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  1 / 33 MEDLINE  
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[PMID]:27900666
[Au] Autor:Jain KK; Kumar S; Deswal D; Kuhad RC
[Ad] Endereço:Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
[Ti] Título:Improved Production of Thermostable Cellulase from Thermoascus aurantiacus RCKK by Fermentation Bioprocessing and Its Application in the Hydrolysis of Office Waste Paper, Algal Pulp, and Biologically Treated Wheat Straw.
[So] Source:Appl Biochem Biotechnol;181(2):784-800, 2017 Feb.
[Is] ISSN:1559-0291
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Thermostable cellulases have wide variety of applications and distinctive advantages, but their low titer becomes the hurdle in their commercialization. In the present work, an assessment of optimum levels of significant factors (temperature, moisture ratio, inoculum size, and ammonium sulfate) and the effect of their interactions on production of thermostable CMCase, FPase, and ß-glucosidase by Thermoascus aurantiacus RCKK under solid-state fermentation (SSF) was carried out using central composite design (CCD) of response surface methodology (RSM). The study revealed 33, 13, and 8 % improvement in FPase, CMCase, and ß-glucosidase production, respectively. Moreover, crude cellulase from T. aurantiacus RCKK efficiently hydrolyzed office waste paper, algal pulp (Gracillaria verulosa), and biologically treated wheat straw at 60 °C with sugar release of about 830 mg/ml, 285 mg/g, and 260 mg/g of the substrate, respectively. The thermostable enzyme from T. aurantiacus RCKK holds potential to be used in biofuel industry.
[Mh] Termos MeSH primário: Celulase/biossíntese
Celulase/química
Resíduos Industriais/prevenção & controle
Papel
Thermoascus/enzimologia
Triticum/química
[Mh] Termos MeSH secundário: Biodegradação Ambiental
Estabilidade Enzimática
Eucariotos/química
Temperatura Alta
Caules de Planta/química
Eliminação de Resíduos/métodos
Especificidade da Espécie
Thermoascus/classificação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Industrial Waste); EC 3.2.1.4 (Cellulase)
[Em] Mês de entrada:1702
[Cu] Atualização por classe:170206
[Lr] Data última revisão:
170206
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161201
[St] Status:MEDLINE
[do] DOI:10.1007/s12010-016-2249-7


  2 / 33 MEDLINE  
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[PMID]:27510979
[Au] Autor:Ozawa K; Iwasa H; Sasaki N; Kinoshita N; Hiratsuka A; Yokoyama K
[Ad] Endereço:Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
[Ti] Título:Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.
[So] Source:Appl Microbiol Biotechnol;101(1):173-183, 2017 Jan.
[Is] ISSN:1432-0614
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T ) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.
[Mh] Termos MeSH primário: Fungos/enzimologia
Glucose Desidrogenase/isolamento & purificação
Glucose Desidrogenase/metabolismo
Temperatura Alta
Talaromyces/enzimologia
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Clonagem Molecular
Coenzimas/análise
Estabilidade Enzimática
Escherichia coli/genética
Escherichia coli/metabolismo
Flavina-Adenina Dinucleotídeo/análise
Fungos/genética
Expressão Gênica
Glucose Desidrogenase/química
Glucose Desidrogenase/genética
Pichia/genética
Pichia/metabolismo
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/isolamento & purificação
Proteínas Recombinantes/metabolismo
Análise Espectral
Talaromyces/genética
Thermoascus/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Coenzymes); 0 (Recombinant Proteins); 146-14-5 (Flavin-Adenine Dinucleotide); EC 1.1.1.- (Glucose Dehydrogenases)
[Em] Mês de entrada:1701
[Cu] Atualização por classe:170620
[Lr] Data última revisão:
170620
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160812
[St] Status:MEDLINE
[do] DOI:10.1007/s00253-016-7754-7


  3 / 33 MEDLINE  
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[PMID]:27554938
[Au] Autor:de Souza AR; de Araújo GC; Zanphorlin LM; Ruller R; Franco FC; Torres FA; Mertens JA; Bowman MJ; Gomes E; Da Silva R
[Ad] Endereço:UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil.
[Ti] Título:Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756.
[So] Source:Int J Biol Macromol;93(Pt A):20-26, 2016 Dec.
[Is] ISSN:1879-0003
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.
[Mh] Termos MeSH primário: Endo-1,4-beta-Xilanases/química
Proteínas Fúngicas/química
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Endo-1,4-beta-Xilanases/genética
Estabilidade Enzimática
Proteínas Fúngicas/genética
Concentração de Íons de Hidrogênio
Simulação de Dinâmica Molecular
Mutagênese Sítio-Dirigida
Estrutura Secundária de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fungal Proteins); EC 3.2.1.8 (Endo-1,4-beta Xylanases)
[Em] Mês de entrada:1704
[Cu] Atualização por classe:170406
[Lr] Data última revisão:
170406
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160825
[St] Status:MEDLINE


  4 / 33 MEDLINE  
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[PMID]:27413773
[Au] Autor:de Oliveira AP; Silvestre MA; Garcia NF; Alves-Prado HF; Rodrigues A; da Paz MF; Fonseca GG; Leite RS
[Ad] Endereço:Laboratory of Enzymology and Fermentation Processes, Faculty of Biological and Environmental Sciences, Federal University of Grande Dourados (FCBA/UFGD), Rodovia Dourados/Itahum, km 12, 79804-970 Dourados, MS, Brazil.
[Ti] Título:Production and Catalytic Properties of Amylases from Lichtheimia ramosa and Thermoascus aurantiacus by Solid-State Fermentation.
[So] Source:ScientificWorldJournal;2016:7323875, 2016.
[Is] ISSN:1537-744X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5-10.5 and pH 4.5-9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product.
[Mh] Termos MeSH primário: Amilases/química
Fermentação
Mucorales/enzimologia
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Hidrólise
Microbiologia Industrial
Amido/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
9005-25-8 (Starch); EC 3.2.1.- (Amylases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160715
[St] Status:MEDLINE
[do] DOI:10.1155/2016/7323875


  5 / 33 MEDLINE  
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[PMID]:27089425
[Au] Autor:Pang ZW; Lu W; Zhang H; Liang ZW; Liang JJ; Du LW; Duan CJ; Feng JX
[Ad] Endereço:State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, People's Republic of China.
[Ti] Título:Butanol production employing fed-batch fermentation by Clostridium acetobutylicum GX01 using alkali-pretreated sugarcane bagasse hydrolysed by enzymes from Thermoascus aurantiacus QS 7-2-4.
[So] Source:Bioresour Technol;212:82-91, 2016 Jul.
[Is] ISSN:1873-2976
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Sugarcane bagasse (SB) is a potential feedstock for butanol production. However, biological production of butanol from SB is less economically viable. In this study, evaluation of eight pretreatments on SB showed that alkali pretreatment efficiently removed lignin from SB while retaining the intact native structure of the released microfibrils. In total, 99% of cellulose and 100% of hemicellulose in alkali-pretreated SB were hydrolysed by enzymes from Thermoascus aurantiacus. The hydrolysate was used to produce butanol in a fed-batch fermentation by Clostridium acetobutylicum. At 60h, 14.17 and 21.11gL(-1) of butanol and acetone-butanol-ethanol (ABE) were produced from 68.89gL(-1) of total sugars, respectively, yielding 0.22 and 0.33gg(-1) of sugars. The maximum yield of butanol and ABE reached 15.4g and 22.9g per 100g raw SB, respectively. This established process may have potential application for butanol production from SB.
[Mh] Termos MeSH primário: Álcalis/farmacologia
Técnicas de Cultura Celular por Lotes/métodos
Butanóis/metabolismo
Celulose/química
Clostridium acetobutylicum/metabolismo
Fermentação
Saccharum/química
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Acetona/metabolismo
Reatores Biológicos/microbiologia
Etanol/metabolismo
Fermentação/efeitos dos fármacos
Hidrólise
Cinética
Saccharum/efeitos dos fármacos
Temperatura Ambiente
Fatores de Tempo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Alkalies); 0 (Butanols); 1364PS73AF (Acetone); 3K9958V90M (Ethanol); 9004-34-6 (Cellulose); 9006-97-7 (bagasse)
[Em] Mês de entrada:1701
[Cu] Atualização por classe:170811
[Lr] Data última revisão:
170811
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160419
[St] Status:MEDLINE


  6 / 33 MEDLINE  
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[PMID]:25435507
[Au] Autor:Xin D; Ge X; Sun Z; Viikari L; Zhang J
[Ad] Endereço:College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China.
[Ti] Título:Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides.
[So] Source:Enzyme Microb Technol;68:62-8, 2015 Jan.
[Is] ISSN:1879-0909
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and ß-glucosidase played an important role in mannan hydrolysis. Addition of 10mg/ml mannan reduced the glucose yield of Avicel (at 20g/l) from 40.1 to 24.3%. No inhibition of ß-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on ß-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.
[Mh] Termos MeSH primário: Proteínas de Bactérias/antagonistas & inibidores
Celulose 1,4-beta-Celobiosidase/antagonistas & inibidores
Mananas/farmacologia
Oligossacarídeos/farmacologia
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Ligação Competitiva
Celulase/metabolismo
Celulose/metabolismo
Hidrólise
Relação Estrutura-Atividade
Trissacarídeos/farmacologia
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Mannans); 0 (Oligosaccharides); 0 (Trisaccharides); 15548-43-3 (mannobiose); 3634-02-4 (mannotetraose); 6817-81-8 (mannotriose); 9004-34-6 (Cellulose); EC 3.2.1.4 (Cellulase); EC 3.2.1.91 (Cellulose 1,4-beta-Cellobiosidase)
[Em] Mês de entrada:1508
[Cu] Atualização por classe:141201
[Lr] Data última revisão:
141201
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:141202
[St] Status:MEDLINE


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[PMID]:25424281
[Au] Autor:Jain KK; Bhanja Dey T; Kumar S; Kuhad RC
[Ad] Endereço:Department of Microbiology, Lignocellulose Biotechnology Laboratory, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
[Ti] Título:Production of thermostable hydrolases (cellulases and xylanase) from Thermoascus aurantiacus RCKK: a potential fungus.
[So] Source:Bioprocess Biosyst Eng;38(4):787-96, 2015 Apr.
[Is] ISSN:1615-7605
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20% inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, ß-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90%, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH(∙) scavenging, FRAP and in vivo antioxidant capacity against H2O2-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.
[Mh] Termos MeSH primário: Celulase/biossíntese
Endo-1,4-beta-Xilanases/biossíntese
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Antioxidantes/química
Biocombustíveis
Biotecnologia
Cromatografia em Camada Delgada
Fermentação
Peróxido de Hidrogênio/química
Concentração de Íons de Hidrogênio
Hidrólise
Ácidos Fosfóricos/química
Filogenia
Saccharomyces cerevisiae/enzimologia
Temperatura Ambiente
Triticum
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Antioxidants); 0 (Biofuels); 0 (Phosphoric Acids); BBX060AN9V (Hydrogen Peroxide); E4GA8884NN (phosphoric acid); EC 3.2.1.4 (Cellulase); EC 3.2.1.8 (Endo-1,4-beta Xylanases)
[Em] Mês de entrada:1512
[Cu] Atualização por classe:150320
[Lr] Data última revisão:
150320
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:141127
[St] Status:MEDLINE
[do] DOI:10.1007/s00449-014-1320-4


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[PMID]:25074836
[Au] Autor:Chawachart N; Anbarasan S; Turunen S; Li H; Khanongnuch C; Hummel M; Sixta H; Granström T; Lumyong S; Turunen O
[Ad] Endereço:Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand.
[Ti] Título:Thermal behaviour and tolerance to ionic liquid [emim]OAc in GH10 xylanase from Thermoascus aurantiacus SL16W.
[So] Source:Extremophiles;18(6):1023-34, 2014 Nov.
[Is] ISSN:1433-4909
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70-75 °C. The enzyme's half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75-80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.
[Mh] Termos MeSH primário: Proteínas de Bactérias/química
Endo-1,4-beta-Xilanases/química
Imidazóis/farmacologia
Líquidos Iônicos/farmacologia
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Proteínas de Bactérias/antagonistas & inibidores
Proteínas de Bactérias/metabolismo
Domínio Catalítico
Endo-1,4-beta-Xilanases/antagonistas & inibidores
Endo-1,4-beta-Xilanases/metabolismo
Estabilidade Enzimática
Temperatura Alta
Simulação de Acoplamento Molecular
Dados de Sequência Molecular
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Imidazoles); 0 (Ionic Liquids); EC 3.2.1.8 (Endo-1,4-beta Xylanases); TVE1D62MAK (1-ethyl-3-methylimidazolium)
[Em] Mês de entrada:1507
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM; S
[Da] Data de entrada para processamento:140731
[St] Status:MEDLINE
[do] DOI:10.1007/s00792-014-0679-0


  9 / 33 MEDLINE  
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[PMID]:24889637
[Au] Autor:Kjaergaard CH; Qayyum MF; Wong SD; Xu F; Hemsworth GR; Walton DJ; Young NA; Davies GJ; Walton PH; Johansen KS; Hodgson KO; Hedman B; Solomon EI
[Ad] Endereço:Department of Chemistry, Stanford University, Stanford, CA 94305;
[Ti] Título:Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.
[So] Source:Proc Natl Acad Sci U S A;111(24):8797-802, 2014 Jun 17.
[Is] ISSN:1091-6490
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.
[Mh] Termos MeSH primário: Cobre/química
Proteínas Fúngicas/química
Oxigenases de Função Mista/química
Oxigênio/química
Polissacarídeos/química
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Catálise
Domínio Catalítico
Quitina/química
Simulação por Computador
Espectroscopia de Ressonância de Spin Eletrônica
Elétrons
Modelos Moleculares
Espectrofotometria
Superóxidos/química
Termodinâmica
Raios X
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S.
[Nm] Nome de substância:
0 (Fungal Proteins); 0 (Polysaccharides); 11062-77-4 (Superoxides); 1398-61-4 (Chitin); 789U1901C5 (Copper); EC 1.- (Mixed Function Oxygenases); S88TT14065 (Oxygen)
[Em] Mês de entrada:1409
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140604
[St] Status:MEDLINE
[do] DOI:10.1073/pnas.1408115111


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[PMID]:24559135
[Au] Autor:Forsberg Z; Røhr AK; Mekasha S; Andersson KK; Eijsink VG; Vaaje-Kolstad G; Sørlie M
[Ad] Endereço:Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences , P.O. Box 5003, N-1432 Ås, Norway.
[Ti] Título:Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases.
[So] Source:Biochemistry;53(10):1647-56, 2014 Mar 18.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.
[Mh] Termos MeSH primário: Bacillus/enzimologia
Proteínas de Bactérias/metabolismo
Celulose/metabolismo
Quitina/metabolismo
Proteínas Fúngicas/metabolismo
Oxigenases de Função Mista/metabolismo
Serratia marcescens/enzimologia
Streptomyces coelicolor/enzimologia
Thermoascus/enzimologia
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Bacillus/química
Bacillus/genética
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Domínio Catalítico
Cobre/metabolismo
Proteínas Fúngicas/química
Proteínas Fúngicas/genética
Oxigenases de Função Mista/química
Oxigenases de Função Mista/genética
Dados de Sequência Molecular
Alinhamento de Sequência
Serratia marcescens/química
Serratia marcescens/genética
Streptomyces coelicolor/química
Streptomyces coelicolor/genética
Thermoascus/química
Thermoascus/genética
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Fungal Proteins); 1398-61-4 (Chitin); 789U1901C5 (Copper); 9004-34-6 (Cellulose); EC 1.- (Mixed Function Oxygenases)
[Em] Mês de entrada:1406
[Cu] Atualização por classe:140318
[Lr] Data última revisão:
140318
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140225
[St] Status:MEDLINE
[do] DOI:10.1021/bi5000433



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