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Artykuły w czasopismach na temat "GH15"
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Wang, Qiong, Mengmeng Xu, Liting Zhao, Lei Chen i Zhongyang Ding. "Novel Insights into the Mechanism Underlying High Polysaccharide Yield in Submerged Culture of Ganoderma lucidum Revealed by Transcriptome and Proteome Analyses". Microorganisms 11, nr 3 (17.03.2023): 772. http://dx.doi.org/10.3390/microorganisms11030772.
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Polysaccharides are crucial dietary supplements and traditional pharmacological components of Ganoderma lucidum; however, the mechanisms responsible for high polysaccharide yields in G. lucidum remain unclear. Therefore, we investigated the mechanisms underlying the high yield of polysaccharides in submerged cultures of G. lucidum using transcriptomic and proteomic analyses. Several glycoside hydrolase (GH) genes and proteins, which are associated with the degradation of fungal cell walls, were significantly upregulated under high polysaccharide yield conditions. They mainly belonged to the GH3, GH5, GH16, GH17, GH18, GH55, GH79, GH128, GH152, and GH154 families. Additionally, the results suggested that the cell wall polysaccharide could be degraded by GHs, which is beneficial for extracting more intracellular polysaccharides from cultured mycelia. Furthermore, some of the degraded polysaccharides were released into the culture broth, which is beneficial for obtaining more extracellular polysaccharides. Our findings provide new insights into the mechanisms underlying the roles that GH family genes play to regulate high polysaccharide yields in G. lucidum.
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Lam, Ming Quan, Nicola C. Oates, Daniel R. Leadbeater, Kian Mau Goh, Adibah Yahya, Madihah Md Salleh, Zaharah Ibrahim, Neil C. Bruce i Chun Shiong Chong. "Genomic Analysis to Elucidate the Lignocellulose Degrading Capability of a New Halophile Robertkochia solimangrovi". Genes 13, nr 11 (17.11.2022): 2135. http://dx.doi.org/10.3390/genes13112135.
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Robertkochia solimangrovi is a proposed marine bacterium isolated from mangrove soil. So far, the study of this bacterium is limited to taxonomy only. In this report, we performed a genomic analysis of R. solimangrovi that revealed its lignocellulose degrading ability. Genome mining of R. solimangrovi revealed a total of 87 lignocellulose degrading enzymes. These enzymes include cellulases (GH3, GH5, GH9 and GH30), xylanases (GH5, GH10, GH43, GH51, GH67, and GH115), mannanases (GH2, GH26, GH27 and GH113) and xyloglucanases (GH2, GH5, GH16, GH29, GH31 and GH95). Most of the lignocellulolytic enzymes encoded in R. solimangrovi were absent in the genome of Robertkochia marina, the closest member from the same genus. Furthermore, current work also demonstrated the ability of R. solimangrovi to produce lignocellulolytic enzymes to deconstruct oil palm empty fruit bunch (EFB), a lignocellulosic waste found abundantly in palm oil industry. The metabolic pathway taken by R. solimangrovi to transport and process the reducing sugars after the action of lignocellulolytic enzymes on EFB was also inferred based on genomic data. Collectively, genomic analysis coupled with experimental studies elucidated R. solimangrovi to serve as a promising candidate in seawater based-biorefinery industry.
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Gu, Jingmin, Xiaohe Liu, Mei Yang, Yue Li, Changjiang Sun, Rong Lu, Jun Song i in. "Genomic characterization of lytic Staphylococcus aureus phage GH15: providing new clues to intron shift in phages". Journal of General Virology 94, nr 4 (1.04.2013): 906–15. http://dx.doi.org/10.1099/vir.0.049197-0.
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Phage GH15 is a polyvalent phage that shows activity against a wide range of Staphylcoccus aureus strains. This study analysed the genome of GH15. The genome size of GH15 (139 806 bp) was found to be larger than that of the known staphylococcal phages, and the G+C content (30.23 mol%) of GH15 was lower than that of any other staphylococcal myovirus phages. By mass spectrometry, ten structural proteins were identified. Analysis revealed that GH15 was closely related to phages G1, ISP, A5W, Sb-1 and K, and was moderately related to Twort. In light of the variability in identity, coverage, G+C content and genome size, coupled with the large number of mosaicisms, there certainly were close evolutionary relationships from K to Sb-1, A5W, ISP, G1 and finally GH15. Interestingly, all the introns and inteins present in the above phages were absent in GH15 and there appeared to be intron loss in GH15 compared with the intron gain seen in other phages. A comparison of the intron- and intein-related genes demonstrated a clear distinction in the location of the insertion site between intron-containing and intron-free alleles, and this might lead to the establishment of a consensus sequence associated with the presence of an intron or intein. The comparative analysis of the GH15 genome sequence with other phages not only provides compelling evidence for the diversity of staphylococcal myovirus phages but also offers new clues to intron shift in phages.
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Gu, Jingmin, Xiaohe Liu, Rong Lu, Yue Li, Jun Song, Liancheng Lei, Changjiang Sun i in. "Complete Genome Sequence of Staphylococcus aureus Bacteriophage GH15". Journal of Virology 86, nr 16 (27.07.2012): 8914–15. http://dx.doi.org/10.1128/jvi.01313-12.
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GH15 is a polyvalent phage that shows activity against a wide range ofStaphylococcus aureusstrains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.
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Sakaguchi, Masayoshi, Satoru Shimodaira, Shin-nosuke Ishida, Miko Amemiya, Shotaro Honda, Yasusato Sugahara, Fumitaka Oyama i Masao Kawakita. "Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range". Applied and Environmental Microbiology 81, nr 15 (15.05.2015): 4920–31. http://dx.doi.org/10.1128/aem.00956-15.
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ABSTRACTTwo glucoamylase-like genes,TVN1315andTa0286, from the archaeaThermoplasma volcaniumandT. acidophilum, respectively, were expressed inEscherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes displayKmvalues for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.
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Janeček, Štefan. "Amylolytic enzymes - focus on the alpha-amylases from Archae and plants". Nova Biotechnologica et Chimica 9, nr 1 (29.11.2021): 5–26. http://dx.doi.org/10.36547/nbc.1284.
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Amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related poly- and oligosaccharides. The three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. In the sequence-based classification of all glycoside hydrolases (GHs) they have therefore been classified into the three independent families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases). Some amylolytic enzymes have been placed to the families GH31 and GH57. The family GH13 together with the families GH70 and GH77 constitutes the clan GH-H, well-known as the α-amylase family. It contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic TIM-barrel fold, retaining reaction mechanism and catalytic triad. Among the GH13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. Despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. These specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from Archaea.
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Dai, Xin, Yan Tian, Jinting Li, Xiaoyun Su, Xuewei Wang, Shengguo Zhao, Li Liu i in. "Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen". Applied and Environmental Microbiology 81, nr 4 (12.12.2014): 1375–86. http://dx.doi.org/10.1128/aem.03682-14.
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ABSTRACTThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ∼1% and ∼0.1% of the total non-rRNAs, respectively. The majority (∼98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized byRuminococcusandFibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the generaRuminococcus,Prevotella, andFibrobacter. Most (∼82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the generaRuminococcus,Fibrobacter, andPrevotellaare predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.
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Zhang, Junhua, Xuehua Yu, Bo Guan, Youzhen Hu, Xu Li, Jun Zeng i Yongqing Ni. "Identification and Characterization of a Novel Cold-Adapted GH15 Family Trehalase from the Psychrotolerant Microbacterium phyllosphaerae LW106". Fermentation 8, nr 10 (21.09.2022): 471. http://dx.doi.org/10.3390/fermentation8100471.
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Psychrophiles inhabiting various cold environments are regarded as having evolved diverse physiological and molecular strategies, such as the accumulation of trehalose to alleviate cold stress. To investigate the possible contributions of trehalose metabolism-related enzymes to cold-adaption in psychrotrophic bacteria and enrich the resource bank of trehalose hydrolysis enzymes, a novel cold-adapted GH15 GA-like trehalase (MpTre15A) from psychrotolerant Microbacteriumphyllosphaerae LW106 isolated from glacier sediments was cloned and characterized. The recombinant MpTre15A from M. phyllosphaerae LW106 was expressed and purified in Escherichia coli BL21(DE3). The purified MpTre15A functioned as a hexamer and displayed maximal activity at pH 5.0 and 50 °C. Substrate specificity assay proved MpTre15A only showed hydrolytic activity toward α,α-trehalose. Site-directed mutation verified the key catalytic sites of Glu392 and Glu557 in MpTre15A. The kcat and kcat/Km values of MpTre15A at 4 °C (104.50 s−1 and 1.6 s−1 mM−1, respectively) were comparable to those observed for thermophilic GH15 trehalases at 50 °C, revealing its typical cold-adaptability. MpTre15A showed a trehalose conversion rate of 100% and 99.4% after 10 min and 15 min of incubation at 50 °C and 37 °C, respectively. In conclusion, this novel cold-adapted α,α-trehalase MpTre15A showed potential application for developing therapeutic enzymes, enzyme-based biosensors, and enzyme additives in the fermentation industry.
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Huyen, Do Thi, Nguyen Minh Giang, Nguyen Thu Nguyet i Truong Nam Hai. "Probe design for mining and selection of genes coding endo 1- 4 xylanase from dna metagenome data". TAP CHI SINH HOC 40, nr 1 (25.01.2018): 39–50. http://dx.doi.org/10.15625/0866-7160/v40n1.9200.
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According to the CAZY classification, endo 1- 4 xylanase belongs to GH 5, 8, 10, 11, 30, 51, 98. However only 03 sequences of GH8, 27 sequences of GH10, 18 sequence of GH11, only one sequence of each GH30 and GH51 from CAZy and NCBI database were thouroughly experimentally studied for biological activity and characteristics of the enzyme. Through the collected sequences, two probes for endo 1- 4 xylanase of GH10 and GH11 were designed, based on the sequence homology. The GH10 probe was 338 amino acids lenghth contained all the conserved amino acid residues (16 conserved residues in all sequences, 13 residues similar in almost sequences, 14 residues conserved in many sequences) with the lowest maxscore of 189, coverage of 88% and identity of 39%. The GH11 probe was 204 amino acids contained all the conserved amino acid residues (54 conserved residues were identity in all sequences, 25 residues similar in almost sequences, 24 residues conserved in many sequences) with the lowest maxscore of 165, coverage of 84% and identity of 50%. Using the two probes, we mined only one sequence (GL0018509) for endo 1- 4 xylanase from metagenomic DNA data of free-living bacteria in Coptotermes termite gut. Prediction of three-dimention structure of GL0018509 sequence by Phyre2 and Swiss Prot showed that this sequence was high similarity (95% by Phyre2 and 93,4% by Swiss Prot) with endo 1- 4 xylanase with the 100% confidence.
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Wu, Xiaofeng, Chijioke O. Elekwachi, Shiping Bai, Yuheng Luo, Keying Zhang i Robert J. Forster. "Characterizing the Alteration in Rumen Microbiome and Carbohydrate-Active Enzymes Profile with Forage of Muskoxen Rumen through Comparative Metatranscriptomics". Microorganisms 10, nr 1 (30.12.2021): 71. http://dx.doi.org/10.3390/microorganisms10010071.
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Muskox (Ovibos moschatus), as the biggest herbivore in the High Arctic, has been enduring the austere arctic nutritional conditions and has evolved to ingest and digest scarce and high lignified forages to support the growth and reproduce, implying probably harbor a distinct microbial reservoir for the deconstruction of plant biomass. Therefore, metagenomics approach was applied to characterize the rumen microbial community and understand the alteration in rumen microbiome of muskoxen fed either triticale straw or brome hay. The difference in the structure of microbial communities including bacteria, archaea, fungi, and protozoa between the two forages was observed at the taxonomic level of genus. Further, although the highly abundant phylotypes in muskoxen rumen fed either triticale straw or brome hay were almost the same, the selective enrichment different phylotypes for fiber degrading, soluble substrates fermenting, electron and hydrogen scavenging through methanogenesis, acetogenesis, propionogenesis, and sulfur-reducing was also noticed. Specifically, triticale straw with higher content of fiber, cellulose selectively enriched more lignocellulolytic taxa and electron transferring taxa, while brome hay with higher nitrogen content selectively enriched more families and genera for degradable substrates-digesting. Intriguingly, the carbohydrate-active enzyme profile suggested an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on triticale straw. The majority of the cellulases belonged to fiver GH families (i.e., GH5, GH6, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus, Piromyces, Neocallimastix, and Fibrobacter. Abundance of major genes coding for hemicellulose digestion was higher than cellulose mainly including GH8, GH10, GH16, GH26, and GH30, and these enzymes were produced by members of the genera Fibrobacter, Ruminococcus, and Clostridium. Oligosaccharides were mainly of the GH1, GH2, GH3, and GH31 types and were associated with the genera Prevotella and Piromyces. Our results strengthen metatranscriptomic evidence in support of the understanding of the microbial community and plant polysaccharide response to changes in the feed type and host animal. The study also establishes these specific microbial consortia procured from triticale straw group can be used further for efficient plant biomass hydrolysis.
Rozprawy doktorskie na temat "GH15"
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Millet, Nicolas. "Etude des familles de Glycoside-Hydrolases GH16, GH17 et GH55 dans la morphogénèse pariétale du pathogène opportuniste, Aspergillus fumigatus". Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC330.
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La paroi cellulaire fongique est une couche externe et robuste composée principalement de polysaccharides, qui protège la cellule fongique de son environnement, médie l'interaction cellule-cellule et est responsable de la forme de la cellule. La paroi cellulaire du pathogène opportuniste Aspergillus fumigatus est essentiellement composée de ß(1,3)glucane qui est synthétisé au niveau de la membrane plasmique par un complexe transmembranaire puis modifié dans l'espace pariétal par des activités de ramification, de réticulation et de dégradation. La paroi cellulaire est une structure hautement dynamique qui subit des changements constants au cours de la morphogenèse fongique. Dans cette étude, nous avons étudié le rôle de trois familles de glycoside-hydrolases (GH16, GH17 et GH55) dans le remodelage du ß(1,3)glucane la paroi cellulaire de ce pathogène fongique par différentes approches. Les activités transglycosidase et glucanase respectivement de AfCrh5p et AsScw11p ont été étudiées en utilisant des protéines recombinantes et la première structure cristallographique de la famille Crh a été résolue. D’autre part, une délétion complète des gènes de chaque famille a été réalisée pour étudier la fonction biologique de ces enzymes et a mis en évidence un rôle à de multiples facettes de ces glycosides-hydrolases dans la morphogenèse du champignon filamenteux, A. fumigatusThe fungal cell wall is an outer and robust layer mainly composed of polysaccharides, which protects the fungal cell from its environment, mediates cell-cell interaction, and is responsible for the shape of the cell. The cell wall of the opportunistic pathogen Aspergillus fumigatus is essentially composed of ß(1,3)glucan which is synthesized at the plasma membrane by a transmembrane complex and then modified in the cell wall space by branching, cross-linking and degradating activities. The cell wall is a highly dynamic structure, which undergoes constant change during cell division, growth and morphogenesis. In this study, we investigated the role of three glycoside-hydrolases families (GH16, GH17 and GH55) in cell wall remodeling of this fungal pathogen by different approaches. Transglycosidase and glucanase activities of respectively AfCrh5p and AsScw11p have been studied by using recombinant proteins and the first crystal structure of the Crh family have been resolved. Furthermore, complete deletion of each family's genes has been performed to study the biological function of these enzymes and highlighted a multi-faceted role of this glycosides-hydrolases in the morphogenesis of the filamentous fungus, A. fumigatus
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Leno, Antoine. "Contribution à l’amélioration des performances en rendement et en stabilité d’impulsion à impulsion des amplificateurs de puissance, conçus à base de transistors en Nitrure de Gallium, pour les applications RADAR en Bande S". Electronic Thesis or Diss., Limoges, 2023. http://www.theses.fr/2023LIMO0022.
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Ces travaux de thèse s’intègrent dans le cadre des études et des recherches pour améliorer les performances conjointes de puissance, de gain, de rendement et stabilité d’impulsion à impulsion des amplificateurs de puissance à base de transistor en technologie GaN pour la mise en œuvre de RADAR à antennes actives en bande S qui constituent actuellement un enjeu primordial au niveau académique comme au niveau industriel. La conception de ces amplificateurs de puissance pour la détection précise et fiable de cibles représente un défi majeur pour les entreprises du domaine, lorsqu’il est associé à des rendements énergétiques ambitieux avec un objectif supérieur à 65%. Une méthode de conception d’amplificateur de puissance en technologie Q-MMIC en boîtier plastique DFN fondé sur l’utilisation des transistors compacts GH15 EU a été développée et utilisée pour concevoir un amplificateur de puissance fonctionnant en bande S [2.9 - 3.3] GHz. L’amplificateur de puissance réalisé a été caractérisé en termes de rendement en puissance ajoutée, de puissance délivrée, de gain et de stabilité d’impulsion à impulsion en présence de signaux radar. L’amplificateur de puissance compact montre des performances très intéressantes comparées à celles obtenue dans la littérature. En effet, à une puissance moyenne disponible du générateur égale à 26dBm, dans la bande [2.8 - 3.3] GHz, la PAE est comprise entre 59% et 66%, la puissance délivrée varie entre 45W et 52W sur la bande considérée et elle est associée à un gain supérieur à 20dB et une stabilité d’impulsion à impulsion calculée égale à -52dB par la méthode RMS. Les résultats de caractérisations de l’amplificateur de puissance à très haut rendement/ haute puissance à base de transistors compact GH15 EU ont démontré l’intérêt de son utilisation dans les nouvelles générations des systèmes radar en termes de performances RF, de stabilité P2P, d’intégration et de coûtThis thesis work is part of the studies and research to improve the joint performance of power, gain, efficiency and pulse-to-pulse stability of transistor-based power amplifiers in GaN technology for the implementation of RADAR with active antennas in S-band, which is currently a major issue at the academic and industrial levels. The design of these power amplifiers for accurate and reliable detection of targets represents a major challenge for companies in the field, when associated with ambitious energy yields with an objective greater than 65%. A design method for a power amplifier in Q-MMIC technology in a DFN plastic package based on the use of GH15 EU compact transistors has been developed and used to design a power amplifier operating in the S-band [2.9 - 3.3] GHz. The realized power amplifier has been characterized in terms of added power efficiency, delivered power, gain and pulse-to-pulse stability in the presence of radar signals. The compact power amplifier shows very interesting performances compared to those obtained in the literature. Indeed, at an average available power of the generator equal to 26dBm, in the band [2.8 - 3.3] GHz, the PAE is between 59% and 66%, the delivered power varies between 45W and 52W on the considered band and it is associated with a gain higher than 20dB and a pulse-to-pulse stability calculated equal to - 52dB by the RMS method The results of the characterization of the GH15 EU compact transistor based high efficiency/high power power amplifier have demonstrated the interest of its use in the new generation of radar systems in terms of RF performance, P2P stability, integration and cost
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Liberato, Marcelo Vizoná. "Caracterização estrutural de endoglucanases da família GH5 e beta-glicosidases da família GH1: interação enzima-substrato". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-28012014-142924/.
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A celulose é o biopolímero de maior abundância no mundo e tem potencial para se tornar fonte de energia renovável através de sua transformação em açúcares fermentáveis, que por sua vez serão transformados em etanol. A recalcitrância da celulose, principal dificuldade encontrada no processo, pode ser superada com o auxílio de enzimas (celulases). Ao menos três enzimas celulolíticas são necessárias para a degradação total da celulose, incluindo as celobioidrolases, que hidrolisam as ligações glicosídicas das extremidades redutoras e não redutoras da cadeia, as endoglucanases, que clivam a cadeia de celulose amorfa randomicamente, e as beta-glicosidases, que produzem glicose através dos celo-oligômeros. Mas para que esse processo se torne financeiramente viável é necessário conhecer o funcionamento, otimizar a atividade e aumentar a produção dessas celulases. Com o intuito de avançar na compreensão da função e estrutura dessas enzimas, o presente trabalho teve como objetivo o estudo estrutural de beta-glicosidases da família GH1 e endoglucanases da família GH5. Na primeira parte do trabalho, a expressão da endoglucanase II de Trichoderma reesei não foi alcançada, mesmo utilizando diferentes organismos e condições de expressão. Porém, na segunda etapa, foi obtida a expressão, purificação e os primeiros ensaios de cristalização de 11 beta-glicosidases bacterianas da família GH1 e 8 endoglucanases bacterianas da família GH5. Dentre elas, três beta-glicosidases e uma endoglucanase de Bacillus licheniformis foram cristalizadas e tiveram sua estrutura resolvida. As beta-glicosidases, apesar de possuírem o enovelamente similar, apresentaram variações no tamanho e posição das alças formadoras da fenda catalítica e divergem em relação a um dos aminoácidos importantes para a estabilização do substrato. Essas diferenças podem ajudar a explicar o mecanismo dessas enzimas para reconhecer substratos distintos. A endoglucanase da família GH5, possuindo dois módulos acessórios, foi cristalizada tanto na forma apo quanto complexada ao substrato celotetraose. O segundo módulo acessório possivelmente é um domínio de ligação à celulose (CBM) e seus resíduos aromáticos, que são responsáveis pela interação com o substrato, parecem complementar o sítio catalítico, sendo assim um novo mecanismo de auxílio enzimático de um CBM. O primeiro módulo acessório não possui um aparente sítio de interação com carboidratos e provavelmente funciona como um conector entre domínio catalítico e o CBM. O posicionamento do substrato no sítio de ligação é parecido com outras estruturas já determinadas, porém, suscita algumas dúvidas sobre a função dos resíduos catalíticos que é conservada na família. O carbono anomérico do substrato possui uma densidade eletrônica contínua com o glutamato da fita β4 (que deveria ser o ácido/base) e está mais próximo dele que do glutamato da fita β7 (que deveria ser o nucleófilo).Cellulose is the most abundant biopolymer in the world and can become a renewable energy source through its transformation in fermentable sugars, which will be converted in bioethanol. The cellulose recalcitrance, main difficulty in the process, can be overcome with the aid of enzymes (cellulases). At least three cellulolytic enzymes are required for complete hydrolysis of cellulose, including cellobiohydrolases for hydrolyzing the glycosidic linkages from the reducing and non-reducing chain ends, endoglucanases for randomly cleaving cellulose chains in the amorphous regions, and beta-glucosidases for producing glucose from the solubilized cello-oligomers. But, to become a financially viable process it is necessary to know the mechanism, optimize the activity and improve the production of these cellulases. In order to advance the understanding of the structure and function of these enzymes, the present work intended to study the structure of beta-glucosidases from family GH1 and endoglucanases from family GH5. In the first part of the work, the expression of endoglucanase II from Trichoderma reesei was not achieved, even using different organisms and expression conditions. However, in the second part, the expression, purification and the crystallization first trials of eleven bacterial beta-glucosidases and eight bacterial endoglucanases were achieved. Among them, three beta-glucosidases and one endoglucanase from Bacillus licheniformis were crystallized and had their structures solved. Beta-glucosidases, although having a similar folding, showed variations in the length and position of the loops that form the catalytic cleft and diverge in relation to one of the amino acids that are important in substrate stabilization. These differences may help explain the mechanism of these enzymes to recognize distinct substrates. The endoglucanase, which has two accessory modules, was crystallized in the apo form and complexed with the substrate celotetraose. The second accessory module probably is a cellulose binding domain (CBM) and its aromatic residues, which are responsible for the substrate interaction, seem to complement the catalytic site. Therefore it can be a new mechanism of CBM assistance in the enzymatic activity. The first accessory module has no apparent interaction site with carbohydrates and probably works as a connector between the catalytic domain and CBM. The positioning of the substrate in the binding site is similar to other structures already solved but raises some questions about the role of the catalytic residues, that are conserved in the family. The anomeric carbon of the substrate has a continuous electron density with glutamate from sheet-β4 (which should be the acid/base) and is closer to it than to glutamate from sheet-β7 (which should be the nucleophile).
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Berto, Gabriela Leila. "Clonagem, expressão e purificação de glicosil hidrolases (GH5 e GH45) provenientes do fungo Gloeophyllum trabeum e estudo da ação das proteínas como auxiliares na hidrólise de polissacarídeos". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/97/97131/tde-14092016-175401/.
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Fungos de podridão branca e fungos de podridrão parda são capazes de degradar a biomassa lignocelulósica utilizando diferentes mecanismos. Os fungos de podridão parda degradam a celulose e a hemicelulose enquanto modificam a lignina, sendo, portanto, as enzimas hidrolíticas ativas em substrato rico em lignina. O arsenal enzimático diferenciado desses fungos torna-os alvos para prospecção de enzimas que podem ser aplicadas em diversos processos biotecnológicos. O fungo Gloeophyllum trabeum é uma das espécies mais compreendidas deste grupo, e sabe-se que é capaz de degradar a celulose sem produzir CBHs. Além disso já foi reportado uma GH5 proveniente desse organismo com caraterísticas processivas. Nossa tentativa de clonagem dessa enzima (GtGH5) em A. nidulans A773 não foi bem sucedida, todavia nosso grupo esta repetindo os passos de clonagem e expressão. No genoma desse organismo esta presente um gene que codifica uma GH45. A GtGH45 foi a clonada e expressa com alto rendimento em A. nidulans cepa A773. A expressão, secreção e acumulaçao da GtGH45 foi positiva após 72 h de incubação em meio líquido (maltose como indutor), confirma por eletroforese SDS-PAGE do extrato enzimático concentrado e dialisado. A massa molar de 18,4 kDa foi consistente com a predição da sequência de 204 amino-ácidos obtída apartir da sequência gênica e corrobora com a caracterização por espectometria de massas (18,9 kDa). A análise filogenética é consistente com estudos anteriores, agrupando a proteína alvo com outras GH45s de basidiomicetos na subfamília C. A enzima purificada foi testada para determinação da especificidade pelo substrato apresentando maior atividade em arabinoxilana, xilana, arabinoxilana e CMC e foi capaz de gero celo-oligômeros a partir da hidrólise de PASC. O pH ótimo em CMC foi 2,5, além se demonstrar ser temoestável em ensaio de Thermoflour. A hidrólise enzimática de substratos lignocelulósicos derivados de cana-de-açúcar mostrou que a GtGH45 foi eficiente para suplementar coqueteis enzimáticos comerciais, principalmente na conversão de xilana.White-rot and brown-rot basidiomycetes are able to transform lignocellulosic materials through different mechanisms. The brown-rot fungi are able to degrade cellulose and hemicellulose meanwhile modifies lignin. The hydrolytic enzymes of these fungi act on a lignin-enriched substrate, which makes them targets to prospect new enzymes for polysaccharides hydrolysis. Gloeophyllum trabeum is one of the best understood fungal species in this group. An interesting processive GH5-endoglucanase (EG) has been described in this species suggesting an unusual pathway for lignocellulosic degradation without cellobiohydrolases (CBH). Our first attempt to clone this GtGH5 was default but our grup is trying a new attempt of heterologous expression of this protein. Furthermore, G. trabeum genome points out for a low molar mass GH45-EG. Here we report on a recombinant high-yield G. trabeum ATCC 11539 GtGH45 production system. Expression and secretion of endoglucanase GtGH45 was positive after 72 h incubation of the transformed A. nidulans stain A773 in stationary liquid cultures (maltose as inductor). The SDS-PAGE electrophoresis of ultra-filtrated extract showed a single-band GtGH45 over expressed band. The molar mass of 18,4 KDa was consistent with the predicted 204 amino acids sequence derived from gene sequence analyses and corroborates with mass spectometry characterization (18,9 KDa). Even more, the phylogenetic analyze is consistent to previews studies, clustering the target protein with others GH45 from basidiomycetes at subfamily C. The purified protein was assayed for substrate specificity showing activity against xylan, arabinoxylan and PASC. GtGH45 was able to produce cello-oligomers from PASC. The optimum pH was 2.5 with CMC as the substrate. Xylan conversion was enhanced when GtGH45 was mixed with commercial enzymes in enzymatic hydrolysis of alkaline-sulfite pretreated sugar cane bagasse.
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Mulinari, Evandro José. "Expressão heteróloga em Aspergillus nidulans e caracterização bioquímica e estrutural de uma endoglucanase de Aspergillus terreus". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-07052015-085141/.
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A degradação enzimática rápida, eficiente e robusta de polissacarídeos derivados de biomassa lignocelulósica é atualmente um grande desafio na produção de biocombustíveis e considerada uma alternativa viável e promissora para se enfrentar a crise energética mundial e diminuir a dependência das fontes fósseis de energia. O bagaço de cana-de-açúcar no Brasil é a principal matéria lignocelulósica sustentável de grande potencial para a produção do etanol de 2ª geração. O principal requisito para a consolidação dessa abordagem é a disponibilidade de enzimas que hidrolisam a celulose, hemicelulose e outros polissacarídeos em açúcares fermentescíveis e em condições adequadas para a utilização industrial. O presente estudo visou à caracterização molecular, estrutural e funcional da endoglucanase GH12 do fungo Aspergillus terreus (AtGH12) por diferentes técnicas. O gene que codifica para essa enzima foi clonado e expressado no fungo filamentoso A. nidulans linhagem A773. A cepa com maior secreção foi selecionada e a sequência da enzima confirmada por espectrometria de massas MALDI TOF MS. Posteriormente, através de estudos funcionais de parametrização enzimática como pH e temperatura ótimos, estabilidade térmica, efeitos supressores e potencializadores de aditivos, a enzima AtGH12 foi caracterizada bioquímica e fisicamente. A espectrometria de massas do substrato hidrolisado pela catálise enzimática foi tomada como uma forma de investigar o padrão de clivagem da hidrólise e estudo do reconhecimento enzima/substrato para a AtGH12. As caracterizações estruturais das enzimas recombinantes obtidas utilizando as técnicas de espalhamento dinâmico de luz, dicroísmo circular, espalhamento de raios X a baixo ângulo e gel nativo serviram para determinação do enovelamento e estado oligomérico em solução da AtGH12. Com o intuito de fornecer subsídios para o desenvolvimento de coquetéis enzimáticos mais eficazes para hidrólise da biomassa lignocelulósica, a atividade da AtGH12 foi avaliada frente ao bagaço de cana-de-açúcar pré-tratados pelos processos hidrotérmicos e organossolve. Posteriormente, o seu grau de sinergismo nesse tipo de substrato foi determinado com o coquetel enzimático comercial Acellerase®.Fast, more efficient and robust enzymatic degradation of lignocellulosic biomassderived polysaccharides is currently a major challenge in the production of biofuels and considered a feasible and promising alternative to confront the global energy crisis and reduce the dependence on fossil energy resources. The sugarcane bagasse in Brazil is the most abundant and sustainable lignocellulosic material for the production of 2nd generation ethanol. The main requirement for the consolidation of this approach is the availability of enzymes that hydrolyze cellulose, hemicelluloses and other polysaccharides into fermentable sugars suitable for industrial use. The present study was aimed at molecular, structural and functional characterization of an endoglucanase from the fungus Aspergillus terreus (AtGH12) using different techniques. The gene encoding this enzyme has been cloned and expressed in the filamentous fungus Aspergillus nidulans strain A773. The strain with increased secretion was selected and the enzyme sequence was confirmed by mass spectroscopy MALDI TOF MS. Later, functional studies such as analysis of optimal pH and temperature, thermal stability, suppression and enhance effects of additives were applied to the AtGH12 characterization. The mass spectrometry of hydrolyzed substrate from the enzyme catalysis was acquired as a way to investigate the cleavage pattern of hydrolysis and the study of the enzyme/substrate interaction. Structural characterization of the recombinant enzymes was obtained using techniques such as dynamic light scattering, circular dichroism as well as small angle X-ray scattering and native gel, aided to determine the folding and oligomeric state of AtGH12 in solution. In order to provide support for the development of more effective enzyme cocktails for hydrolysis of lignocellulosic biomass, the activity of AtGH12 was evaluated using sugarcane bagasse pretreated by hydrothermal and organosolv processes. Subsequently, the degree of synergism in this type of substrate was measured using a commercial enzyme cocktail Acellerase®.
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Molina, Gustavo Avelar. "Caracterização biofísica da dinâmica catalítica de uma xilanase GH11". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/59/59138/tde-17042016-155242/.
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A dinâmica estrutural fundamentando a função das xilanases GH11 ainda não está clara. Novo conhecimento sobre a dinâmica catalítica dessas enzimas é crucial para a engenharia de novas enzimas melhoradas beneficiando, assim, diversas indústrias biotecnológicas e de química verde. Com base nesse fato, esse trabalho teve por objetivo obter novas informações acerca da dinâmica catalítica de uma xilanase GH11, através do uso de um conjunto de diversas técnicas avançadas de biofísica molecular em nível bulk e em nível de molécula única (inglês single molecule ou sm). Para isso, foram projetadas xilanases GH11 de Bacillus subtilis ssp. subtilis 168 (XynA) com mutações únicas de cisteína para a marcação dos resíduos D119 e R122 no domínio polegar, do resíduo N54 no domínio dedos, e do resíduo N151 na alfa-hélice, seguidas pela sua construção e produção por métodos de biologia molecular. Esses mutantes foram marcados em seus respectivos grupos tióis com a sonda fluorescente sensível à polaridade Acrylodan, com a sonda de spin MTSSL, e com a sonda fluorescente fotoestável AttoOxa11. A xilanase tipo selvagem for marcada em seu N-terminal com a sonda fotoestável Alexa Fluor® 488 5-SDP Ester. Foram utilizados ensaios de espectrofotometria de fluorescência em nível bulk e de espectroscopia de ressonância paramagnética eletrônica para investigar como a dinâmica do domínio polegar da xilanase GH11, temperatura, e ligação ao substrato se correlacionam um com o outro. Os resultados atestaram que um estado do domínio polegar controlado por temperatura, aberto, dinâmico e flexível tem mais chances de se ligar efetivamente ao substrato de uma maneira produtiva, o que está em completo acordo com estudos anteriores de simulação de dinâmica molecular, cristalografia, desnaturação térmica, e análise funcional por desenho racional de mutantes de domínio polegar de xilanases GH11. Com base nas evidências adquiridas e em estudos anteriores, nós propomos um conjunto de hipóteses e modelos para a dinâmica catalítica da xilanase, focando no papel do domínio polegar nesse processo. No intuito de determinar a constante de afinidade da xilanase por seu substrato e os tempos de relaxamento e constantes de velocidade dos movimentos do domínio polegar, foram feitas medidas de espectroscopia de correlação de fluorescência simples e combinada com transferência eletrônica fotoinduzida, usando as xilanases marcadas com as sondas fluorescentes fotoestáveis, na presença e na ausência de substrato. Os resultados mostraram tempos de difusão muito maiores para as xilanases na presença de substrato, como efeito da afinidade da enzima pelo mesmo. Entretanto, não foi verificada nenhuma curva de decaimento como efeito de supressão dinâmica da sonda por PET. Esses mesmos conjugados foram aplicados com sucesso em microscopia por imagem de tempo de vida de fluorescência, no intuito de analisar sistematicamente a afinidade da xilanase por partículas insolúveis e filmes de substrato, e por fragmentos insolúveis de frações de processos de deslignificação e desestruturação de bagaço de cana-de-açúcar, assim como para a análise da composição, estrutura e topologia desses materiais. Foi possível verificar a presença de xilano na maioria das frações desse bagaço tratado, mas em quantidades variáveisThe structural dynamics underlying the function of GH11 xylanases is still unclear. New insights into the catalytic dynamics of these enzymes are crucial for engineering novel improved enzymes benefiting biotechnological and green chemistry industries. The objective of this work was to obtain new information concerning the catalytic dynamics of a GH11 xylanase, by using a combination of advanced molecular biophysics techniques, both at the bulk level and at the single molecule level (sm). Mutant GH11 xylanases from Bacillus subtilis ssp. subtilis 168 (XynA) were designed with single point cysteine mutations for labeling the residues D119 and R122 on the thumb domain, N54 on the fingers domain, and N151 on the alpha helix, followed by their construction and production by molecular biology methods. These mutants were labeled at their respective thiol groups by the polarity sensitive fluorescent probe Acrylodan, by the electron spin probe MTSSL, and by the photostable fluorescent probe AttoOxa11. The wild-type xylanase was labeled at its N-terminus by the photostable fluorescent probe Alexa Fluor® 488 5-SDP Ester. Bulk fluorescence spectrophotometry and electron paramagnetic resonance assays were used to investigate how the thumb domain dynamics of the GH11 xylanase, temperature and substrate binding were correlated. These results demonstrated that a temperature controlled, open, dynamical and flexible thumb domain state is more likely to effectively bind the substrate in a productive way, which is in complete agreement with previous studies from molecular dynamics simulations, crystallography, thermal denaturation, and function analysis by the rational design of thumb mutants for GH11 xylanases. Based on this evidence and previous studies, we proposed a hypothesis for the xylanase catalytic dynamics, focusing on the role of the thumb domain. In order to determine the xylanase affinity constant for its substrate and the relaxation times and rate constants of the thumb domain movements, fluorescence correlation spectroscopy measurements were performed. Both simple and combined measurements with photoinduced electron transfer were performed, using the xylanases labeled with photostable fluorescent probes, in the presence and absence of substrate. The results have shown longer diffusion times for the xylanases in the presence of substrate, as an effect of the enzyme affinity for it. However, it was not verified any decay curve as an effect of the dynamic suppression of the probe via PET. The same conjugates were successfully applied to fluorescence-lifetime imaging microscopy, aiming to systematically analyze the affinity for xylanase of substrates in the form of insoluble particles and films, and for water insoluble fractions from sugarcane bagasse delignification processes. In addition, the composition, structure and topology of these materials was examined. It was possible to verify the presence of xylan in most fractions of this treated bagasse, although in variable quantities
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Wang, Yang. "Exploring glycoside hydrolase family 5 (GH5) enzymes". Licentiate thesis, KTH, Glykovetenskap, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-121537.
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In 1990, the classification of carbohydrate-active enzymes (CAZymes) was introduced by the scientist Bernard Henrissat. According to sequence similarity, these enzymes were separated into families with conserved structures and reaction mechanisms. One interesting class of CAZymes is the group of glycoside hydrolases (GHs) containing more than 138000 modules divided into 131 families as of February 2013. One of the most versatile and the largest of these GH families, containing enzymes with numerous biomass-deconstructing activities, is glycoside hydrolase family 5 (GH5). However, for large and diverse families like the GH5 family, another layer of classification is required to get a better understanding of the evolution of diverse enzyme activities. In Paper I, a new subfamily classification of GH5 is presented in order to sort the family members into distinct groups with predictive power. In total, 51 subfamilies were defined. Despite the fact that several hundred GH5 enzymes have been characterized, 20 subfamilies lacking biochemically characterized enzymes and 38 subfamilies without structural data were identified. These highlighted subfamilies contain interesting targets for future investigation. The GH5 family includes endo-β-mannanases catalyzing the hydrolysis of the β-1,4-linked backbone of mannan polysaccharides, which are common hemicelluloses found as storage and structural polymers in plant cell walls. Mannans are commonly utilized as raw biomaterials in food, feed, paper, textile and cosmetic industries, and mannanases are often applied for modifying and controlling the property of mannan polysaccharides in such applications. The overwhelming majority of characterized mannanases are from microbial origin. The situation for plant mannanases is quite different, as the catalytic properties for only a handful have been determined. Paper II describes the first characterization of a heterologously expressed Arabidopsis β-mannanase.År 1990 introducerade forskaren Bernard Henrissat en klassificering av kolhydrataktiva enzymer (CAZymer), enligt vilken enzymerna - baserat på sekvenslikhet - delades in i familjer med konserverade strukturer och reaktionsmekanismer. En intressant CAZym-klass är glykosidhydrolaserna (GH), en klass som i februari 2013 innehöll fler än 138000 katalytiska moduler indelade i 131 olika familjer. En av de största och mest varierade av GH-familjerna är glykosidhydrolasfamilj 5 (GH5), vilken innehåller en mångfald av identifierade enzymaktiviteter relevanta för nedbrytning av biomassa. För stora och diversifierade familjer som GH5 krävs det dock ytterligare en klassificeringsnivå för att bättre förstå evolutionen och uppkomsten av de många förekommande enzymaktiviteterna. I manuskript I presenteras en ny uppdelning av GH5 enzymer i subfamiljer med syfte att dela upp familjemedlemmarna i distinkta grupper som representerar olika funktioner. Utifrån denna klassificering kan sedan ett enzyms funktion förutsägas baserat på vilken subfamilj det tillhör. Totalt definierades 51 subfamiljer. Trots att hundratals GH5 enzymer har karaktäristerats så visade det sig att 20 av subfamiljerna helt saknar biokemiskt karaktäriserade enzymer och 38 av dem saknar publicerade proteinstrukturer. Dessa subfamiljer är särskilt intressanta för framtida studier. GH5-familjen inkluderar endo-β-mannanaser som katalyserar hydrolysen av den β-1,4-länkade huvudkedjan i mannanpolysackarider. Dessa växtpolymerer som ingår i hemicellulosagruppen är vanligt förekommande i cellväggarna, där de fungerar som energilagringsmolekyler eller har en strukturell funktion. Mannaner används ofta som råmaterial för industriell livs- och djurfodersproduktion, papper, textilier och kosmetika. I dessa processer behövs ofta mannanaser för modifiering och kontroll av egenskaperna hos dessa polysackarider. Den överväldigande majoriteten av alla karaktäriserade mannanaser kommer från mikroorganismer. Endast för ett fåtal växtmannanaser har de katalytiska egenskaperna analyserats. Manuskript II beskriver den första karaktäriseringen av ett heterologt uttryckt β-mannanas från Arabidopsis.
QC 20130506
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Dias, Bruno Augusto [UNESP]. "Caracterização funcional e estrutural de uma ?-glucanase GH12 de Aspergillus terreus". Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/108896.
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A conversão enzimática dos polissacarídeos da biomassa é um fator chave no desenvolvimento de bioetanol de segunda geração. A recalcitrância da lignocelulose para a degradação enzimática e o custo elevado de enzimas hidrolíticas necessárias para a despolimerização de polissacarídeos encontrados na parede celular da planta são barreiras significativas para a produção em larga escala e para a comercialização de biocombustíveis e bioprodutos derivados da biomassa vegetal. A fim de aumentar rapidamente a produção de biocombustíveis celulósicos e bioprodutos, existe a necessidade de desenvolver coquetéis enzimáticos mais eficientes e de menor custo para a conversão de biomassa em açúcares fermentáveis. Nesse contexto, o estudo de enzimas degradadoras da parede celular é essencial. No presente trabalho a enzima endo-1,4-?-D-glucanase de Aspergillus terreus (ATEG_09894) foi clonada para expressão em A. nidulans. O teste de expressão mostrou a expressão solúvel da proteína. Sua identidade foi confirmada por espectrometria de massas. O pH ótimo e a temperatura ótima para sua atividade enzimática foram de 5,0 e 55ºC, respectivamente. A desnaturação térmica mostrou que a partir de 60ºC a enzima começa a perder estrutura. Interessantemente a enzima apresenta atividades ?-glucanase e xiloglucanase, tendo preferência por ?-glucano. A caracterização estrutural mostrou que ATEG_09894 foi expressa corretamente e os resultados de SEC e SAXS mostram que a proteína é monomérica em solução e o modelo de sua estrutura tridimensional indica que a superfície eletrostática da molécula contribui para um monômero estável. Os dados apresentados nesse trabalho são importantes pois identificam peculiaridades da enzima ATEG_09894, que atua na degradação da biomassa, sugerem os determinantes de sua seletividade enzimática e apresenta a degradação, não usual, de ?-glucanos...
The enzymatic conversion of polysaccharides from biomass is a key factor in the development of second generation bioethanol. The recalcitrance of lignocellulose to enzymatic degradation and the high cost of hydrolytic enzymes necessary for the depolymerization of polysaccharides found in plant cell wall are significant barriers to large-scale production and commercialization of biofuels and bioproducts derived from plant biomass. In order to rapidly increase the production of biofuel and byproducts cellulosic, a need exists to develop more efficient and lower cost enzymatic cocktails for the conversion of biomass into fermentable sugars. In this context, the study of cell wall degrading enzymes is essential. In this work the enzyme endo-1,4-?-D-glucanase from Aspergillus terreus (ATEG_09894) was cloned for expression in A. nidulans. The expression test showed the expression of a soluble protein. Its identity was confirmed by mass spectrometry. The optimum pH and optimum temperature for the enzyme activity were 5.0 and 55 ºC, respectively. The thermal denaturation showed that above 60 ºC the enzyme starts to lose structure. Interestingly, the enzyme has ?-glucanase and xiloglucanase activities, preferring ?-glucan. Structural characterization showed that ATEG_09894 was expressed correctly folded and the results of SEC and SAXS show that the protein is monomeric in solution and the model of its three dimensional structure indicates that the electrostatic surface molecule contributes to a stable monomer. The data presented in this study are important because they identify peculiarities of ATEG_09894, which acts in the degradation of biomass, suggest the determinants of its selectivity and enzymatic degradation presents, unusual, of ?-glucans and xyloglucans, promising feature for degradation of lignocellulosic material
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Dias, Bruno Augusto. "Caracterização funcional e estrutural de uma β-glucanase GH12 de Aspergillus terreus /". São José do Rio Preto, 2013. http://hdl.handle.net/11449/108896.
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Orientador: Eleni GomesCoorientador: Fábio Márcio Squina
Banca: Roberto da Silva
Banca: Henrique Ferreira
Banca: Leandro Cristante de Oliveira
Banca: André Ricardo de Lima Damásio
Resumo: A conversão enzimática dos polissacarídeos da biomassa é um fator chave no desenvolvimento de bioetanol de segunda geração. A recalcitrância da lignocelulose para a degradação enzimática e o custo elevado de enzimas hidrolíticas necessárias para a despolimerização de polissacarídeos encontrados na parede celular da planta são barreiras significativas para a produção em larga escala e para a comercialização de biocombustíveis e bioprodutos derivados da biomassa vegetal. A fim de aumentar rapidamente a produção de biocombustíveis celulósicos e bioprodutos, existe a necessidade de desenvolver coquetéis enzimáticos mais eficientes e de menor custo para a conversão de biomassa em açúcares fermentáveis. Nesse contexto, o estudo de enzimas degradadoras da parede celular é essencial. No presente trabalho a enzima endo-1,4-β-D-glucanase de Aspergillus terreus (ATEG_09894) foi clonada para expressão em A. nidulans. O teste de expressão mostrou a expressão solúvel da proteína. Sua identidade foi confirmada por espectrometria de massas. O pH ótimo e a temperatura ótima para sua atividade enzimática foram de 5,0 e 55ºC, respectivamente. A desnaturação térmica mostrou que a partir de 60ºC a enzima começa a perder estrutura. Interessantemente a enzima apresenta atividades β-glucanase e xiloglucanase, tendo preferência por β-glucano. A caracterização estrutural mostrou que ATEG_09894 foi expressa corretamente e os resultados de SEC e SAXS mostram que a proteína é monomérica em solução e o modelo de sua estrutura tridimensional indica que a superfície eletrostática da molécula contribui para um monômero estável. Os dados apresentados nesse trabalho são importantes pois identificam peculiaridades da enzima ATEG_09894, que atua na degradação da biomassa, sugerem os determinantes de sua seletividade enzimática e apresenta a degradação, não usual, de β-glucanos...
Abstract: The enzymatic conversion of polysaccharides from biomass is a key factor in the development of second generation bioethanol. The recalcitrance of lignocellulose to enzymatic degradation and the high cost of hydrolytic enzymes necessary for the depolymerization of polysaccharides found in plant cell wall are significant barriers to large-scale production and commercialization of biofuels and bioproducts derived from plant biomass. In order to rapidly increase the production of biofuel and byproducts cellulosic, a need exists to develop more efficient and lower cost enzymatic cocktails for the conversion of biomass into fermentable sugars. In this context, the study of cell wall degrading enzymes is essential. In this work the enzyme endo-1,4-β-D-glucanase from Aspergillus terreus (ATEG_09894) was cloned for expression in A. nidulans. The expression test showed the expression of a soluble protein. Its identity was confirmed by mass spectrometry. The optimum pH and optimum temperature for the enzyme activity were 5.0 and 55 ºC, respectively. The thermal denaturation showed that above 60 ºC the enzyme starts to lose structure. Interestingly, the enzyme has β-glucanase and xiloglucanase activities, preferring β-glucan. Structural characterization showed that ATEG_09894 was expressed correctly folded and the results of SEC and SAXS show that the protein is monomeric in solution and the model of its three dimensional structure indicates that the electrostatic surface molecule contributes to a stable monomer. The data presented in this study are important because they identify peculiarities of ATEG_09894, which acts in the degradation of biomass, suggest the determinants of its selectivity and enzymatic degradation presents, unusual, of β-glucans and xyloglucans, promising feature for degradation of lignocellulosic material
Doutor
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Alsina, Verdú Cristina. "Enginyeria de glicosintases derivades de quitinases GH18 per a la polimerització de quitooligosacàrids". Doctoral thesis, Universitat Ramon Llull, 2019. http://hdl.handle.net/10803/667340.
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Els quitosans i quitooligosacàrids (COS), obtinguts per enzims modificadors de la quitina, presenten un elevat interès biotecnològic a causa de l’important nombre d’aplicacions en àrees tant diverses com són l’agricultura, el tractament d’aigües, la indústria alimentària, la biomedicina i la cosmètica, entre d’altres. D’entre les funcions biològiques dins la indústria medicofarmacèutica destaquen activitats antiinflamatòries, immunoestimulants, antimicrobianes, antitumorals, de prevenció de l’obesitat i control del colesterol, com a vectors per a teràpia gènica i de promoció de la cicatrització i regeneració de la pell. Aquestes propietats biològiques dels COS no només són dependents del grau de polimerització i acetilació sinó que possiblement també depenen del patró d’acetilació d’aquests.
Actualment la síntesi de COS presenta dos problemes principals: poca reproductibilitat entre lots i l’origen animal dels productes, que dificulten la seva utilització en la indústria medicofarmacèutica. Amb l’objectiu de sobreposar-se a aquestes limitacions s’ha pretès desenvolupar una plataforma biotecnològica per a la producció de COS de baix pes molecular amb seqüències definides. El projecte pretén utilitzar l’activitat transglicosidasa de les quitinases com a eina sintètica per a obtenir oligòmers de quitina i quitosà definits amb patrons d’acetilació repetitius.
Les quitinases són glicosil hidrolases que catalitzen la hidròlisi d’enllaços glicosídics β1,4 de polímers de quitina i quitosà. Algunes quitinases presenten també activitat de transglicosidació (TG) mitjançant la qual són capaces d’introduir nous enllaços glicosídics entre un molècula donadora i una acceptora amb la conseqüent generació de COS oligomèrics. Aquestes quitinases poden utilitzar-se per a la polimerització in vitro de COS, però l’activitat hidrolítica que presenten tendeix a despolimeritzar els productes de TG ràpidament.
Amb l’objectiu d’augmentar l’activitat de TG de quitinases per a la obtenció de nous COS estructuralment definits, en aquesta tesi s’ha aplicat l’estratègia glicosintasa (GS) sobre diferents quitinases de la família GH18 per mutació del residu assistent i l’ús d’un derivat oxazolina com a donador, amb les que s’ha aconseguit una important disminució de l’activitat hidrolítica i un increment de l’activitat de TG. Tot i l’increment de l’activitat de TG, l’activitat hidrolítica residual que presenten dona lloc a la hidròlisi dels productes GS i de TG formats que impedeix augmentar el rendiment. Amb aquest propòsit s’ha optat per la modificació d’un dels enzims seleccionats per enginyeria de proteïnes. Mitjançant mutagènesi dirigida s’ha aconseguit incrementar el rendiment en polímer fins a un 60% (p/p) amb l’ús d’un derivat oxazolina d’un COS de quitina (el 60% del qual correspon al producte de la reacció GS), i també la obtenció d’oligòmers/polímers de quitosà amb l’ús de derivats oxazolina de quitooligosacàrids parcialment desacetilats, estructuralment definits.Los quitosanos y quitooligosacáridos (COS), obtenidos por enzimas modificadores de la quitina, presentan un elevado interés biotecnológico debido al importante número de aplicaciones en áreas tan dispares como son la agricultura, el tratamiento de aguas, la industria alimenticia, la biomedicina y la cosmética, entre otras. Entre las funciones biológicas dentro de la industria médico-farmacéutica destacan actividades antiinflamatorias, inmunoestimulantes, antimicrobianas, antitumorales, de prevención de la obesidad y control del colesterol, como vectores de terapia génica y de promoción de la cicatrización y regeneración de la piel. Estas propiedades biológicas de los COS no solo son dependientes del grado de polimerización y acetilación sino que posiblemente también dependen del patrón de acetilación de estos. Actualmente la síntesis de COS presenta dos problemas principales: poca reproducibilidad entre lotes y el origen animal de los productos, que dificultan su utilización en la industria médico-farmacéutica. Con el objetivo de sobreponerse a estas limitaciones se ha pretendido desarrollar una plataforma biotecnológica para la producción de COS de bajo peso molecular con secuencias definidas. El proyecto pretende utilizar la actividad transglicosidasa de las quitinasas como herramienta sintética para obtener oligómeros de quitina y quitosano definidos con patrones de acetilación repetitivos. Las quitinasas son glicosil hidrolasas que catalizan la hidrólisis de enlaces glicosídicos β1,4 de polímeros de quitina y quitosano. Algunas quitinasas presentan también actividad de transglicosidación (TG) mediante la cual son capaces de introducir nuevos enlaces glicosídicos entre una molécula donadora y una aceptora con la consecuente generación de COS oligoméricos. Estas quitinasas pueden utilizarse para la polimerización in vitro de COS, pero la actividad de hidrólisis que presentan tiende a despolimerizar los productos de TG rápidamente. Con el objetivo de aumentar la actividad de TG de quitinasas para la obtención de nuevos COS estructuralmente definidos, en esta tesis se ha aplicado la estrategia glicosintasa (GS) sobre diferentes quitinasas de la familia GH18 por mutación del residuo asistente y el uso de un derivado oxazolina como donador, con las que se ha logrado una importante disminución de la actividad de hidrólisis y un incremento de la actividad de TG. A pesar del incremento de la actividad de TG, la actividad hidrolasa residual que presentan da lugar a la hidrólisis de los productos GS y de TG formados que impide el aumento del rendimiento. Con este propósito se ha optado por la modificación de una de las enzimas seleccionadas por ingeniería de proteínas. Mediante mutagénesis dirigida se ha logrado incrementar el rendimiento en polímero hasta un 60% (p/p) con el uso de un derivado oxazolina de un COS de quitina (el 60% del cual corresponde al producto de la reacción GS), y también la obtención de oligómeros/polímeros de quitosano con el uso de derivados oxazolina de quitooligosacáridos parcialmente desacetilados, estructuralmente definidos.
Chitosan and chitooligosaccharides (COS), obtained by chitin-modifying enzymes, are of interest to a wide variety of areas such as agriculture, water treatment, food industry, biomedicine and cosmetics, among others, due to their important number of applications. Among the biological roles on the medical-pharmaceutical industry are amply demonstrated anti-inflammatory, immunostimulants, antimicrobians and antitumorals activities, obesity prevention and cholesterol control, ability of gene and drug delivery, wound healing and skin regeneration activities. These biological properties are not only related to their degree of polymerization and acetylation, but possibly they are also dependent on their pattern of acetylation. Nowadays the synthesis of COS has two main hurdles: a poor reproducibility between batches and the animal origin of the products, which difficult their usage in the medical-pharmaceutical industry. With the goal of overcome these limitations, we were aiming at the development of a biotechnological platform for the production of sequence-defined low molecular weight chitosans. The project addresses the use of the transglicosidase activity of chitinases as a synthetic tool to obtain well-defined oligomers with repeating patterns of acetylation. Chitinases are glycoside hydrolases that catalyse the hydrolysis of β1,4 glycosidic bonds of chitin and chitosan polymers. Some chitinases have also transglycosydase activity (TG), allowing them to introduce new glycosidic bonds between donor and acceptor sugar molecules with the consequent generation of oligomeric COS. Such transglycosylating chitinases can be used for the in vitro polymerization of COS, but the hydrolytic activity of these enzymes tends to depolymerise the TG products quickly. With the main goal of increase TG activity of chitinases to obtain new well-defined COS, in the present work we use the glycosynthase technology (GS) on different GH18 chitinases by mutation of the assisting residue and the use of an activated glycosyl donor (an oxazoline derivative), with which an important diminish of the hydrolytic activity and an increase of the TG activity have been obtained. Despite the higher TG activity, the residual hydrolytic activity of the assisting residue mutants results in the hydrolysis of the GS and TG products that not allow the increase of the polymer yield. Protein engineering (rational approach) was used to modify one of the selected enzymes. By site-directed mutagenesis it has been possible to increase the polymer yield up to 60% (w/w) using a chitin oligomer oxazoline derivative (the 60% of which corresponds to the product of the GS reaction), and it has also been possible to obtain chitosan oligomers/polymers using structurally defined partially deacetylated COS oxazoline derivatives.
Książki na temat "GH15"
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Thorpe, David. Panasonic GH5 Menu System Simplified. Independently Published, 2017.
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Keast, Greg. Panasonic GH5: Video Quick Start and Basic Reference Guide. Independently Published, 2019.
Znajdź pełny tekst źródłaCzęści książek na temat "GH15"
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André-Leroux, Gwénaëlle. "Structural Insights: Toward a Molecular Comprehension of the GH13 Amylase Specificity". W ACS Symposium Series, 170–85. Washington, DC: American Chemical Society, 2006. http://dx.doi.org/10.1021/bk-2006-0930.ch009.
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Qiu, Haiyan, Zhongyuan Li, Hui Wang, Shuang Li i Tongcun Zhang. "A Novel GH10 Xylanase Xyn13-3 from Alkaline Soil: Gene Cloning and Heterogenous Expression". W Lecture Notes in Electrical Engineering, 97–103. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4801-2_10.
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Trovalim Jordão, Felipe, Aline Diniz Cabral, Felipe Baena Garcia, Edmar Silva Santos, Rodrigo Buzinaro Suzuki, Max Mario Fuhlendorf i Márcia Aparecida Sperança. "Chitinase from Basal Trypanosomatids and Its Relation to Marine Environment: New Insights on Leishmania Genus Evolutionary Theories". W Chitin-Chitosan - Isolation, Properties, and Applications [Working Title]. IntechOpen, 2023. http://dx.doi.org/10.5772/intechopen.111471.
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Leishmaniasis, an infectious disease that affects humans, domestic dogs, and wild animals, is caused by 20 of the 53 Leishmania genus species and is transmitted by sandflies. Despite its significant impact, the disease is often neglected. Leishmania genus, belong to Trypanosomatide Family and Kinetoplastida Order, are grouped in five subgroups according to biogeographic and evolution history of parasites and hosts. The GH18 Leishmania chitinase is encoded by a specie-specific single copy gene, conserved in basal groups of trypanosomatids, and is absent in the genus Trypanosoma. Preservation of the chitinase genomic locus in the aquatic free-living protozoan Bodo saltans, discloses a primitive common origin. Trypanosomatid chitinase amino acid sequence comparative analysis revealed high similarity with chitinase from sea living prokaryotes and protozoan microorganisms, indicating a probable marine origin. Amino acid sequence comparative analysis revealed that perhaps the trypanosomatid chitinase derived from a water living Kinetoplastida ancestor and its phylogenetic reconstruction corroborates the Supercontinent Origins theory for Leishmania. The chitinase-encoding gene was effective for differential molecular diagnosis among Leishmania clinical important species worldwide.
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"m na atg io n n if s i . edNw at hue ra nlphlaazcaerddsinre th su eltco in n te si xgtnioff ic d an ev telloosps in ogfn af aftliiocn te adl by drought request life and serious economic, environmental, and social sodes are conm ot m re upno it r y te odr . dTohnuorasgso istance from the inter impacts that greatly retard the development process. those that occurred in Austra s l , ias , eBvveerrendmr ents, these epi Figure 1.1 illustrates the trend of major natural dis England, the United States, and mra az niyl, ou o C th agnhatdsa , suS ch as asters between 1963 and 1992, expressed as the num in recent years are not included in these sta etriscto ic usnp tr aiiens , tboetraloafndniu sa a sters affecting 1 per cent or more of the . these disaste lrsgrboyss ty npaet , i o il n lu asltrparto in dgutcht. atFd ig ro u u re gh1t . , 2flroaondkss , aon cc dutrrro in p g ic d al ursitnogrm th siswpeerreiotdh . eTm he osCtefn re tr qeufeonrtRde isasters Drough in the Epidemiology of Disasters (Blaikie et al. s1e9a9 rc 4 h ) acfofm ec ptlienxtbiustc le onsidered by many to be the mo g more a st puenodpe le r sto th oadn of aan ll y na o tu th ra elr ha hzaazradsst , sgh ro ouwpnedthnaattutrhaeldniu sa m st beerrococfud rr reonucgehb ts y d in eccraedae and has (Hagman 1984). For example, in sub-Saharan Afri rd 62 in the 1960s to 237 during the 1980s. H se odwe fr voemr, t th oehdarvoeugahdtvseo rs fet ly heaefa fe rl cytetdommiodr -e 19t8h0asn ar 4e0 re m po il rltcead , tohneeseoff ig u th re esfm or osdtrouungdhet are misleading. Drought is people (Office of Foreign Disaster Assistance 199 ion because the sources of mos rtreopfotrhteesdesn ta attiu st riaclsad re is a in st teerrs Tmh il e li o1n99p1e -o 2plderoaungdhtre in su s lt oeudthienrnaAd fr eifciaciat ff oefc te cde02 ) 0 . national aid or donor organisations. Unless countries supplies of more than 6.7 million tonnes (SAD r C ea C l * = 1 % or more of total annual GNP". W Droughts, 34–35. Routledge, 2016. http://dx.doi.org/10.4324/9781315830896-25.
Pełny tekst źródłaStreszczenia konferencji na temat "GH15"
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Maati, A., G. Mouchon, J. Belluot, P. Augoyat, M. Dinari, T. Huet, V. Serru, M. Camiade i A. Katz. "Design and Characterization of a Ka Band 40 W RF Chain Based on GH15-10 GaN Technology for Space Solid State Power Amplifier Applications". W 2020 50th European Microwave Conference (EuMC). IEEE, 2021. http://dx.doi.org/10.23919/eumc48046.2021.9338161.
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Кулакова, Е. Г., Е. В. Нагаева i О. Б. Безлепкина. "МУТАЦИЯ В ГЕНЕ GH1 - ОПИСАНИЕ СЕМЕЙНОГО СЛУЧАЯ". W III Конференция по орфанным и детским эндокринным заболеваниям «Молекулярно-генетические исследования в практике детского эндокринолога. ФГБУ «НМИЦ эндокринологии» Минздрава России, 2023. http://dx.doi.org/10.14341/mgsppe-2023-51.
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Floriot, D., V. Brunel, M. Camiade, C. Chang, B. Lambert, Z. Ouarch-Provost, H. Blanck i in. "GH25-10: New qualified power GaN HEMT process from technology to product overview". W 2014 9th European Microwave Integrated Circuits Conference (EuMIC). IEEE, 2014. http://dx.doi.org/10.1109/eumic.2014.6997833.
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Dong, Changsheng, Minlin Zhong, Dongye Zhang, Hongjun Zhang i Wenjin Liu. "High temperature performance of laser deposition GH105 layers on nickel base super alloy blade". W PICALO 2010: 4th Pacific International Conference on Laser Materials Processing, Micro, Nano and Ultrafast Fabrication. Laser Institute of America, 2010. http://dx.doi.org/10.2351/1.5057218.
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de Almeida Scarcella, Ana Sílvia, Ana Vici, Liliane Ribeiro, Aline Polizeli i Maria de Lourdes Teixeira de Moraes Polizeli. "HIPEREXPRESSÃO DE UMA XILANASE DA FAMÍLIA GH10 DE Malbranchea pulchella EM Aspergillus nidulans UTILIZANDO MEIO DE CULTIVO DE BAIXO CUSTO EM BIORREATOR". W Simpósio Nacional de Bioprocessos e Simpósio de Hidrólise Enzimática de Biomassa. Campinas - SP, Brazil: Galoá, 2015. http://dx.doi.org/10.17648/sinaferm-2015-33516.
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LAZEBNAYA, I. V., O. E. LAZEBNY i YU A. STOLPOVSKY. "DISTRIBUTION OF GH1, GHR, AND PRL GENE POLYMORPHISMS IN TWO TURANO MONGOLIAN CATTLE BREEDS FROM RUSSIA, CHINA, AND MONGOLIA". W 5TH MOSCOW INTERNATIONAL CONFERENCE "MOLECULAR PHYLOGENETICSAND BIODIVERSITY BIOBANKING". TORUS PRESS, 2018. http://dx.doi.org/10.30826/molphy2018-27.
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de Colstoun, F. Brown, C. W. Lowry, G. Khitrova, H. M. Gibbs, A. E. Paul, S. W. Koch, T. M. Brennan i B. E. Hammons. "Asymmetric Gain in a Vertical-Cavity Surface-Emitting Laser". W Quantum Optoelectronics. Washington, D.C.: Optica Publishing Group, 1993. http://dx.doi.org/10.1364/qo.1993.qwa.5.
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The gain spectrum of a semiconductor can be locally modified by a strong nearly resonant laser field through population pulsations.1,2 Rayleigh-gain, an asymmetric modification of the gain profile of an atom, has been investigated previously.3,4 Laser diode longitudinal modes have been used to show gain asymmetry1,5, but the mode spacing (75 GHz5) has limited the observation to the periphery of the curve. Mapping the more closely spaced gain peak and dip is important because they can significantly modify the output spectrum of a laser (creating new lasing lines at the new gain peak and extinguishing the original lasing with the gain dip). Pump/probe experiments in traveling wave amplifiers (TWA)6 have shown the gain to be asymmetric, but the gain peak and dip were broadened because the injected signal grew in power (from ≃ 87 μW to 4.4mW6) during propagation. Using a high Q vertical-cavity surface-emitting laser (VCSEL) as the gain medium we have intracavity powers at the injected frequency that do not grow with propagation and are much higher than the TWA experiments (up to 75 mW in a 5 μm spot), pushing the gain peak as far as 36.5 GHz from the injected frequency. Rather than using longitudinal modes to probe discretely the gain asymmetry, when the gain extrema are within 10 GHz of the injected frequency we directly observe localized dips and peaks in the continuous ouput spectrum of the VCSEL. As increased injected power pushes the gain extrema out further, we observe modification of the line shape and pushing of the lasing. We find that the detuning of the gain extrema from the injected frequency increases linearly with the intracavity intensity at the injected frequency (in contrast with relaxation oscillations and other effects that scale with the field level). Understanding local modifications of the cw gain profile is of crucial importance to elucidate instabilities in semiconductors lasers.