, 2010.
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2
Chin-u, Yi. Sŏmyuso punhae hyoso saengsan kyunju rŭl iyong han wanggyŏ wa ssalgyŏ ŭi chaehwaryong kisul kaebal =: Development of technology for utilization of rice hull and rice bran by microorganism produced cellulase. [Seoul]: Nongnimbu, 2007.
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3
Yi, Chin-u. Sŏmyuso punhae hyoso saengsan kyunju rŭl iyong han wanggyŏ wa ssalgyŏ ŭi chaehwaryong kisul kaebal =: Development of technology for utilization of rice hull and rice bran by microorganism produced cellulase. [Seoul]: Nongnimbu, 2007.
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4
Chin-u, Yi. Sŏmyuso punhae hyoso saengsan kyunju rŭl iyong han wanggyŏ wa ssalgyŏ ŭi chaehwaryong kisul kaebal =: Development of technology for utilization of rice hull and rice bran by microorganism produced cellulase. [Seoul]: Nongnimbu, 2007.
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5
Primrose, S. B. Modern biotechnology. Oxford [Oxfordshire]: Blackwell Scientific Publications, 1987.
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6
Necrosis: Methods and protocols. New York: Humana Press, 2013.
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7
Tissue remodeling and epithelial morphogenesis. San Diego: Elsevier/Academic Press, 2009.
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8
Edwards, M. J. ATCC microbes & cells at work: An index to ATCC strains with special applications. Rockville, Md: American Type Culture Collection, 1988.
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9
Bacterial growth and division: Biochemistry and regulation of prokaryotic and eukaryotic division cycles. San Diego: Academic Press, 1991.
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10
Srivastava, Manish, P. K. Mishra, Neha Srivastava, Ram Lakhan Singh e P. W. Ramteke. New and Future Developments in Microbial Biotechnology and Bioengineering: From Cellulose to Cellulase - Strategies to Improve Biofuel Production. Elsevier, 2019.
Cerca il testo completoCapitoli di libri sul tema "Cellulose – Microbiology"
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Degli-Innocenti, F., G. Goglino, G. Bellia, M. Tosin, P. Monciardini e L. Cavaletti. "Isolation and Characterization of Thermophilic Microorganisms Able to Grow on Cellulose Acetate". In Microbiology of Composting, 273–86. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-662-08724-4_23.
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Nozhevnikova, A. N., e M. V. Simankova. "Interspecies Transport of Hydrogen in Thermophilic Anaerobic Cellulose Decomposition". In Microbiology and Biochemistry of Strict Anaerobes Involved in Interspecies Hydrogen Transfer, 427–29. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4613-0613-9_51.
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Himmel, Michael E., John O. Baker, William S. Adney e Stephen R. Decker. "Cellulases, Hemicellulases, and Pectinases". In Methods for General and Molecular Microbiology, 596–610. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555817497.ch24.
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El Nawawy, A. S., E. El-Rayes, R. D. Al Hussaini e A. Tawheed. "Bioconversion of Cellulosic Wastes". In Perspectives in Biotechnology and Applied Microbiology, 223–30. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4321-6_16.
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Tsao, G. T. "Structures of Cellulosic Materials and their Hydrolysis by Enzymes". In Perspectives in Biotechnology and Applied Microbiology, 205–12. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4321-6_14.
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Sukan, S. Suha. "Challenges in Bioconversion of Cellulosic and Partially Soluble Plant Materials in Submerged Culture". In Developments in Food Microbiology—3, 109–40. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-1085-3_5.
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Bu’lock, John D. "Biodegradation of Non-Cellulosic Waste for Environmental Conservation and Fuel Production". In Perspectives in Biotechnology and Applied Microbiology, 171. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4321-6_12.
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Moo-Young, M., J. Lamptey e P. Girard. "Bioconversion of Cellulosic Waste into Protein and Fuel Products: A Case Study of the Technoeconomic Potentials". In Perspectives in Biotechnology and Applied Microbiology, 183–201. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4321-6_13.
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Wilson, D. B. "Cellulases". In Encyclopedia of Microbiology, 252–58. Elsevier, 2009. http://dx.doi.org/10.1016/b978-012373944-5.00138-3.
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Lamed, Raphael, e Edward A. Bayer. "The Cellulosome of Clostridium thermocellum". In Advances in Applied Microbiology, 1–46. Elsevier, 1988. http://dx.doi.org/10.1016/s0065-2164(08)70203-x.
Testo completoAtti di convegni sul tema "Cellulose – Microbiology"
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Snevajsova, P., J. Vytrasova e J. Remesova. "Effect of oxidized cellulose on probiotic bacteria". In Proceedings of the III International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2009). WORLD SCIENTIFIC, 2010. http://dx.doi.org/10.1142/9789814322119_0068.
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Kvesitadze, E., L. Kutateladze, M. Jobava, N. Zakariashvili e I. Khokhashvili. "Xylanase and cellulose free xylanase preparations from microscopic fungi isolated in the South Caucasus". In Proceedings of the II International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2007). WORLD SCIENTIFIC, 2009. http://dx.doi.org/10.1142/9789812837554_0098.
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Vasiliauskiene, Dovile, Andrijana Danytė, Giedrius Balčiūnas e Jaunius Urbonavičius. "The cellulase activity of the fungi that grow on the bio-based thermal insulation composite materials". In 1st International Electronic Conference on Microbiology. Basel, Switzerland: MDPI, 2020. http://dx.doi.org/10.3390/ecm2020-07141.
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Ueda, Junko, Keiko Watanabe, Shuichi Yamamoto e Norio Kurosawa. "Isolation and characterization of cellulase producing bacteria from pruning tree compost and soil". In Proceedings of the III International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2009). WORLD SCIENTIFIC, 2010. http://dx.doi.org/10.1142/9789814322119_0070.
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Pujiati, M. W. Ardhi, E. Muktiani, N. K. Dewi, N. Jadid e E. N. Prasetyo. "The Effect of Incubation Time on Various Type of Local Agricultural Waste in Madiun, Indonesia to Produce Cellulases Using Trichoderma viride". In 10th International Seminar and 12th Congress of Indonesian Society for Microbiology (ISISM 2019). Paris, France: Atlantis Press, 2021. http://dx.doi.org/10.2991/absr.k.210810.030.
Testo completoRapporti di organizzazioni sul tema "Cellulose – Microbiology"
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Peck, Jr., H. D., L. G. Ljungdahl, L. E. Mortenson e J. K. W. Wiegel. The microbiology and physiology of anaerobic fermentations of cellulose: Progress report, November 1988--July 1989. Office of Scientific and Technical Information (OSTI), gennaio 1989. http://dx.doi.org/10.2172/5961636.
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Ljungdahl, L. G., J. Wiegel, H. D. Jr Peck e L. E. Mortenson. Microbiology and physiology of anaerobic fermentation of cellulose. Annual report for 1990, 1992, 1993 and final report. Office of Scientific and Technical Information (OSTI), agosto 1993. http://dx.doi.org/10.2172/90164.
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Ljungdahl, L. G. Microbiology and physiology of anaerobic fermentation of cellulose. Progress report (4/30/91--4/30/92) and outline of work for the period 9/1/92--9/1/93. Office of Scientific and Technical Information (OSTI), dicembre 1992. http://dx.doi.org/10.2172/90165.
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